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SAB 2013

Sociedad Argentina de Biofsica







XLII Reunin Anual
2 - 4 de Diciembre 2013
Villa Carlos Paz, Crdoba
ARGENTINA



SAB 2013







Fanani, Laura
XLII Reunin Anual de la Sociedad Argentina de Biofsica / Laura Fanani ; Natalia
Wilke ; Gerardo Fidelio. - 1a ed. - Buenos Aires : SAB - Sociedad Argentina de
Biofsica, 2013.
146 p. ; 30x21 cm.

ISBN 978-987-27591-2-4

1. Biologa.Investigacin. I. Wilke, Natalia II. Fidelio, Gerardo III. Ttulo
CDD 570.711



Fecha de catalogacin: 07/11/2013


Quedan prohibidos, dentro de los lmites establecidos en la ley y bajo apercibimiento
legalmente previsto, la reproduccin total o parcial de esta obra por cualquier medio o
procedimientos ya sea electrnico o mecnico, el tratamiento informtico, el alquiler o
cualquier otra forma de cesin de la obra sin la autorizacin previa y por escrito de los
titulares del copyright.


Diagramacin y Edicin: Laura Fanani, Natalia Wilke.
Diseo de Tapa: Agustin Mangiarotti, Gerardo Fidelio
Asistencia tcnica web: Marcos Solovey


Impreso en Crdoba, Argentina, Noviembre 2013.


SAB 2013


Comisin Directiva de la Sociedad Argentina de Biofsica Ao 2013
Presidente Gerardo Fidelio
Universidad Nacional de Crdoba

Vicepresidente Gabriela Amodeo
Universidad de Buenos Aires

Presidente saliente Luis Gonzlez Flecha
Universidad de Buenos Aires

Secretario Mauricio Sica
Centro Atmico Bariloche

Tesorera La Pietrasanta
Universidad de Buenos Aires

Vocales Titulares Karina Alleva
Universidad de Buenos Aires

Rosana Chehn
Universidad Nacional de Tucumn

Vocales suplentes Rodolfo Rassia
Universidad Nacional de Rosario

Florencia Martini
Universidad de Buenos Aires

Comit Cientfico SAB 2013 Comit Organizador y Logstica SAB 2013

Dr. Gerardo D Fidelio (UNC)

Dr. Gerardo D Fidelio (UNC)
Dr. Guillermo Montich (UNC)

Dr. Ernesto Ambroggio (UNC)
Dra. Laura Fanani (UNC)

Dr. Benjamn Caruso (UNC)
Dra. Graciela Borioli (UNC)

Dr. Ernesto Grasso (UNC)
Dr. Rafael Oliveira (UNC)

Dra. Soledad Bazn (UNC)
Dr. Ernesto Ambroggio (UNC)

Lic. Pablo Yunes Quartino (UNC)
Dr. Marcos Villarreal (UNC)

Lic. Maria Elisa Mariani (UNC)
Dra. Natalia Wilke (UNC)

Lic. Julio Pusterla (UNC)
Dra. Karina Alleva (UBA)

Lic. Ignacio Gallea (UNC)
Dr. Rodolfo Gonzlez Lebrero (UBA) Lic. Agustin Mangiarotti (UNC)

SAB 2013



Agradecimientos por financiamiento / Acknowledgements for funding





























C I Q U I B I C
C O N I C E T
U N C


SAB 2013



Palabras de Agradecimientos / Words of Acknowledgements
Desde su fundacin, hace 42
aos, SAB organiza su reunin anual en
forma ininterrumpida, congregando a
cientficos nacionales e internacionales
que trabajan el rea de la Biofsica.
En nombre de SAB y como
responsables organizadores de la
Reunin Anual 2013 de la Sociedad, es
para nosotros un honor dar la bienvenida
en Crdoba a los participantes de la XLII
Reunin Anual de SAB. Hacemos
extensivos estos saludos de bienvenida a
los alumnos y docentes/investigadores
asistentes al VI Curso POSLATAM co-
organizado con LAFeBS y auspiciado por
IUPAB.
Queremos agradecer muy
especialmente a los conferencistas y
simposistas que aceptaron venir a
Crdoba y honramos su muestra de
gratitud. Al grupo de Logstica del
Congreso que puso todo de s para una
buena organizacin.
Queremos incluir en los
agradecimientos a las autoridades de
LAFeBs que depositaron su confianza
para organizar el VI Curso POSLATAM
de la regin en Crdoba para 2013.
Por ltimo, agradecemos a la
Facultad de Ciencias Qumicas y a la
Universidad Nacional por su ayuda en
infraestructura y financiacin; a CONICET
y a FONCYT de Agencia Nacional de
Promocin Cientfica y Tecnolgica por
su ayuda financiera. A IUPAB que, a
travs de LAFeBS, ha financiado con
becas el traslado de doctorandos para el
VI Curso POSLATAM. Tengan todos
Ustedes una grata estada en Crdoba.
Since its founding, 42 years ago,
SAB organizes its annual meeting without
interruption, bringing together national
and international scientists working in the
field of biophysics.
On behalf of SAB and as
organizers of the 2013 Annual Meeting of
the Society is an honor to welcome to the
participants of the XLII Annual Meeting of
the SAB held in Crdoba. We want to
extend these greetings to welcome also to
students and teachers/researchers
attending the VI POSLATAM Course co-
organized with LAFeBS and sponsored by
IUPAB.
We want to especially thank to the
speakers who agreed to come to Cordoba
and we honor their selfless displays of
gratitude, and to the Congress Logistics
group for its great labor in the
organization of the Meeting.
In the acknowledgments we want
to include to LAFeBs authorities who
placed their trust in us to organize the VI
POSLATAM Course of the region in 2013.
Finally, we thank to the School of
Chemical Sciences and the National
University of Crdoba for their
infrastructure and financial help; to both
CONICET and the National Agency for
Promoting Science and Technology for
financial assistance. To IUPAB, that
throughout LAFeBS has financed the
transfer of doctoral students to assist to VI
POSLATAM Course.
We wish to all of you a pleasant
stay in Crdoba.

Organizadores / Organizers SAB 2013
SAB 2013


400 palabras sobre la historia de la UNC (http://www.400.unc.edu.ar/)
El origen de la Universidad Nacional de Crdoba fue la promesa de un capital
del Obispo Trejo y Sanabria al padre jesuita Diego de Torres en 1613; independiente
de la autoridad real, aquella Universidad, se gobern y se financi a s misma. La
Compaa de Jess opt por formar lites y clero en un modelo tradicional de
conocimiento, alterado cuando la Orden Franciscana se hizo cargo de la Universidad e
introdujo obras de Descartes, Newton y Leibnitz.
Durante la disolucin del Estado nacional, la Universidad qued en la rbita
provincial hasta su nacionalizacin en 1856. La Ley Universitaria data de 1885. En esa
ltima poca, la UNC se moderniz: elimin la Teologa e incorpor las Facultades de
Fsico-Matemticas y Medicina.
Para 1918, la UNC acumulaba las tensiones de una sociedad en
transformacin y los estudiantes plantearon un conjunto de demandas que fueron
paulatinamente atendidas por el gobierno de H. Yrigoyen.
La UNC no fue ajena a los vaivenes de la poltica: en 1930 como en cada uno
de los cinco golpes militares posteriores padeci intervenciones y restricciones a la
autonoma.
Durante las presidencias de Juan D. Pern las universidades ampliaron su
matrcula y jerarquizaron nuevas reas de conocimiento, que en Crdoba incluy
Ciencias Econmicas, y Filosofa y Humanidades. Luego se incorporaron nuevos
campos como Arquitectura y Urbanismo, Trabajo Social, Matemtica, Astronoma y
Fsica, Agronoma y Comunicacin.
Durante la primavera democrtica de 1973 las expectativas se concentraron en
la renovacin curricular y el compromiso intelectual con el cambio social pero el
proceso se trunc en 1975, cuando la presidenta Pern envi la intervencin a las
universidades nacionales. Desde 1976, la dictadura militar profundiz un proyecto de
universidad elitista y funcional a sus objetivos restringiendo el ingreso, la libertad de
ctedra y anulando el co-gobierno.
El retorno a la democracia en 1983 permiti recuperar la institucionalidad de la UNC
restituyendo el co-gobierno y la autonoma, y renovando la idea de Universidad
comprometida con la sociedad.
La Ley de Educacin Superior de 1995, modific la vida universitaria en aspectos
como la produccin de conocimientos y el rol de las Universidades en las sociedades.
El siglo XXI conforma un escenario diferente en el que hay nuevos horizontes para
disear e implementar polticas de educacin superior que jerarquicen a las
universidades como productoras de ciencia y tecnologa atentas a las demandas del
desarrollo productivo y social en los valores ciudadanos propios de una sociedad
democrtica.

SAB 2013


Indice/Index


VI Curso POSLATAM / VI POSLATAM Course.

i
Programa del Congreso / Meeting Program........ 1
Conferencias Plenarias / Plenary Lectures... 7
Mini-conferencias / Short lectures.. 14
Simposios / Symposia.......... 17
Resumenes de posters / Poster abstracts: ......... 27
Biofsica de lpidos y membranas / Biophysics of lipid and membranes (BLM)... 29
Biofsica de protenas y cidos nuclicos / Biophysics of proteins and nucleic acids
(BPA) ...
52
Enzimologa/ Enzymology (ENZ).. 74
Bioenergtica y transferencia electrnica / Bioenergetics and transfer (BTE) 79
Teora y modelado de sistemas biolgicos / Theory and modeling of biological
systems (TMSB)
81
Transportadores, receptores y canales / Transporters, receptors and channels
(TRC) ...
94
Sealizacin y Dinmica Intracelular / Signaling and Intracellular dynamics (SDI).. 104
Nuevas tcnicas en biofsica / New techniques in biophysics (NTB) 108
Biofsica: Aplicaciones biotecnolgicas / Biophysics: Biotechnological applications
(BBA) ...
111
Indice por Autores / Authors index.. 119
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LATIN AMERICAN FEDERATION OF BIOPHYSICAL SOCIETIES
VI POSLATAM COURSE SCHEDULE
Biophysical approaches to study systems of biological interest.
28
th
November to 4
th
December 2013, Crdoba, Argentina
Dia /Day
Tiempo/Time
Profesor/Procedencia
Professor/Affiliations
Tpicos/Topics
Mircoles 27 de Noviembre / Wednesday 27
th
November LLEGADA DE ESTUDIANTES/ARRIVAL OF STUDENTS.

Lugar de desarrollo del Curso del 28 de noviembre al 1 de Diciembre de 2013 en la Facultad de Ciencias Qumicas, Ciudad Universitaria, Universidad Nacional de Crdoba
(www.fcq.unc.edu.ar). Las conferencias relacionadas al Meeting del 2 al 4 de diciembre 2013 se llevaran a cabo en el hotel sede en Villa Carlos Paz, Crdoba (www.portaldelago.com.ar).
Course development from November 28
th
to December 1
st
2013 will be held at the School of Chemical Sciences, Campus, National University of Cordoba, Crdoba (400
th
Anniversary)
Meeting-related conferences from 2
nd
-
4
th
December 2013 will be held at the Congress Hotel in Villa Carlos Paz, Crdoba (www.portaldelago.com.ar).
Coordinador/Coordinator: Dr. Gerardo Fidelio (mails: gfidelio@mail.fcq.unc.edu.ar ; gerardo.fidelio@gmail.com) web: http://sab2013.fcq.unc.edu.ar/

Jueves 28 de Noviembre/
Thursday 28
th
November

Total Tiempo estimado/Estimated Total time: 6 hs

Maana/Morning (Primera Parte/First Part of Dr. Disalvo 3 hs)
Primera Clase/First Lecture: 9.00-10.30
Caf/Coffe Break: 10.30-10.55
Segunda Clase/Second Lecture: 11.00-12.30


Dr. Anbal Disalvo
Universidad Nacional deSantiago del
Estero. Argentina

MEMBRANE HYDRATION AND BIOLOGICAL FUNCTIONS (First Part)
Thermodynamics of lipid self-assembly in water. Structural and physicochemical
properties of water. Swelling processes: area, thickness and interlamellar space.
Methods and criteria. Hydration Water. Excluded volume and hydration forces.
Densities and distribution of water and hydration centers in membranes. Influence of
the topology on hydration and water states.
The membrane-solution interphase. Criteria for a new model for membranes.
Surface potentials. Determination of zeta potential. Limitations. Electrophoretic
mobility. Isotherms of adsorption of charged amino acids and peptides.
Synergism and cooperativity. Partition of amino acids and polyols: Corrections
to the Wiener -White and Butler-Barclay rules in relation to the water content.
Almuerzo/Lunch 13.00/14.30
Jueves 28 de Noviembre/Thursday 28
th
November

Tarde/Afternoon Maana/Morning (Segunda Parte/Second Part
of Dr. Disalvo 3 hs)

Primera Clase/First Lecture: 15.00-16.30
Caf/Coffe Break: 16.30-16.55
Segunda Clase/Second Lecture: 17.00-18.30

MEMBRANE HYDRATION AND BIOLOGICAL FUNCTIONS (Second Part)
Water activity in membranes. Water activity and surface pressure. Defay-
Prigogine model. Effect of hydrogen bonding compounds on water activity in
membranes. Relationship between dipolar potential and hydration water.
Aqueous domains. Hydration water and confined water. Water species and
phase states. Effect of lipid composition. Lipidomics and aquaomics.
Lyotropic phenomena. Expansion and contraction of lipid membranes. Water
penetration and dielectric properties. Generation of defects by osmosis and
electrical fields. Surface changes in hypertonic and hypotonic processes.
Kinetics of dehydration and rehydration. Relaxation processes in the insertion
of peptides. Influence of the order parameter and fluctuations. Chemical
potential of water and water activity by peptide insertion. Kinks and water
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species. Translocons and waterons.
Tiempo Libre. Free time

Viernes 29 de Noviembre/Friday 29
th
November

Maana/Morning
Tiempo Estimado/Estimated Time: 3 hs.
Primera Clase/First Lecture: 9.00-10.30
Caf/Coffe Break: 10.30-10.55
Segunda Clase/Second Lecture: 11.00-12.30

Dra. Natalia Wilke
Universidad Nacional deCrdoba.
Argentina

MEMBRANE RHEOLOGY AND ELECTROSTATICS.
Membranes with phasecoexistence: Distribution of the phases at the plane of the
membrane: domain size, domain shape, number of domains. Domain growth, Oswald
ripening, equilibrium and non-equilibrium domain distributions and sizes. Line
tension. Intra and inter-domains long-range interactions.
Membranerheologyand electrostatics: Shear in membranes: free diffusion, solid
obstacles, interacting obstacles. Bending and compression of membranes.
Experimental approaches: Model membranes.
Active and pasive methods in membrane rheology. Manipulation of membranes using
electric fields and optical tweezers.
Almuerzo/Lunch 13.00/14.30
Viernes 29 de Noviembre/Friday 29
th
November

Tarde/Afternoon
Tiempo Estimado/Estimated Time: 3 hs.
Primera Clase/First Lecture: 15.00-16.30
Caf/Coffe Break: 16.30-16.55
Segunda Clase/Second Lecture: 17.00-18.30

Dr. Luis BAGATOLLI
MembraneBiophysics and Biophotonics
Group/MEMPHYS - Center for
BiomembranePhysics, Department of
Biochemistry and Molecular Biology,
University of Southern Denmark,
Denmark
FLUORESCENCE MICROSCOPY FOR BIOPHYSICAL STUDIES IN
BIOMEMBRANES
Modern Fluorescence microscopy instrumentation. Spectral properties of more popular
probes. Epi-, confocal and two photon excitation fluorescence microscopy. Giant
unilamellar vesicles (GUVs) as tool to study lateral lipid organization and membrane
perturbation: lipid-lipid interaction, peptide, protein and lipolytic enzymes interacting
with organized lipids.
Tiempo Libre. Free time
Sbado 30 de Noviembre/Saturday 30
th
November

Maana/Morning
Tiempo Estimado/Estimated Time: 3 hs.
Primera Clase/First Lecture: 9.00-10.30
Caf/Coffe Break: 10.30-10.55
Segunda Clase/Second Lecture: 11.00-12.30

Dr. Benoit SORRE
Center for Studies in Physics and
Biology, TheRockefeller University,
New York, NY., USA

BIOPHYSICS OF MEMBRANE CURVATURE

Fundamentals of membrame curvature. The mean-field theoretical description of
membrane mechanics (Helfrich Hamiltionian). Artificial systems to study membranes
mechanical properties, (GUVs, micropipette aspiration, membrane fluctuations
methods). Ways to pull membrane nanotubes (optical tweezers) to study biology
inspired questions like lipid sorting and membrane deformation by proteins
(amphiphysin, dynamin).


Almuerzo/Lunch 13.30/15.00
Tarde/Afternoon 3/4 hs

Tarde/Afternoon
Tiempo Estimado/Estimated Time: 3 hs.
Primera Clase/First Lecture: 15.00-16.30
Caf/Coffe Break: 16.30-16.55
Segunda Clase/Second Lecture: 17.00-19.00

Dr. Frederic CARRIRE
Laboratory of Enzymology at I nterfaces
and Physiology of Lipolysis E.I .P.L
C.N.R.S., MARSEI LLE, France

INTERFACIAL ENZYMOLOGY: THE CASE STUDY OF LIPOLYTIC
ENZYMESFundamentals of interfacial enzymology. Modes of action of lipolytic
enzymes (lipases and phospholipases) and kinetic models. Monomolecular films as
model interfaces for studying lipase-lipid interactions (adsorption/penetration) and
interfacial activity (the "zero-order" trough and the barostat technique). Structure-
function relationships deduced from X-ray crystallography. Probing conformational
changes using site-directed spin-labeling coupled to EPR spectroscopy, kinectics.
Surface spectroscopy for studying lipase adsorption at various interfaces (TIRF, ATR-
FTIR).
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Tiempo Libre. Free Time

Domingo 1ro Diciembre / Sunday 1
st
December


Maana/Morning
Tiempo Estimado/Estimated Time: 3 hs.
Primera Clase/First Lecture: 9.00-10.30
Caf/Coffe Break: 10.30-10.55
Segunda Clase/Second Lecture: 11.00-12.30

Dra. Maria Elena Carrizo
Universidad Nacional de
Crdoba. Argentina

PROTEIN CRYSTALLOGRAPHY
Protein crystallization. Principles and techniques. Diffraction data collection.
Fundamentals of the theory of X-ray diffraction by a crystal. X-ray sources and
detectors. Facilities at the Brazilian Synchrotron Light Laboratory. From diffraction
data to electron density. Electron density as a function of intensities and phases.
Phase determination and improvement. Electron density maps. From electron density
maps to molecular models. Electron density map interpretation. Model building.
Structure refinement. PDB files.

Almuerzo/Lunch 13.30/15.00


Tarde/Afternoon
Tiempo Estimado/Estimated Time: 3/4 hs.

Primera Clase/First Lecture: 15.00-16.30
Caf/Coffe Break: 16.30-16.55
Segunda Clase/Second Lecture: 17.00-19.00


Dr. Paulo Mascarello Bisch
Laboratrio deFsica Biolgica
UnidadeMultidisciplinar deGenmica
I nstituto deBiofsica Carlos Chagas
FilhoUniversidadeFederal do Rio de
J aneiro, Brazil


PROTEIN FOLDING AND STRUCTURE MODELING


1. Protein Folding General Concepts; 2. Protein folding and misfolding, the
funel theory; 3. Molecular Dynamic Simulations of Protein folding; 4.
Comparative Molecular Modeling.

Tiempo Libre. Free time
Lunes 2 de Diciembre/Monday 2
nd
December
Maana/Morning
Traslado de Estudiantes del Curso al Hotel del Meeting / Transfer of the students to the Hotel's Meeting


MAIN LECTURES FROM THE XLII ARGENTINIAN BIOPHYSICAL SOCIETY MEETING ASSOCIATED TO VI POSLATAM COURSE
From 2
nd
-4
th
December 2013. (Place: Meeting Hotel)

Lunes 2 de Diciembre / Monday 2
nd
December

15.00-16.00 CONFERENCE 1

Dr. John SEDDON
Hydrostatic Pressure Effects on the Structure and Stability of Lipid Membranes and
Lyotropic Mesophases. Membrane Biophysics group, Department of Chemistry,
Imperial College London, Imperial College, London. UK.

19.00-20.00 CONFERENCE 2

Dr. Frdric CARRIRE
I nterfacial enzymology: investigating themodeof action of lipases requires a combination
of various biophysical approaches. Laboratory of Enzymology at Interfaces and
Physiology of Lipolysis E.I.P.L, C.N.R.S. MARSEILLE, FRANCE FRANCIA.
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Martes 3 de Diciembre / Tuesday 3
rd
December

8.30-9.30 CONFERENCE 3

Dr. Benoit SORRE
"Dynamics of TGF-beta signaling: how positional information can belearned froma
changing morphogen gradient". Center for Studies in Physics and Biology, The
Rockefeller University, New York, NY, USA.

9.30-10.30 CONFERENCE 4

Dr. Luis BAGATOLLI


"Do liposomes penetrateskin". MembraneBiophysics and Biophotonics Group/MEMPHYS
- Center for BiomembranePhysics, Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Denmark


17.30-18.30 CONFERENCE 5

Dr. Tibor Lszl PLI
"On therotary mechanismof thevacuolar proton-ATPase". Institute of Biophysics,
Biological Research Centre, P.O. Box 521, H-6701 Szeged, HUNGARY.


19.30-20.30 CONFERENCE 6

Dr. Manuel PRIETO
Ceramideand Glucosylceramideimpact on membranebiophysical properties: frommodel
to cell membranes. Instituto Superior Tcnico, Universidade Tcnica de Lisboa, Lisboa,
PORTUGAL.
Mircoles 4 de Diciembre / Wednesday 4
th
December

17.30-18.30 CONFERENCE 7

Dr. Pedro ARAMENDA

Single molecule and single nanoparticle fluorescence microscopy. Fac. Ciencias Exactas
y Naturales, UBA. CIBION-CONICET, ARGENTINA.


19.30-20.30 CONFERENCE 8

Dr. Flix GOI

Membrane properties of the simple sphingolipids. Unidad de Biofsica (CSIC-
UPV/EHU), Pas Vasco, ESPAA, SPAIN.

Fin de las Actividades / END OF ACTIVITIES

SAB 2013
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PROGRAMA/PROGRAM


Lunes 2 de Diciembre / Monday, December 2
nd



8.30-13.00 Llegada de Participantes y Registro/Arrival of Participants and Registration

9.00-12.30 Reunin de Representantes del Ncleo Disciplinar de Biofsica AUGM
(Asociacin Universidades Grupo Montevideo) / Meeting of
Representatives of Biophysics Discipline from AUGM (Association of
Universities of Montevideo Group)

13.00-14.30 Almuerzo. Colocacin de posters impares de todas las secciones/Lunch.
Placing of posters. Odd boards: All Topics

14.45-15.00 Apertura del Congreso / Opening Ceremony.
Bienvenida / Welcome by Dr. Gerardo Fidelio

15.00-16.00 CONFERENCIA DE APERTURA / OPENING LECTURE.

Dr. John Seddon: Hydrostatic Pressure Effects on the Structure and Stability of Lipid
Membranes and Lyotropic Mesophases. Membrane Biophysics Group,
Department of Chemistry, Imperial College London, Imperial College, London.
UK.
Chair: Bruno Maggio.

16.00-18.00 SIMPOSIO 1 / SYMPOSIUM 1: BIOFSICA DE BIOMEMBRANAS E
INTERACCIN LPIDO-PROTENA / LIPID-PROTEIN INTERACTION AND
MEMBRANE BIOPHYSICS.
Chairs: Ernesto Ambroggio and Natalia Wilke

16:00-16:30: Betina Crsico.Novel lipid binding proteins from helminth parasites. Structural
and functional analysis. INIBIOLP-La Plata. Argentina
16:30-17:00: Larisa Cybulski The power of being at the interface: mechanism of DesK
thermosensing. IBR-CONICET-Rosario. Argentina
17:00-17:30: Beln Decca. Conformation of peripherally bound membrane proteins: the
influence of the lipid phase state.CIQUIBIC-Crdoba. Argentina
17:30-18:00: Luis Gonzalez Flecha.Phospholipid modulation of membrane protein thermal
stability. IQUIFIB-Buenos Aires. Argentina


18.00-18.25 Caf / Coffee Break

18.30-19.00 SECCIN JOVEN I / YOUNG SECTION I

Mini-conferencia I / Short-lecture I
PREMIO MEJOR TESIS SAB / BEST THESIS AWARD SAB

Dra. Leticia Llarrul:Insights on the Molecular Events that unleash resistance to b-lactam
antibiotics in Staphylococcus aureus. IBR- CONICET-Rosario, Argentina.
Chair: Mario Ermcora

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2

19.00-20.00 CONFERENCIA PLENARIA 2 / PLENARY LECTURE 2

Dr. Frdric Carrire: Interfacial Enzymology: investigating the mode of action of lipases
requires a combination of various biophysical approaches. Laboratory of
Enzymology at Interfaces and Physiology of Lipolysis E.I.P.L,
C.N.R.S.MARSEILLE, FRANCE.
Chair: Gerardo Fidelio

20.30-23.00 SECCIN DE POSTERS (Posters impares: Todas las Secciones) /
POSTER SECTION (Odd boards: All Topics)
(cena, snack / dinner, snack)




Martes 3 de Diciembre / Tuesday, December 3
rd




8.30-9.30 CONFERENCIA PLENARIA 3 / PLENARY LECTURE 3

Dr. Benoit Sorre: Dynamics of TGF-beta signaling: how positional information can
be learned from a changing morphogen gradient. Center for Studies in
Physics and Biology, The Rockefeller University, New York, NY
Chair: Ernesto Ambroggio

9.30-10.30 CONFERENCIA PLENARIA 4 / PLENARY LECTURE 4

Dr. Luis Bagattoli: Do liposomes penetrate skin? Membrane Biophysics and Biophotonics
group. MEMPHYS Center for biomembrane Physics, Department of
Biochemistry and Molecular Biology, University of Southern Denmark.
Chair: Felix Goi


10.30-10.55 Caf/ Coffee Break

11.00-13.00 SIMPOSIO 2 / SYMPOSIUM 2. ESTRUCTURA Y FUNCIN DE PROTENAS /
PROTEIN STRUCTURE AND FUNCTION.
Chair: Gerardo Fidelio

11:00-11:30 Sonia Longhi. Structural disoder and induced folding in the nucleoproteins
and phosphoproteins of paramyxoviruses. Group Leader "Structural Disorder
and molecular Recognition" Architecture et Fonction des Macromolecules
Biologiques (AFMB) UMR 7257 CNRS et Universitd'Aix-Marseille, FRANCE.
11:30-12:00 Gorka Basaez. Elucidating the mechanisms of action of BCL-2 family
proteins in apoptosis using in vitro reconstituted systems. Unidad de Biofsica
(CSIC-UPV/EHU), Pas Vasco, Espaa, Spain.
12:00-12:30 Mauricio Sica. Equilibrium Unfolding of the PDZ Domain of b2-Syntrophin
Departamento de Ciencia y Tecnologa, UNQ, Buenos Aires, y Laboratorio de
Bioenergas, IEDS, CONICET, Centro Atmico Bariloche, Ro Negro,
Argentina.
12:30-13:00 Paulo Mascarello Bisch: "A large scale search for protein sequence-structure-
function relationship". Laboratrio de Fsica Biolgica, Unidade
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Multidisciplinar e Genmica, Instituto de Biofsica Carlos Chagas Filho
Universidade Federal do Rio de Janeiro, Brazil.


13.00-14.30 Almuerzo. Colocacin de posters pares de todas las secciones / Lunch.
Placing of posters. Even boards: All Topics


15.00-17.00 SIMPOSIO 3 / SYMPOSIUM 3: TRANSPORTADORES Y CANALES DE
MEMBRANA / TRANSPORTERS AND CHANNELS IN MEMBRANES.
Chairs: Rodolfo Gonzlez Lebrero and Karina Alleva.

15:00-15:30 David Naranjo.Small and Large conductance postassium channels: Where is
the difference? Centro Interdisciplinario de Neurociencias de Valparaso,
Universidad de Chile
15:30-16:00 Luciano Moffatt.Achieving maximal speed of solution exchange for patch
clamp experiments in purinergic receptors. INQUIMAE, UBA, Argentina.
16:00-16:30 Daniel Peluffo.Cationic Amino Acid Transporters: insights from a non-
transportable enantiomer. Universidad de la Repblica, Regional Norte,
Uruguay.
16:30-17:00 Josh Berlin."Conformational changes in transmembrane alpha helices of the
H-ATPase, AHA2". Department of Pharmacology and Physiology, UMDNJ-
New Jersey Medical School, USA


17.00-17.25 Caf/ Coffee Break


17.30-18.30 CONFERENCIA PLENARIA 5 / PLENARY LECTURE 5

Dr. Tibor Lszl Pli: On the rotary mechanism of the vacuolar proton-ATPase. Institute
of Biophysics, Biological Research Centre, Szeged, Hungary
Chair: Guillermo Montich

18.30 a 19.30 CONFERENCIA PLENARIA 6 / PLENARY LECTURE 6

Dr. Manuel Prieto: Ceramide and Glucosylceramide impact on membrane biophysical
properties: from model to cell membranes Instituto Superior Tcnico,
Universidade Tcnica de Lisboa, Lisboa, Portugal.
Chair: Laura Fanani

19.30-20.00 Reunin LAFeBS-POSLATAM / LAFeBS-POSLATAM Meeting

20.00 Asamblea Anual de Socios de SAB / Annual meeting of SAB members

20.30-23.00 SECCIN DE POSTERS (Posters pares: Todas las Secciones) / POSTER
SECTION (even boards: All Topics)

(cena, snack / dinner, snack)





SAB 2013

4

Miercoles 4 de diciembre / Wednesday, December 4
th




8.30-10.30 SIMPOSIO 4 / SYMPOSIUM 4: MODELADO BIOMOLECULAR /
BIOMOLECULAR MODELING
Chair: Marcos Villarreal.

8:309:00 Xavier Ambroggio. Strategies for the de novo design of protein-protein
interactions. Rosetta Design Group LLC, Virginia, USA.
9:009:30 Sergio Pantano. Botulinum neurotoxins and SNARE complexes: A new
structural view from modeling and simulations.Institut Pasteur, Montevideo,
Uruguay.
9:3010:00 Roberto Lins.Predictive Biomolecular Modeling Applied to Protein Engineering
and Proteomics.Department of Fundamental Chemistry, Federal University of
Pernambuco, Recife, PE, Brazil
10:0010:30 Ernesto A. Romn. Study of Frataxin folding. IQUIFIB-Buenos Aires.
Argentina

10.30-10.55 Caf /Coffee Break

11.00-13.00 SECCIN JOVEN II / YOUNG SECTION II

11.00-11.30 Mini-conferencia II / Short-lecture II

Leandro C. Tabares: One enzyme two pathways: Single molecules studies on Nitrite
Reductase". CEA Saclay, France.
Chair: Rodolfo Rasia

11.30-13.00 Exposicin Oral de Posters Seleccionados /Oral Presentation of Selected
Posters.
Chairs: Laura Fanani and Natalia Wilke

11.30-11.50 BLM39_Surface and hysteresis properties of lipid interphases composed by
head group substituted phosphatidylethanolamines. Salcedo, C.L., Bouchet,
A.M., Nazareno, M.A., Disalvo, E.A., Frias, M.A.
11.50-12.10 BPA25_Growth Hormone Releasing Hexapeptide is able to form Nanotubes: a
complementary study by Small Angle X-Ray Scattering, Transmission
Electron Microscopy and Molecular Dynamic Simulations. Barbosa, LRS,
Santana, H, Avila, CL, Cabrera, I, Pez, R, Falcn, V, Pessoa, Jr. A, Ventosa, N,
Veciana, J, Itri, R.
12.10-12.30 TMSB1_Stochastic and Algebraic Methods for Modeling the Binding of
Transcription Factor Cohorts to Cis-acting Gene Regulatory Modules. Mauricio
Bustos and Fernando Levstein.

12.30-12.50 TRC15_The S6 transmembrane segment modulates channel opening in BK
channels. Carrasquel-Ursulaez, W., Contreras, G. F., Seplveda, R., Aguayo, D.,
Gonzlez-Nilo, F., Gonzlez, C. and Latorre, R.

13.00-14.30 Almuerzo /Lunch

SAB 2013

5

15.00-17.00 SIMPOSIO 5 / SYMPOSIUM 5: DIFRACCIN DE RAYOS X Y SAXS EN
BIOESTRUCTURAS / X-RAYS DIFFRACTION AND SAXS TO STUDY
BIOSTRUCTURES.
Chairs: Rafael Oliveira and Graciela Borioli.

15:00-15:30 Sebastin Klinke. Structural studies on a two-component system activated by
blue light in Brucellaabortus, FundacinInstituto Leloir, Buenos Aires,
Argentina.
15:30-16:00 Leide Pasos Cavalcanti."Small Angle X ray Scattering to study liposomes for
gene therapy", Laboratorio Nacional de Luz Sincrotron, Campinas, Brazil.
16:00-16:30 Mario Ermcora.Structure and function of ICA2, a receptor involved in insulin
secretion Structural Biology and Biotechnology Group, IMBICE, UNQ-
Conicet, Argentina.
16:30-17:00 Marcelo Ceolin. "Synchrotron radiation experiments on the biomineralization
of ferritin", INIFTA, Univ. Nac. de La Plata, Argentina.

17.00-17.30 Caf / Coffee Break


17.30-18.30 CONFERENCIA PLENARIA 7 CONFERENCIA GREGORIO WEBER /
GREGORIO WEBER CONFERENCE PLENARY LECTURE 7.

Pedro Aramenda: Single molecule and single nanoparticle fluorescence microscopy.
Fac. Ciencias Exactas y Naturales, UBA. CIBION-CONICET, Argentina.
Chair: Luis Bagatolli.

19.30-20.30 CONFERENCIA DE CLAUSURA / CLOSING LECTURE

Flix Goi: Membrane properties of the simple sphingolipids Unidad de Biofsica (CSIC-
UPV/EHU), Pas Vasco, Espaa, Spain.
Chair: Gerardo D. Fidelio.

20:30 Anuncio del Premio al Mejor Poster / Announcement of Best Poster Award
20:40 Palabras sobre PosLatam y LAFeBS / Words about PosLatam and LAFeBS.
Dr. Silvia Alonso, Dr. Pietro Ciancaglini, Dr. Marcelo Morales.

20:55 Palabras de Cierre por el Dr. G. Fidelio / Closing words by Dr. G. Fidelio
21.00 Cena de Cierre / Closing Dinner







SAB 2013

6











SAB2013 Conferencias Plenarias (plenary lectures)
7


CONFERENCIAS PLENARIAS / PLENARY LECTURES

P1_Hydrostatic Pressure Effects on the Structure and Stability of Lipid Membranes and
Lyotropic Mesophases
Seddon, J. M.
Membrane Biophysics Group, Chemistry Department and Institute of Chemical Biology, Imperial College
London, Exhibition Road, London SW7 2AZ, UK.

Lyotropic liquid crystals of 1-, 2-, or 3-dimensional periodicity spontaneously assemble when lipids are
mixed with solvent under various conditions of temperature, pressure and hydration. The mesophases
formed include the 1-D fluid lamellar (L), 2-D hexagonal (H
I
/H
II
) and 3-D cubic phases (Q
I
/Q
II
). Although
the flat fluid lamellar phase is the structure on which biomembranes are generally based, there is
increasing evidence that curved structures such as the inverse cubic phases may be present in cell
membranes, and/or may facilitate various cellular processes such as endo- and exocytosis, membrane
budding, and fusion, as these all involve changes in membrane topology. Previous studies of lyotropic
phase transitions have mainly concentrated on transformations between lamellar phases and from
lamellar to inverse hexagonal structures, with little work done on transitions involving cubic phases.
However, a complete understanding of the physical processes governing such transitions, including the
nature of any intermediates formed, and the mechanistic routes taken, is essential if we are to further our
knowledge of their possible roles in fundamental cellular processes involving membranes. We have
therefore been using high pressure and pressure-jump X-ray diffraction to investigate lyotropic phases
and transitions in a range of lipid systems. The use of pressure to trigger transitions has several
advantages: 1) the solvent properties are not significantly altered; 2) pressure propagates rapidly meaning
that equilibrium is achieved rapidly; and 3) pressure-jumps can be both in the pressurisation and
depressurisation directions. We have studied the effects of pressure on the gel-fluid transition in
sphingomyelin bilayer membranes, and have found that the ordering of the chains and the development of
the ripples on forming the gel phase occur on different timescales. We have previously shown that by
addition of weakly-polar amphiphiles such as diacylglycerols to phospholipids, one can tune the interfacial
curvature to be strongly inverse, leading to the formation of a discontinuous cubic phase of spacegroup
Fd3m, with a structure based upon a complex close packing of two types of inverse micelle of different
diameters. We have recently investigated the effect of hydrostatic pressure on the structure and stability of
this phase, and have discovered a number of novel effects. We discovered a lyotropic liquid crystal phase
of space group P6
3
/mmc, whose structure is based upon a hexagonal close packing of identical quasi-
spherical inverse micelles. The system consists of a hydrated mixture of dioleoylphosphatidylcholine,
dioleoylglycerol, and cholesterol. This novel phase has a number of unique features which may render it
useful for a wide range of applications. We have studied the effect of chain branching on glycolipid
thermotropic and lyotropic phases for a series of synthetic -D-glucosides derived from Guerbet alcohols,
whose total hydrocarbon chain length ranged from C
8
to C
24
. A wide range of liquid-crystalline phases was
observed, with the C
16
Guerbet glucoside (i.e. -Glc-C
10
C
6
) forming an inverse bicontinuous cubic phase
of space group Ia3d in excess water, which is very unusual behaviour.



Conferencias Plenarias (plenary lectures) SAB2013
8



P2_Interfacial enzymology: investigating the mode of action of lipases
requires a combination of various biophysical approaches

Carrire, F.

CNRS, Aix Marseille Universit, UMR7282 Enzymology at Intefaces and Physiology of Lipolysis,
Marseille, France



Many enzymes are active at interfaces in the living world (such as in signaling processes at the surface of
cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental
enzymology remains largely focused on the interactions between enzymes and soluble substrates. The
biochemical and kinetic characterization of lipases has opened up however new paths of research in the
field of interfacial enzymology and specific interfacial kinetic models have been developed (1). In order to
hydrolyze their insoluble substrate, triglycerides, lipases have first to bind the lipid-water interface
(adsorption step) before forming an enzyme-substrate complex in a two dimensional space. This process
is often associated with conformational changes allowing lipases to interact with lipids/amphiphiles while
preserving these enzymes from total unfolding. It is still difficult to characterize the successive steps of
enzymatic lipolysis in a single experimental set-up and access to kinetic constants. Individual steps can
however be studied separately using various biophysical methods. Conformational changes have been
studied using first X-ray crystallography, and more recently site-directed spin labelling coupled to electron
paramagnetic resonance spectroscopy has allowed monitoring the opening of the amphiphilic lid covering
the active site of pancreatic lipase in the presence of amphiphiles and lipids (2, 3). The adsorption step or
lipase-lipid interactions are usually approached using other means such as monomolecular films and
surface pressure measurements (4, 5) or surface spectroscopy like total internal fluorescence
spectroscopy (TIRF; (6)). Examples taken from structure-function studies of pancreatic and gastric
lipases, as well as some microbial lipases, will be presented here to illustrate the complex mode of action
of lipolytic enzymes.

1 Aloulou et al.: Biochim. Biophys. Acta - Molecular and Cell Biology of Lipids. 2006. 995.
2 Belle et al.: Biochemistry. 2007. 2205.
3 Ranaldi et al.: Biochemistry. 2010. 2140.
4 Point et al. Biochimie. 2013. 51.
5 Bnarouche et al.: Colloids and Surfaces B: Biointerfaces. 2013. 306.
6 Chahinian et al.: Biochemistry. 2006. 993.









SAB2013 Conferencias Plenarias (plenary lectures)
9






P3_Dynamics of TGF-beta signaling: how positional information can be learned from a
changing morphogen gradient.


Benoit Sorre
1,2
, Aryeh Warmflash
1,2
, Ali H. Brivanlou
1
& Eric D. Siggia
2



1 - Laboratory of Molecular Vertebrate Embryology
The Rockefeller University. New York, USA.
2 - Laboratory of Theoretical Condensed Matter Physics
The Rockefeller University. New York, USA.




Genetics and biochemistry have defined the components and wiring of the signaling pathways that pattern
the embryo. Many of these pathways have the potential to behave as morphogens: in vitro experiments
have clearly established that these molecules can dictate cell fate in a concentration dependent manner.
How morphogens convey positional information in a developing embryo, where signal levels are changing
with time, is less understood. Recently we showed that the evolutionarily conserved TGF-beta pathway
responds transiently and adaptively to a step in ligand stimulation. Building on previous work, here we use
integrated microfluidic cell culture to stimulate the cells with well-defined temporal profile of morphogen
(TGF-) and timelapse microscopy to record their response in real-time, we demonstrate that the speed of
ligand presentation has a key and previously unexpected influence on signaling outcomes. Slowly
increasing the ligand concentration diminishes the response while well-spaced pulses of ligand combine
additively resulting in greater pathway output than is possible with constant stimulation. Our results
suggest that in an embryonic context, an adaptive pathway can naturally extract positional information as
ligand spreads dynamically from a fixed source, thereby providing an alternative to the static morphogen
model where the rate of change of ligand concentration, rather than its level, is the meaningful signal for
patterning.













Conferencias Plenarias (plenary lectures) SAB2013
10





P4_Do liposomes penetrate skin?

Bagatolli Luis A.

Membrane Biophysics and Biophotonics group/MEMPHYS Center for biomembrane Physics,
Department of Biochemistry and Molecular Biology, University of Southern Denmark. Campusvej 55, DK
5230, Odense, Denmark. E-mail bagatolli@bmb.sdu.dk




A multiphoton excitation based fluorescence fluctuation spectroscopy method, Raster Image
Correlation Spectroscopy (RICS), was used to measure the local diffusion coefficients of distinct model
fluorescent substances in excised human skin. In combination with structural information obtained by
multiphoton excitation fluorescence microscopy imaging, the acquired diffusion information was processed
to construct spatially resolved diffusion maps at different depths of the stratum corneum (SC).
Experiments using amphiphilic and hydrophilic fluorescently labeled molecules show that their diffusion in
SC is very heterogeneous on a microscopic scale. This diffusion-based strategy was further exploited to
investigate the integrity of liposomes during transdermal penetration. Specifically, the diffusion of dual
color fluorescently labeled liposomes -containing an amphiphilic fluorophore in the lipid bilayer and a
hydrophilic fluorophore encapsulated in the liposome lumen- was measured using cross correlation RICS.
This type of experiment allows discrimination between separate (uncorrelated) and joint (correlated)
diffusion of the two different fluorescent probes, giving information about liposome integrity. Independent
of the liposome composition (phospholipids or transfersomes), our results show a clear lack of cross-
correlation below the skin surface, indicating that the penetration of intact liposomes is highly
compromised by the skin barrier.




1 J. Brewer, J. Kubiak J, M. Bloksgaard, J. A. Srensen and L.A. Bagatolli. J. Investig.
Dermatol.(2013).133:2601268










SAB2013 Conferencias Plenarias (plenary lectures)
11



P5_On the rotary mechanism of the vacuolar proton-ATPase

Pli, T.

Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged,
Hungary

The internal compartments of eukaryotic cells are more acidic than the cytoplasm. The transport protein
complex that is responsible for the acidification is Nature's most universal proton pump, the vacuolar
proton-ATPase (V-ATPase). The V-ATPase is a membrane-bound molecular rotary engine, which
converts the chemical energy from ATP hydrolysis to the rotation of the rotor domain via a torque between
specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and
rotor domains. We have estimated the rate of rotation of the rotor in the yeast V-ATPase, relative to the
stator or steady parts of the enzyme, in native vacuolar membrane vesicles from Saccharomyces
cerevisiae under standardised conditions, in two ways:

(A) The fraction of the total ATPase activity originating from the V-ATPase was determined by using the
potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in
the vacuolar membrane vesicle system was assayed spectrophotometrically for different concanamycin A
concentrations. A fit of the quadratic binding equation to the inhibitor titration curve determined the
concentration of the enzyme. Combining this data with the known ATP/rotation stoichiometry of the V-
ATPase has led to an average rate of ~10 Hz for full 360 rotation, as a lower-limit estimate (1).

(B) We have tested the effect of alternating electric (AC) field on V-ATPase activity in the same yeast
vacuolar vesicle system. This was the first of its kind of experiment on V-ATPase, and we got strikingly
different results from previous studies on other proteins: both low and high frequency AC field reduced
ATPase activity in a wide frequency range, and a sharp resonance was seen at ~88 Hz, where the
ATPase activity reached or exceeded the control (no AC) level. Assuming that the AC field interacts with
the proton movements, and considering the estimated geometry of the proton binding sites and the
hydrophilic proton channels, we conclude that the resonance frequency corresponds to that of the 60
rotor steps. Therefore the rotation rate of the rotor is ~15 Hz, which agrees very well with the above lower-
limit estimate.

To our knowledge, we are the first to report the rotation rate in a V-ATPase that is not subjected to genetic
or chemical modification and is not fixed to a solid support, instead it is functioning in its native membrane
environment.


Acknowledgement: This work was supported by Hungarian National Science Fund (OTKA) grants K68804
and K101633.

1 Ferencz, C., Petrovszki, P., Kota, Z., Fodor-Ayaydin, E., Haracska, L., Bota, A., Varga, Z., Der, A.,
Marsh, D. and Pali, T. European Biophysics Journal 42(2-3). 2003. 147.



Conferencias Plenarias (plenary lectures) SAB2013
12


P6_ Ceramide and Glucosylceramide impact on membrane biophysical properties: from
model to cell membranes


Varela A.R.
a,b,c
, Pinto, S.N.
c
, Gonalves da Silva, A.M.P.S.
d
, Futerman A.
b
, Silva L.C.
a
and Prieto M.
c



a
iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Faculdade de Farmcia,
Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
b
Department of Biological Chemistry, Weizmann Institute of Sciences, Rehovot 76100, Israel
c
Centro de Qumica-Fsica Molecular & Institute of Nanoscience and Nanotechnology and
d
Centro de
Qumica Estrutural, Instituto Superior Tcnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001
Lisboa, Portugal




Sphingolipids (SLs) have emerged as an important class of lipids due to their bioactive role in a number of
cellular events and in disease. The evidence that several SL species participate in the formation of lipid
domains and that this might underlie their biological mechanism of action has fostered research in the
biophysical aspects of bioactive SLs. This has been one of the aims being pursued in my research group.
In this talk I will focus on two important SLs ceramide and glucosylceramide and their interplay with
other lipid components in simple and complex membrane models. Using a combination of biophysical
methodologies that include fluorescence spectroscopy, confocal and two-photon microscopy, surface
pressure-area measurements, allowed elucidating their effects on the biophysical properties of
membranes composed of a variety of lipids and displaying different phase properties. In addition, I will
describe how these interactions can be modulated by alterations in the membrane environment, such as
changes in pH. It will be highlighted how the small structural differences of these lipids influence their
packing properties, membrane shaping and lateral organization. Inferences will be made regarding the
importance of the headgroup, acyl chain length and unsaturation on the modulation of membrane
properties.
Finally, I will emphasize the significance of these model membrane studies to predict the biophysical and
biological implications of these lipids in cellular membranes and will show examples of how the
observations obtained from model membranes are translated into the cell level.



Supported by FCT (Portugal) grants PTDC/BBB-BQB/0506/2012, PTDC/QUI-BIQ/111411/2009,
SFRH/BD/69982/2010 to ARV, SFRH/BD/46296/2008 to SNP, Compromisso para a Cincia 2008 to LCS.








SAB2013 Conferencias Plenarias (plenary lectures)
13



P7_Single molecule and single nanoparticle fluorescence microscopy


Aramenda, P. F.



Dept. Qumica Inorgnica. FCEN. Univ. Buenos Aires and CIBION-CONICET. Godoy Cruz 2390. 1425
Ciudad de Buenos Aires. Argentina. pedro@qi.fcen.uba.ar.




Single molecule fluorescence detection has found many applications in materials science and biology
since its first report in 1990. It offers unique possibilities because of its ultimate detection limit, the ability to
detect and follow in time single events, and the access to the distribution of behaviors versus the average
value of conventional bulk detection methods. At the same time, it attains a time resolution in the
milliseconds range and a spatial resolution of hundreds of nanometers, in conventional detection, and
tens of nanometers in super resolution techniques. More recently, metallic nanoparticles (MNP) have been
extensively used to enhance the performance of molecular fluorescence in bulk and single molecule
applications. The interaction between MNP and fluorophores opens new perspectives in fluorescence
microscopy, based on the interaction of the plasmonic band of the nanostructure and the molecular
electronic states. These interactions allow to detect low intrinsic fluorescent molecules, by an
enhancement in emission brightness, and they also provide an increase in the total number of emitted
photons and in the monitoring time before photo bleaching. In the last six years our laboratory has been
performing research in single molecule techniques, including the use of gold NP (AuNP). In this lecture I
will illustrate experiments in fluorescence microscopy using AuNP to detect low emission quantum yield
molecules, to increase the monitoring time in cellular environments, to provide protection against
photobleaching, and to enhance the performance of a fluorescent photochromic system in super
resolution localization.













SAB2013 Conferencias (lectures)

14


Mini-conferencia I / Short conference I: Premio a la mejor tesis SAB / best thesis
award

Insights on the Molecular Events that Unleash Resistance to -lactam antibiotics in
Staphylococcus aureus


Llarrull, L.I.

Instituto de Biologa Molecular y Celular de Rosario (IBR), Ocampo y Esmeralda, Predio CONICET,
2000, Rosario.



Staphylococcus aureus is the main cause of hospital- and community-associated infections.
1
The
expression of the BlaZ-lactamase and of the PBP2a DD-transpeptidase renders S. aureus
resistant to -lactam antibiotics. The expression of these gens is regulated by two related systems,
composed of a membrane associated sensor protein (BlaR1 or MecR1, respectively) and a
repressor (BlaI or MecI, respectively). The sensor proteins and PBP2a itself are promising targets
for the design of inhibitors that would restore the efficiency of -lactam antibiotics. We have used a
combination of spectroscopic techniques, biochemical techniques, molecular modeling and organic
chemistry to characterize different aspects of these two systems. We have documented a lysine N

-
decarboxylation switch that arrests the sensor domain of BlaR1 in an activated state required for
signal transduction,
2,3
we have characterized BlaI binding to its operator region, and we have shown
that the in vivo concentrations account for the basal level transcription of the resistance genes.
4
We
have also presented evidence that support the hypothesis that BlaR1 fragmentation is a means for
turnover,
5
a process required for recovery from induction of resistance in S. aureus in the absence
of the antibiotic challenge, and that BlaR1 is indeed a metallo-protease that degrades the gene
repressor BlaI.
6
Regarding the DD-transpeptidase PBP2a, we have recently reported the
identification of an allosteric binding site that regulates the opening of the active site to permit
substrate entry, through a multiresidue conformational change.
7
In my group, we are currently
working on the elucidation of the topology and structure of the sensor proteins BlaR1 and MecR1.


Acknowledgements: The PEW Charitable Trusts, NIH, ANPCyT, CONICET

1 Llarrull LI, Fisher JF, Mobashery S. Antimicrob. Agents Chemother.2009. 4051.
2 Borbulevych O, Kumarasiri M, Wilson B, Llarrull LI, et al. J. Biol. Chem. 2011. 31466
3 Kumarasiri M, Llarrull LI, et al. Journal of Biological Chemistry. 2012. 8232.
4 Llarrull LI, Prorok M, Mobashery S. Biochemistry. 2010. 7975
5 Llarrull LI, Toth M, Champion MM, Mobashery S. Journal of Biological Chemistry. 2011. 38148
6 Llarrull LI, Mobashery S. Biochemistry, 2012. 4642.
7 Otero LH, Rojas-Altuve A, Llarrull LI, et al. Proc. Natl. Acad. Sci. USA. 2013. 16808.




Conferencias (lectures) SAB2013
15



Mini-conferencia II / Short conference II



One enzyme two pathways: Single molecules studies on Nitrite Reductase

Tabares LC



Service de BionergtiqueBiologieStructuraleetMcanismes, CEA-Saclay, France.
Leiden Institute of Physics, University of Leiden, The Netherlands.

Single enzymes measurements have changed the way we look to these molecular machines mode
of operation, providing new, previously unobtainable, information. However, most of these
measurements were restricted to enzymes which undergo conformational changes, have
fluorescent cofactors or use/produce fluorescent compounds. We have developed a new method
for monitoring the redox state of a metalloprotein based on fluorescence resonance energy transfer
(FRET) between a fluorescent label and the protein metal center. The method was applied to the
study of copper-containing nitrite reductase from Alcaligenesxylosoxidans. Nitrite reductase is a key
enzyme in bacterial denitrification and plays an important role in the global nitrogen cycle. In our
single molecule studies we found new evidence showing a heterogenic distribution in two
populations of molecules which correspond to two distinct catalytic pathways [1]. In order to obtain
the kinetics parameter we apply a microfluidic trapping device to allow, for the first time,
measurement of single enzymes in solution [2]. Our results reconcile a long-standing dispute about
the mode of action of this enzyme bringing together the previously proposed binding-first,
reduction-first and random mechanisms.


References
1 Tabares LC, Kostrz D, Elmalk A, Andreoni A, Dennison C, Aartsma TJ, Canters GW. Chem. Eur.
J. (2011) 17:12015-9.
2Goldsmith RH, Tabares LC, Kostrz D, Dennison C, Aartsma TJ, Canters GW*, Moerner WE*.
Proc. Natl. Acad. Sci. (2011) 108:17269-74.



SAB2013

16












S2: Estructura y funcin de protenas (protein structure and function)

17

SIMPOSIOS / SYMPOSIA

S1: Biofsica de biomembranas e interaccin lpido-protena / lipid-protein
interaction and membrane biophysics

S1.1_ Novel lipid binding proteins from helminth parasites. Structural and functional
analysis.
Marina Ibez Shimabukuro*, Florencia Rey*, Gisela R. Franchini*, Malcolm W. Kennedy
#
, Alan
Cooper
#
, Brian O. Smith
#
and Betina Crsico*
* Instituto de Investigaciones Bioqumicas de La Plata (CONICET-UNLP), Facultad de de Ciencias
Mdicas, UNLP. Argentina
# Institute of Biomedical & Life Sciences, University of Glasgow. UK

Parasitic helminths express lipid-binding proteins (LBPs) that are structurally distinct from host
LBPs. These proteins bind a wide range of lipid classes such as fatty acids, retinoids, eicosanoids,
triglycerides, phospholipids and cholesterol. Due to helminths limited lipid metabolism, LBPs have
been proposed to participate in parasites development and in the interaction with the host. To
understand the mechanisms involved, we have selected three important types of LBPs from highly
pathogenic helminth parasites: a) a novel class of fatty acid and retinol binding proteins with a
structure that has no known counterpart, b) relatives of the fatty acid binding protein family,
including members that are structurally modified in ways that are unique to nematodes, and c)
nematode polyprotein allergens. The atomic structures are under analysis employing NMR
spectroscopy, for which we already have obtained high quality data and full structure determination
is in progress. Protein's interactions with ligands employing NMR spectra show the changes
registered during the binding process when stripped and reloaded samples are compared. We are
also analyzing their ligand-binding parameters employing fluorescence-based systems. The studies
confirm these LBPs bind natural ligands and fluorescent analogues in the sub-micromolar range.
Structural and functional studies will enhance our understanding of the unique features of helminth
LBPs that may be related to the survival of the organisms and could be used as potential drug
targets.

S1.2_The power of being at the interface: mechanism of DesK thermosensing

Cybulski L
Instituto de Biologa Molecular y Celular de Rosario (IBR)- CONICET and Departamento de
Microbiologa, Facultad de Ciencias Bioqumicas y Farmacuticas, UNR, Rosario, Argentina.

The thermosensor DesK is a five-pass transmembrane (TM) histidine kinase that senses
and signals temperature changes in Bacillus. Temperature sensing involves a built-in instability
caused by two motifs of hydrophilic residues located at both, the N-terminus and C-terminus of the
TM domain. The N-terminus has two hydrophilic amino acids (K10 and N12) below the lipid/water
interface, and the C-terminus has a hydrophilic motif composed of three serines located on one side
of the helix. These interfacial hydrophilic motifs render the protein sensitive to membrane thickness
and to the extent of interfacial hydration, which would in turn depend upon temperature changes. A
conformational changein the linker connecting the TM sensing domain with the cytoplasmic catalytic
domain is triggered by the interplay of these interfacial motifs to control DesK activity.

S2: Estructura y funcin de protenas (protein structure and function)

18

S1.3_ Conformation of peripherally bound membrane proteins: the influence of the
lipid phase state
Mara Beln Decca
Departamento de Qumica Biolgica-CIQUIBIC, Facultad de Ciencias Qumicas, Universidad
Nacional de Crdoba. Haya de la Torre y Medina Allende,5000, Crdoba, Argentina.
The transfer of soluble proteins into the interface between the lipid membrane and the aqueous
phase is recognized as a key step for several cellular processes. This translocation represents a
major change in the protein environment that can stabilize different protein conformations with
possible consequences on its biological activity. Using as a model the peripherally bound protein L-
BABP we found that conformation can be modulated by the phase state of the lipid membrane.
When L-BABP was bound to lipids in the gel phase, the secondary structure was similar to the
native structure in solution, membrane transition to the liquid-crystalline phase produced the partial
unfolding of the protein. This was observed with anionic phospholipids with different polar
headgroup and different melting transition temperature, and it was sensitive to the ionic strength.
We explored changes in surface potential as possible triggers of protein unfolding at the interface.
We measured membrane electrokinetic potential at different temperatures and we found a
correlation with protein conformation: membrane-bound, native-like protein occurred under
conditions in which lipid vesicles have low surface potential and unfolded state was observed in
membranes with higher values of surface potential. Therefore, changes in protein conformation
coupled to lipid phase transitions can result as a consequence of the modification of electrostatic
surface potential during lipid melting. We demonstrate the linkage between lipid organization,
protein conformation, strength of binding, and membrane electrostatic surface potential.

S1.4_Phospholipid modulation of membrane protein thermal stability
Santiago Martnez, Diego I. Cattoni
1,2
, Jos M Argello
3
and F. Luis Gonzlez Flecha
1

1
Laboratorio de Biofisica Molecular. IQUIFIB , Universidad de Buenos Aires-CONICET, Argentina
2
Centre de Biochimie Structurale, INSERM, Universit de Montpellier, France.
3
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, USA

Despite recent progress in understanding membrane protein folding, little is known about the
mechanisms stabilizing these proteins. Here we characterize the effects of phospholipids on the
kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu
+
-ATPase from Archaeoglobus
fulgidus. The enzyme was purified and reconstituted in mixed micelles composed by detergent
(DDM) and different phospholipids. In all the conditions CopA retained its thermophilic
characteristics with maximum activity at 75 C. Incubation of CopA in the absence of substrates at
temperatures in the 66-85 C range led to an irreversible exponential decrease in enzyme activity
suggesting a two-state process involving fully-active and inactive molecules. The lowest thermal
stability was obtained for CopA reconstituted in detergent micelles, and the highest for the enzyme
located in E coli membranes. Remarkably, the activation energy was similar for all the reconstitution
systems assayed. Transition state theory analysis of the kinetic data allowed to evaluate the
enthalpic and entropic contributions. S
#
values were similar in membranes and mixed micelles, but
higher than those obtained for CopA reconstituted in detergent micelles, whereas H
#
followed an
inverse order respect to that observed for the kinetic coefficients. These results suggest that
phospholipids promotes charge and H-bonds distributions between the native and the transition
state and increased the degrees of freedom of the protein solvent system in the transition state.

With grants from UBACyT and ANPCyT

S2: Estructura y funcin de protenas (protein structure and function)

19

S2: Estructura y funcin de protenas / protein structure and function

S2.1_Structural disorder and induced folding in the nucleoproteins and
phosphoproteins of paramyxoviruses
Longhi, S
1
, Habchi, J
1
, Blocquel, D
1
, Beltrandi, M
1
, Erales, J
1
, Dosnon, M
1
, Papageorgiou, N
1
,
Blangy, S
1
, Communi, G
2,3
, Ringkjobing-Jensen M
2
, Blackledge, M
2
, Ruigrok, RWH
3

1
AFMB, UMR 7257, CNRS and Aix-Marseille University, Marseille, France,
2
Institut de Biologie
Structurale, Grenoble, France,
3
UVHCI, Univ Grenoble Alpes-EMBL-CNRS, Grenoble, France
In the last decade there has been an increasing amount of experimental and computational
evidence pointing out that the proteome of eukaryotes and viruses is enriched in intrinsically
disordered proteins (IDPs) and/or intrinsically disordered regions (IDRs). IDPs/IDRs are ubiquitous
functional proteins that lack stable II and III structures under physiological conditions in the absence
of a partner and that rather exist as highly dynamic conformational ensembles. IDPs are often
involved in biological processes implying manifold protein-protein interactions, such as cellular
regulation, transcription and signal transduction.
In the course of the structural and functional characterization of the measles virus replicative
complex, we discovered that the nucleoprotein (N) and the phosphoprotein (P) contain long (up to
230 residues) disordered regions possessing sequence and biochemical features that typify IDPs.
More recently, by combining computational and experimental approaches, we extended these
results to the N and P proteins from the newly emerged Nipah and Hendra viruses. My talk will
focus on (i) the identification and characterization of disordered regions of the N and P proteins of
these paramyxoviruses, (ii) the assessment of their structural state in the context of the full-length N
and P proteins, (iii) the investigation of the molecular mechanisms underlying the induced folding
events triggered by binding partners. Finally, the functional implications of disorder within the
replicative complex of these viruses will be discussed.


S2.2_ Elucidating the mechanisms of action of BCL2 family proteins in apoptosis
using in vitro reconstituted systems
Landeta, O, Landajuela A, Garcia-Valero J, Bustillo, I, Flores-Romero H, Terrones, O, Basaez, G
Unidad de Biofsica, Consejo Superior de Investigaciones Cientficas - Universidad del Pas
Vasco/Euskal Herriko Unibertsitatea (CSIC-UPV/EHU), Barrio Sarriena s/n, Leioa, 48940, Spain,

During apoptosis, mitochondrial membranes undergo dramatic changes in permeability and
morphology. The principal components involved in these processes are the BCL2 family proteins,
with the assistance of an increasing number of mitochondrial protein/lipid effectors. Despite the
remarkable progress made in uncovering the molecular underpinnings of apoptotic cell death in the
last decade, the precise mechanisms by which BCL2 family proteins regulate the structure and
functioning of mitochondrial membranes remains a key and controversial issue in the field of cell
death. Given the inherent complexity of the cellular apoptotic network, we use in vitro reconstituted
systems bearing physiological relevance to try elucidating the mode of action of specific members
of the BCL2 family and/or their effectors at the membrane level, using a multidisciplinary approach
based on biophysical, biochemical, and molecular biology techniques. Here, I will explain our recent
progress in the role of apoptosis-related mitochondrial lipids on BCL2 family protein function. I will
also discuss the mechanism by which BAX and BAK form the lethal mitocondrial apoptotic pore.


S2: Estructura y funcin de protenas (protein structure and function)

20


S2.3_Equilibrium Unfolding of the PDZ Domain of b2-Syntrophin
Torchio, G
1,2
; Burgos, I
3,2
; Fidelio, G
3,2
; Arn, M
4,2
; Gallo, M
4,2
; Ermcora, M
1,2
; Sica, M
5,2
.
1
.Laboratorio de Plegado y Expresin de Protenas, Univ. Nac. de Quilmes-IMBICECIC, Argentina.
2
.CONICET, Argentina.
3
.Centro de Investigaciones de Qumica Biolgica,Univ. Nac. de Crdoba,
Argentina.
4.
Fundacin Instituto Leloir, Argentina.
5
.Laboratorio de Bioenergas. IEDS. Centro
Atmico Bariloche, Argentina.

2-syntrophin, a dystrophin-associated protein, plays a pivotal role in the insulin secretion by
pancreatic -cells. It contains a PDZ domain (2S-PDZ) that, in a complex with protein-tyrosine
phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation
state of 2-syntrophin allosterically regulates the affinity of 2S-PDZ for ICA512, and the disruption
of the complex triggers the mobilization of the insulin granule stores. We have investigated the
thermal unfolding of 2S-PDZ at different conditions and applying various techniques. Our results
indicate that, unlike other PDZ domains, 2S-PDZ is marginally stable. Thermal denaturation
experiments show broad transitions and cold denaturation, and a two-state model fit reveals a
significant unfolded fraction under physiological condition. Furthermore, Tm and Tmax denaturant-
dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest
that two-state and three-state models fail to explain properly the equilibrium data and are in better
agreement with downhill scenario. DSC and NMR experiments are also consistent with this view.
Additionally, a higher stability at pH above 9, and molecular dynamics simulations indicate that this
behavior of 2S-PDZ might be related to its charge distribution. All together, our results suggest a
link between the conformational plasticity of the native ensemble this PDZ domain and the
regulation of the insulin secretion.

S2.4_ A large scale search for protein sequence- structure -function relationship.
Bisch, PM.
Instituto de Biofsica Carlos Chagas Filho Universidade Federal do Rio de Janeiro, Brazil.
In the last years high throughput sequencing of DNA has produced an increasing amount of
biological data. Analyzing gene sequence similarities and putative homology, an inference about
biological function can be assigned to some of the new sequences. Although, it still has a lot of
sequences classified as hypothetical or unknown function. Further, it is well accepted that structures
of proteins are close related to the genes function, unfortunately experimental determination of
protein structure still be a very difficult task. So, now days, for a millions of know protein sequences
only thousands of structures are in the structure data bank. It seems that to overcome this question
a theoretical and computational effort are in order. The emerging System Biology field should
provide a means to overcame this difficult task by exploiting the concept of sequence-structure-
function relationship in a new computational high throughput approach. Fortunately, computational
means and new information technologies have also a big increase in last years. Combined with new
concepts we should deal with a large number of data and increase the knowledge about genes and
their interaction relationship. We show a few examples where we have combine the use
bioinformatics analysis of a large number of unknown sequences, construction of structural models
and molecular dynamics simulations to revel their biological functions.
Acknowledgements; CNPq, CAPES, FAPERJ, FINEP.
1 Campos ,RA et al.: Genet Mol Res. 2012. 2122.
2 Maranho, AG et al.: Infect Genet Evol.. 2012. 1397.
3 Lery, LMS et al.: BMC Genomics. 2010. S7.
S3: Transportadores y canales de membrana (Transporters and channels in membranes)

21

S3: Transportadores y canales de membrana / Transporters and channels in
membranes

S3.1_Small and Large conductance potassium channels: Where is the difference?
Diaz-Franulic, I (1)., Navarro, N.(1), Gonzlez-Nilo, F.(2), Seplveda, R.(2), and Naranjo, D:(1).
(1) Centro Interdisciplinario de Neurociencia, Universidad de Valparaso, Valparaso, Chile, (2)
Center for Bioinformatics and Integrative Biology (CBIB), Universidad Andrs Bello, Santiago, Chile.

Potassium channels are membrane proteins that allow the passage of K+ ions across the
hydrophobic core of the membrane. They display an extremely conserved signature sequence
capable of eliciting high ion transport rates with exquisite K+ selectivity among ions with similar
radii. Despite of this conservation, closely related potassium channels display differences of up to
100-fold in their single channel conductance, suggesting that the ion transport rate limiting step is
somewhere else in the pore. Because the Pro475Asp substitution -near the internal entrance
dramatically increases Shaker K+ transport rate by 7-8 fold, we suggested that such anrise could
result from higher pore occupancy. Then, to test this hypothesis, we introduced charged residues
along the pore of Shaker to fill the permeation pathway and compared their maximal single channel
conductance to that of BK channels (600pS). Fully occupied Shaker variants (as tested with
Molecular Dynamic simulations) were still far below of BK single channel conductance. A possible
explanation for this finding could be that the inner entrance dimensions limit the maximal ion
transport rate. To test this idea we estimated the radius of capture Shaker variants by measuring
the diffusion limited currents in solutions containing additional 2M of sucrose to increase viscosity.
Our result shows that Kv channels have a smaller inner entrance than large conductance K-
channels which imposes an upper limit for the maximal transport rate of K-channels.
This work was supported by FONDECYT 1120818 (DN), 1131003 (FGN), and CINV (Millenium
Initiative, 09-022-F). RS and IDF are CONICYT and MECESUP doctoral fellows, respectively.

S3.2_Achieving maximal speed of solution exchange for patch clamp experiments
in purinergic receptors
Auzmendi, JA; Moffatt L.
INQUIMAE, FCEN, UBA CONICET

Purinergic receptors are cationic channels comprised by three subunits; they form a pore with only
six transmembrane domains. The crystal structure of zP2X4 has been recently determined both in
the closed and the open state
1
and efforts are being made to study the molecular dynamics of the
coupling mechanism of binding and gating. In this context, the ability to obtain kinetic information of
high quality would be inestimable to experimentally ground the possible molecular mechanisms. As
this coupling occurs in the tens to hundreds of microseconds, we focus our latest efforts in the
development of the experimental ability to expose the patch clamp preparation to the agonist during
shorter and shorter periods of time. We developed the ability of applying pulses of 25 microseconds
measured at the open tip of the patch pipette
2
. In this way, the brief intermediate states that occur
between the binding of the agonist and the opening of the pore would be accessible to experimental
study not only for purinergic but also for fast open channels like AMPA and Ach receptors.

This study has been funded by the ANPCyT (PICT 06 1902) and UBACyT (20020100100636)

1 Hattoriet al.Nature 2012: 207
2 Auzmendi et al. PLoS ONE 2012: e42275

S3: Transportadores y canales de membrana (Transporters and channels in membranes)

22

S3.3_Cationic Amino Acid Transporters: insights from a non-transportable
enantiomer
Peluffo, R. D.
Universidad de la Repblica, Regional Norte, Salto, Uruguay.
Cationic amino acid transporters are highly selective for L-enantiomers such as L-arginine (L-Arg).
Because of this stereoselectivity, little is known about the interaction of these transporters with D-
isomers. To study whether these compounds provide information on the molecular mechanism of
transport, inward currents activated by L-Arg with low apparent affinity were measured in whole-cell
voltage-clamped cardiomyocytes as a function of extracellular L-Arg and D-Arg concentrations. D-
Arg inhibited L-Arg currents in a membrane potential (V
M
)-dependent competitive manner, indicating
the presence of D-Arg binding sites in the carrier. Accordingly, D-Arg-dependent charge movements
were also detected in these cells. Analysis of steady-state currents showed that L- and D-Arg
binding reactions dissipate a similar small fraction of the membrane electric field. Since D-Arg is not
transported, these results suggest that enantiomer recognition occurs at conformational transitions
that prepare amino acid translocation. Simulations of the V
M
dependence of maximal current levels
with a four-state alternating model suggest that inward currents arise from the outward movement of
a negative charge in the unliganded transporter. Translocation of the L-Arg-bound complex, on the
other hand, appears to be an electroneutral process. To our knowledge, this study provides first
quantitative data on electrogenic reactions that accompany low-affinity L-Arg transport.
This work was supported by Award Number R01HL076392 from the National Heart, Lung, and
Blood Institute (R.D.P.).

S3.4_ Flexibility in the ion transport pathway of P-type ATPases?
Berlin, J.R.
Dept. of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark,
New Jersey, USA

P-type ATPases are a large family of enzymes responsible for the active transport of ions and
phospholipids across cell membranes. In this family of enzymes, biochemical, mutagenesis and
structural data for the sacroplasmic/endoplasmic reticulum Ca
2+
-ATPase (SERCA) have led to
detailed mechanistic proposals for how biochemical reactions, ATP binding, enzyme
phosphorylation, and P
i
hydrolysis drive vectorial transport of Ca
2+
across the membrane domain of
this enzyme. There seems little doubt that these biochemical reactions are highly conserved
across the P-type ATPase family. However, less data exist as to whether the mechanism of
vectorial transport is also highly conserved. In order to address this question, we have begun to
study a plant P-type H
+
-ATPase from Arabidopsis thaliana, AHA2. The question that we are
investigating is whether, during H
+
transport, conformational changes of the alpha helices located in
the membrane domain of AHA2 could be consistent with those postulated to occur during Ca
2+

transport by SERCA. Cysteine scanning mutagenesis experiments were performed with AHA2
expressed in Saccharomyces cerevisiae. Accessibility of amino acids substituted with cysteine to
extracellular thiol-reactive reagents was tested and H
+
transport rate was measured. Accessibility
of residues in the first transmembrane alpha helix did change with different AHA2 turnover rates;
however, all together, the data suggested that vectorial H
+
transport by AHA2 could follow a
different ion transport pathway than has been postulated for Ca
2+
in SERCA or for Na
+
in the Na,K-
ATPase. These results lead us to postulate an alternative transport pathway for H
+
through the
membrane domain of AHA2.
S4: Modelado molecular (Biomolecular modeling)

23

S4: Modelado molecular / Biomolecular modeling

S4.1_Strategies for the de novo design of protein-protein interactions
Nir London
a,b,
Xavier Ambroggio
a

a
Rosetta Design Group LLC, Burlington, Vermont, USA
b
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA

The great chemical diversity of amino acid side-chain functional groups coupled to the flexibility of
protein backbones makes the computational design of proteins for specific functions a challenging
feat. Early successes in computational design tackled the problem of designing amino acid
sequences that would adopt a specified target conformation, also known as the inverse protein
folding problem, by employing algorithms to sample side-chain conformations and model, with
sufficient accuracy, the physiochemical forces of a folded protein. These successes laid the
groundwork for general computational design algorithms and methodology, however, in order to de
novo design in complex functions, such as protein-protein interactions or protein-ligand interactions,
new strategies have emerged. The strategy of using well-defined models for the desired binding
interaction modes and the development of new methodologies for incorporating those models into
the context of nave scaffolds has led to many recent successes in the de novo design of protein-
protein interactions. We present here an overview of these exciting studies and the strategies they
employed along with some of our experiences in the design of protein-protein interactions and
supramolecular assemblies.

S4.2_ Botulinum neurotoxins and SNARE complexes: A new structural view from
modeling and simulations.
Sergio Pantano
Institut Pasteur de Montevideo, Uruguay. spantano@pasteur.edu.uy
The very high affinity and specificity of Botulinum neurotoxins for SNARE proteins lead to the
paralysis of neuromuscular junctions; making these toxins attractive for therapeutics, cosmetics and
even bioterrorism. However, the molecular details of the neurotransmitter release apparatus remain
still elusive.
Comparison of the mode of actions between different Botulin serotypes suggests that multiple
SNARE complexes associate on a radial super complex. Modelling and simulations are used to
derive 3D information of this nanomachine and identify protein-protein contacts within this
quaternary arrangement. The role in neuroexocytosis of amino acids at the putative inter SNARE
surface is confirmed by electrophysiology measurements on neuromuscular junctions of transgenic
flies, providing support for a radial arrangement of SNARE complexes and furnishing novel insights
on the self-assembly and regulation of biomolecular nanomachines.
- Pantano S and Montecucco C. The Blockade of the Neurotransmitter Release Apparatus by
Botulinum Neurotoxins. Cell. Mol. Life Sci. 2013, DOI:10.1007/s00018-013-1380-7.
- Megighian A, et al. Evidence for a radial SNARE super-complex mediating neurotransmitter
release at the Drosophila neuromuscular junction. J. Cell. Sci., 2013, 136: 3134.
- Megighian A, et al. Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila
melanogaster neuromuscular junction. J Cell Sci. 2010, 123:3276.
- Montecucco C, Schiavo G, Pantano S. SNARE Complexes and Neuroexocytosis: How Many, How
Close? Trends Biochem. Sci. 2005, 30:367.
S4: Modelado molecular (Biomolecular modeling)

24

S4.3_Predictive Biomolecular Modeling Applied to Protein Engineering and
Proteomics
Isabelle F. T. Viana
1,2
, Ranieri V. Carvalho
3
and Roberto D. Lins
1

1
Department of Fundamental Chemistry, Federal University of Pernambuco, Recife, PE, 50740-560,
Brazil;
2
Department of Infectious Diseases and Microbiology, University of Pittsburg, 9022 BST3,
Pittsburgh, PA, 15261, USA;
3
Center of Informatics, Federal University of Pernambuco, Recife, PE,
50740-560, Brazil

Atomic-scale biomolecular modeling is predominantly directed towards finding properties of a
specific system. This presentation will focus on the use of computational biophysics techniques to
address issues in a global and predictive manner. Such approach will be showcased by studies
capable of i. predicting the primary sequence of peptides based on calculated ion mobility mass
spectrometry data; and, ii. de novo design of gp41-based conformation-specific HIV-1 epitopes
grafted onto highly-stable scaffolds aimed to point-of-care diagnostic kits and vaccines.
Keywords: molecular dynamics, protein engineering, ion mobility spectrometry

This work is supported by FACEPE, CNPq, NanoBiotec-BR/CAPES and nBioNet and STINT.
Computer allocation was provided by the Environmental Molecular Sciences Laboratory located at
the Pacific Northwest National Laboratory and Argonne National Laboratory.

S4.4_Study of Frataxin folding
Romn E.A.

IQUIFIB-Buenos Aires, Argentina.
Frataxin is globular protein that appears in all the three biological kingdoms and is related to the
iron intracellular homeostasis. In humans, the lack of this protein yields Friedreich's Ataxia. This
pathology is associated to the cellular redox equillibrium and ATP synthesis. The absence of
functional frataxin results in an increase in the free radical content and also problems in iron
delivery to other protein targets.
In our laboratory, we are interested in the study of the mechanisms of frataxin iron binding, and in
the relation between the stability and functionality of this protein. In this sense, we faced ligand
binding studies, and the stability and folding mechanism analysis and study of this variant.
Previous experimental results suggest that the carboxi-terminal region of human frataxin could be
participating as a limitant step in the folding process of the human variant. Moreover, other
laboratory studies showed that this region is closely related to the global stability of this protein.
In this talk, we will introduce the computational simulation results where we studied by coarse
grained techniques the folding process of human frataxin. From these experiments we obtained
information on the global stability of this variant which could be related to its structural topology.
Also, we inferred the presence of, at least, one folding intermediate that could be related to
structural and energetically to destabilized variants that appear in patients with Friedreich's Ataxia.
The analysis of these results and experimental folding kinetics determinations would make us able
to perform a detailed characterization of this folding process.

S5: Difraccin de rayos X y SAXS en bioestructuras (X-Ray difraction and SAXS to study biostructures)

25

S5: Difraccin de rayos X y SAXS en bioestructuras / X-Ray difraction and SAXS to
study biostructures

S5.1_Structural studies on a two-component system activated by blue light in
Brucella abortus
Klinke S., Rinaldi J. J., Sycz G., Paris G. & Goldbaum F. A.
Fundacin Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435 (C1405BWE), Ciudad de
Buenos Aires, Argentina. E-mail: sklinke@leloir.org.ar
Brucella abortus is an intracellular pathogen that causes a worldwide zoonosis called brucellosis,
an endemic disease that causes abortion and infertility in cattle with consequent huge economic
losses. One of the projects in our lab focuses on the study of a particular two-component signal
transduction system (TCS) in Brucella that is activated by blue light and was shown to be a key
virulence factor [1]. This TCS is composed by (i) the sensor histidine kinase LOV-PAS-HK, which is
a three-domain protein able to sense blue light through a bound FMN molecule, and (ii) two cognate
response regulators called PhyR and CheY.
In this talk, we will present our latest results regarding the structural description of this system using
protein X-ray crystallography. Explicitly, we were able to solve the crystal structure of the isolated
LOV [2] and HK domains in the sensor histidine kinase, as well as the structure of the response
regulator PhyR.We will also describe our present strategies for the resolution of multi-domain
constructs and complex structures. To finish, we will show the technical aspects of synchrotron
radiation application for fast diffraction data collection and automated structure solving of the
proteins described here, according to our experience at the SOLEIL synchrotron in France.
Overall, the structural information on this TCS, complemented with biochemical studies that are
being performed in our lab, correspond to an excellent starting point for the understanding of the
signal transduction effect between the different domainsin LOV-PAS-HK and the general activation
of histidine kinases.
Acknowledgements: CONICET and MINCyT (funding). SOLEIL and Institut Pasteur Montevideo
(access to X-ray data collection) [1] Swartz, T.E. et al. (2007) Science317, 1090-1093. [2] Rinaldi,
J.J. et al. (2012) J. Mol. Biol.420, 112-127.

S5.2_Small Angle X ray Scattering to study liposomes for gene therapy
Balbino, T.
1
, Gasperini, A.
2
, Oliveira, C.
3
, Azzoni, A.
3
, Cavalcanti, L.
2
, de La Torre, L.
1

1
Univ. Campinas,
2
Brazilian Synchrotron Light Lab (LNLS),
3
Univ. So Paulo, BRAZIL

In this talk we will present a characterization study of complexes formed by cationic liposomes (CL)
and pDNA with main application in gene delivery systems. We found that conventional physico-
chemical properties were nearly unaffected at the studied ranges of molar charge ratio between
pDNA and CL, for which the results from in vitro transfection showed significant differences. We
then used small angle x-ray scattering (SAXS) to determine the lipoplex structural modifications
trying to comprehend the transfection properties. The SAXS results revealed that pDNA/CL
complexes can be described as being composed of single bilayers, double bilayers and multiple
bilayers, depending on the charge balance between pDNA and CL
1
. These results were used to
explain the observed transfection differences and allowed proper correlation of the physico-
chemical and structural properties of pDNA/CL complexes with the in vitro transfection, contributing
to a better understanding of the gene delivery process.
Acknowledgements: The SAXS experiments were made at LNLS. The authors TB and AG are
granted by FAPESP agency. 1 Balbino et al, Langmuir (2012) 28:11535
S5: Difraccin de rayos X y SAXS en bioestructuras (X-Ray difraction and SAXS to study biostructures)

26

S5.3_Structure and function of ICA2, a receptor involved in insulin secretion
Ermcora Mario R.
Structural Biology and Biotechnology Group, Imbice, UNQ-Conicet
ICA512 is a type 1 membrane protein located in pancreaticcell, insulinsecretory granules and in
electrodense granules of other neuroendocrine cells. One of its intracellular domains is a tyrosine
phosphatase (PTP) and the protein as a whole is a receptor PTP (RPTP).
ICA512 was discovered in the '90, as an autoantigen associated to autoimmune diabetes, and it
has since been utilized for the precocious diagnosis of the disease. Latter, it became notorious
because of the discovery of its involvement and multiple roles in the process of insulin secretion:
ICA512 modulates the mobility of secretory granules, as well as the expression of insulin and other
genes related to the granular integrity and cell proliferation.
Beside its role in the endocrine pancreas, ICA512 participates in the secretion of pituitary hormones
and its deficiency causes infertility. Today, the receptor and interacting proteins are considered
promising targets for the development of new medicines and as attractive subjects of basic and
medical investigations.
The mature ectodomain of ICA512 (MPEICA512) may be involved in oligomerization process and
cell signalling. Our laboratory solved the structure of MPEICA512 by Xray crystallography under
different conditions relevant for the granulogenesis process. The structural information, along with
evidence obtained in experiments in vivo established the basis for the preparation of 3D model of
the entire receptor. In the presentation, the progress in the preparation of such model will be
discussed.

S5.4_ Synchrotron radiation experiments on the biomineralization of ferritin
Ceoln, M.
Instituto de Investigaciones fsico-Qumicas Tericas y Aplicadas (INIFTA, UNLP-CONICET).
Diagonal 113 y 64 (1900) La Plata

Transmission Electron Microscopy (TEM), X-ray Absorption Near Edge Spectroscopy (XANES),
Electron Energy-Loss Spectroscopy (EELS), Small-Angle X-ray Scattering (SAXS), and SQUID
magnetic studies were performed in a batch of horse spleen ferritins from which iron had been
gradually removed, yielding samples containing 2200, 1200, 500, and 200 iron atoms. Taken
together, findings obtained demonstrate that the ferritin iron core consists of a polyphasic structure
(ferrihydrite, magnetite, hematite) and that the proportion of phases is modified by iron removal.
Thus, the relative amount of magnetite in ferritin containing 2200 to 200 iron atoms rose steadily
from 20% to 70% whereas the percentage of ferrihydrite fell from 60% to 20%. These results
indicate a ferrihydrite-magnetite core-shell structure. It was also found that the magnetite in the
ferritin iron core is not a source of free toxic ferrous iron, as previously believed. Therefore, the
presence of magnetite in the ferritin cores of patients with Alzheimers disease is not a cause of
their increased brain iron(II) concentration.

The author is in debt to CONICET (Argentina) and LNLS (Brazil). The participation of several co-
authors as part of the project (Dr. J:M.Dominguez-Vera and his crew) is also deeply acknowledged.
1 N.Galvez, B.Fernandez, P.Sanchez, R.Cuesta, M.Ceolin, M.C.Leon, S.Trasobares, M.Lopez-
Haro, J.Calvino, O.Stephan and J.M.Dominguez-Vera. J.American Chemical Society (2008) 130
8062.
SAB 2013




Resmenes de posters/Poster abstracts


Biofsica de lpidos y membranas / Biophysics of lipid and membranes (BLM)
Biofsica de protenas y cidos nuclicos / Biophysics of proteins and nucleic acids (BPA)
Enzimologa/ Enzymology (ENZ)
Bioenergtica y transferencia electrnica / Bioenergetics and transfer (BTE)
Teora y modelado de sistemas biolgicos / Theory and modeling of biological systems (TMSB)
Transportadores, receptores y canales / Transporters, receptors and channels (TRC)
Sealizacin y Dinmica Intracelular / Signaling and Intracellular dynamics (SDI)
Nuevas tcnicas en biofsica / New techniques in biophysics (NTB)
Biofsica: Aplicaciones biotecnolgicas / Biophysics: Biotechnological applications (BBA)








SAB 2013


SAB 2013 Biofsica de lpidos y membranas (Biophysics of lipid and membranes)
29

BLM1_Mechanical properties of membranes from spheroplast using optical tweezers.
Colque, A., Gonzalez Montoro, M.A., Valdez-Taubas, J. y Wilke, N.
Instituto de Qumica Biolgica de Crdoba (CIQUIBIC-CONICET), Departamento de Qumica Biolgica,
Facultad de Ciencias Qumicas, Universidad nacional de Crdoba.
Laser tweezers and optical microscopy have been used for the determination of the rheological properties
of model biomembranes
1
and of membranes of mammal cells
2
. Here, we used the technique to explore
the deformability of membranes of spheroplast of yeast Saccharomyces cerevisiae. It was reported that
the shear viscosity of this membrane is very different from mammal cells but the out-of-plane
deformability has not been studied yet. The wall of Saccharomyces cerevisiae cells was digested with
zymoliase and 200 L of a suspension of spheroplasts were placed on a glass cover-slide previously
treated with piranha solution for 2 h and poly-lysine in bufferborate overnight. A 10L-drop of an aqueous
solution of micro-spheres (anionic, 3 m diameter or cationic, 1 m diameter) was added to the
spheroplasts. After an hour, some beads were attached to the spheroplast surface, permitting to catch the
bead with the optical trap and move it apart from the spheroplast. This movement generated a membrane
nanotube between the cell and the bead, which allowed to test the membrane deformability, by tracking
the bead position relative to the spheroplast in time after the trap was turned off. The nanotube relaxation
process suggested that the spheroplast membrane was more elastic than the membrane of mammal
cells.
Acknowledgements: Sistema Nacional de Microscopa, SeCyT-UNC, FONCyT, CONICET.
1- Sorre Betal.PNAS. 2012. 173-178. 2- Pascoal P.et al. Lab Chip. 2010. 2235-2241.3- Valdez-Taubas J.
et al. Curr. Biol2003 1636-1640.



BLM2_Distribution of Salmon Calcitonin in Phospholipid Membranes. A Thermodynamic
study with models of Langmuir-Blodgett (LB-films) and Atomic Force Microscopy (AFM)
Meneses K.
,1
, Giordani C.
,,2
, Giraldo M.
,3

Grupo de Investigacin Biofsica (GIBF), Instituto de Fsica, Universidad de Antioquia, Medelln,
Antioquia, Colombia.

Departimento de Tecnologie e Salute, Istituto Superiore di Sanit, Viale Regina Elena 299, 00161 Roma,
Italy.
1
kayumesi@fisica.udea.edu.co,
2
cristiano@fisica.udea.edu.co,
3
m.a.giraldo@fisica.udea.edu.co

Nowadays over 30 diseases for which no cure is available (e.g. Alzheimer`s and Parkinson`s diseases)
are associated with amyloid-forming proteins. Up-to-date, research activities suggest that amyloid
oligomers may be the toxic species. Although the target has not been yet identified, the oligomer
properties suggest that neurons may be annihilated by unregulated permeabilization of the membrane,
possibly through the formation of a pore amyloid. The studies of Schubert and coworkers showed that
Calcitonin, in analogy with other amyloid proteins, show an aggregative behavior that is toxic to cultured
cells. These observations as well as the slow aggregation rate of the calcitonin, suggest the usage of
salmon calcitonin, sCT, as a "probe" to study the formation of amyloid neurotoxicity. This work aims to
study the molecular mechanism of the interaction of sCT with lipid membrane models which mimic the so-
called lipid rafts through the use of thermodynamic techniques (via the Langmuir-Blodgett trough or also
known as LB-trough) and atomic force microscopy (AFM).
BLM SAB 2013
30

BLM3_Studies of the Interaction of a Galleria mellonella Peptide with Molecular Models of
Gram-Negative Bacteria Membranes
Villanueva G.
1
, Correa W.
1
, Oate J.
1
, Patio E
1
., Espejo. F.
2
, Brandenburg K.
3

Manrique-Moreno M
1
.
1
Instituto de Qumica, Universidad de Antioquia, Medelln, Colombia.
e-mail: mmanrique@exactas.udea.edu.co, mmanriqm@hotmail.com
2
Escuela de Qumica, Facultad de Ciencias, Universidad Nacional, Medelln, Colombia
3
Forschungszentrum Borstel, LG Biophysik, parkalle 10, D-23845 Borstel Germany
Antimicrobial peptides are important components of the immune system of several living organisms, and
they are considered promising alternatives to conventional antibiotics [1]. The aim of this study was to
evaluate the interactions of a G. mellonella peptide with bacterial membranes. Previously we studied the
antimicrobial activity of a synthetic G. mellonella peptide against S. typhimurium and E. coli. Based on
these results we carried out peptide-membrane studies using liposomes of the main lipid components of
Gram-negative bacteria membranes (lipopolysaccharides, dimyristoylphosphatidylglycerol and
dimyristoylphosphatidylethanolamine) [2]. Experiments were performed by Fourier-transform infrared
spectroscopy and Fster resonance energy transfer spectroscopy. Results showed that the peptide
affected the liposome stabilities and, therefore the bacterial membranes. Our lab is presently focused on
the design and synthesis of potential antimicrobial peptides based on the G. mellonella amino acid
sequence in order to improve their biological activity.
[1] M. Pushpanathan, P. Gunasekaran, J. Rajendhran. International Journal of Peptides, 2013, 1.
[2] J.M. Sanderson. Organic and Biomolecular Chemistry, 2005, 201.


BLM4_Effect of the presence of domains on the stiffness of lipid monolayers.
Wilke N., Caruso B., Mangiarotti A.
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC), Departamento de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Pabelln Argentina, Ciudad
Universitaria. X5000HUA Crdoba.
The compressibility of monolayers composed of two lipids with low lateral miscibility was determined with
the aim of investigating the effect of the mechanical properties of each phase on the stiffness of the
composite. In nine different systems, the stiffness of each phase and the texture at the plane of the
monolayer were studied and the results are discussed in the light of the following two hypotheses: a. The
stiffness of the composite is a weighted sum of the stiffness of each phase, regardless of the distribution
of the phases in the plane of the monolayer; b. The stiffness of the composite is dominated by the
mechanical properties of the continuous phase. Our results were better explained by this latter proposal,
as in all the analyzed mixtures it was found that the mechanical properties of the percolating phase were
the determining factors
1
.
On the other hand, the effect of the presence of domains in the dilute regime did not follow a common
trend. This different behavior did not correlate with different domaindomain interactions or differences in
the shape/size of the domains between the systems. We hypothesize that the differences are a
consequence of the cooperativity of the percolation process in each monolayer.
Acknowledgements: SeCyT-UNC, FONCyT, CONICET.
1- Caruso B., Mangiarotti A., Wilke N. Langmuir 2013, 29, 10807-10816.


SAB 2013 BLM
31

BLM5_Interaction of magnetic nanoparticles with phospholipids in Langmuir monolayers
Menzaque, A.
a
, Lanterna, A.
a
, Maggio, B
b
., Granados, A.
a
, Krause, R.
c
, Vico, R.
a
a
INFIQC-CONICET, Departamento de Qumica Orgnica,
b
CIQUIBIC-CONICET, Departamento de
Qumica Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba.
c
Medicinal
Organic and Nanomaterials Chemistry, Department of Chemistry, Rhodes University.
A growing number of innovations have emerged in the fields of nanobiotechnology/nanomedicine and
new engineered nanomaterials are posing new challenges to understand the full spectrum of interactions
at the nanobio interface. Among them, magnetic nanoparticles (MNP) are greatly promising because of
their potential application in fields such us drug delivery, magnetic resonance imaging (MRI), heat source
in magnetic fluid hyperthermia.
1

We are studying the interaction of MNPs with phospholipids in Langmuir monolayers with the aim of
identify the influence of nanoparticle surface functionalization in their biomolecular properties. Here we
present the results of the interaction of MNPs covered with Oleic Acid (MNP@OA) and Stearic Acid
(MNP@SA) with dipalmitoylphosphatidylcholine (DPPC). The presence of either MNP cause phase
condensation of DPPC with respect to the pure phospholipid and significantly modify the surface
topography of DPPC (observed by BAM) along the whole isotherm, even if the MNP constitute only a
small mole fraction of the mixture.

1
Mout, R., Moyano, D., Rana, S., Rotello, V. Chem. Soc. Rev. 2012, 2539.



BLM6_Phase coexistence in films composed of DLPC and DPPC: a comparison between
different model membrane systems.
Mangiarotti A., Caruso B., Wilke N.
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC), Departamento de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Pabelln Argentina, Ciudad
Universitaria. X5000HUA Crdoba.

For the biophysical studies of membranes, a variety of model systems are used to measure different
parameters and to extract general principles of processes that occur in cellular membranes. However,
there are very few reports in which the results obtained with the different models are compared. In this
work, we quantitatively compared the phase coexistence in Langmuir monolayers, freestanding bilayers
and supported films composed of a lipid mixture of DLPC and DPPC. Two-phase segregation was
observed in most of the systems for a wide range of lipid proportions using fluorescent microscopy. The
lipid composition of the coexisting phases was determined and the distribution coefficient of the
fluorescent probe in each phase was quantified, in order to explore their thermodynamic properties. The
comparison between systems was carried out at 30 mN/m since it is accepted that at this or higher lateral
pressures, the mean molecular area in bilayers is equivalent to that observed in monolayers.
Our study showed that Langmuir monolayers and GUVs have a similar phase behavior. On the other
hand, supported films showed a different composition of the phases and the distribution coefficient of the
fluorescent probe was close to one. Our results suggest that in supported membranes, the presence of
the rigid substrate could be leading to a stiffening of the liquid-expanded phase due to a loss in the
degrees of freedom of the lipids when interact with the supporting material and this impacts on the
properties of the coexisting phases.
Acknowledgements: SeCyT-UNC, FONCyT, CONICET.
BLM SAB 2013
32

BLM7_Study of hemorheological properties of hypercholesterolemic rats treated with
proantocianidine extract of Ligaria cuneifolia (Lc)
Garca G
1
, Crosetti D
1
, Dominighini A
1
, Urli L
1
, Galliano S
1
, Gonzalvez J
1
, Monti J
2
, Ronco MT
2
, Wagner
M
3
, Carnovale CE
2
, Luquita A
1

1
Biofsica, Fac. Cs. Mdicas, UNR,
2
Fisiologa, Fac. Cs. Bioq. y Farm.-IFISE-UNR-CONICET,
3
Farmacobotnica, Fac. Farm. y Bioq.-UBA
Lc or creole mistletoe is an Argentine semiparasitic plant. We have previously demonstrated that crude
Lc extract decreases plasma Cholesterol (Cho), increases blood viscosity and reduces erythrocyte
deformability. Objective: to analyze the effect of treatment with an enriched fraction in proanthocyanidine
(PLc) on Cho plasma concentration and erythrocyte rheological properties in hypercholesterolemic rats.
Adult male Wistar rats (70 days), care according International Rules, were fed 28 days with Standard diet
plus Cho (97% purity) 0.8g/100g of diet (28% corn oil wt/wt diet). Control group (C) n=12 received ip
saline. Experimental group (E) n=12 received ip PLc 3mg/100g BWt every 24h, during 10 days. On day
11 the rats were anesthetized (Na-pentobarbital 50 mg/kg BWt) to obtain blood samples by cardiac
puncture. Results: (mean SE). Plasma Cho (enzymatic method)(mg%): C: 119.741.26, E: 68.501.86*,
HDLCho: C: 28.800.62, E:23.170.86*; LDLCho: C.25.300.84, E:18.160.59*. Blood: Morphological
Index (light microscopy): C:-2.350.48, E:-1.98 0.29*; SIII(%): C:40.1214.23, E:27.2611.74**; Rigidity
Index (nucleopore membrane filter): C:8.010.96; E:6.300.92**; Membrane erytrocyte Cho (mg%):
C:1.050.15, E: 0.770.23*.(**p<0.001 and *p<0.05 vs. C). Conclusion: PLc treatment diminishes plasma
Cho, HDLCho and LDLCho. Decrease of Cho from erythrocyte membrane leads to a morphological
change from discocyte to stomatocyte III. In agreement with other reports the morphological change
could point out to an interaction between PLc and the inner layer of the erythrocyte lipid bilayer
membrane altering its curvature; this could explain the erythrocyte deformability increasement i.e.,
Rigidity Index decreases in hypercholesterolemic rats.

BLM8_Molecular amphipathicity impacts on peptide surface behaviour
Ernesto Ambroggio and Gerardo Fidelio

CIQUIBIC, CONICET, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Ciudad
Universitaria, X5000HUA-Crdoba, Argentina.)

Protein primary structure has all the information for a specific protein/peptide folding. This sequence can
define specific amphiphilic regions in the molecules important for the interaction with interfaces. In order
to shed light how amphipathicity has a role in the surface properties of proteins, we designed three pairs
of peptides where all six type of molecules have the same hydrophobic residues but different hydrophilic
amino acids: positively, negatively and non-charged. The sequence of the peptides is inverted in
comparison to their pair. This inversion provokes that one kind of peptide has the N-terminal region
hydrophilic and the C-terminus hydrophobic and the other kind of peptide, with the same type of amino
acids, the opposite distribution. We evaluated how amphiphilicity has a role in peptide lateral stability at
the air-water interface by using the Langmuir monolayer technique. We also performed peptide/peptide
mixtures to understand whether there is a coupling within opposed charges and if amphipathicity may
contribute to the global lateral stability of the peptide film. In addition we measured membrane
destabilization (leakage of a fluorescent solution trapped inside liposomes) and membrane association of
these molecules. Finally we analyzed the peptide secondary structure by ATR-FTIR. Our results show a
distinctive surface behaviour for each kind of molecule whereas all presented the same secondary
structure.

SAB 2013 BLM
33

BLM9_Role of phase coexistence and composition of ternary lipid dispersion containing
cholesterol and ceramide on spontaneous curvature and structural stability.
Giudice F., Maggio B., Fanani M.L.,
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC), Departamento de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba, Haya de la Torre y Medina
Allende, Ciudad Universitaria, X5000HUA, Crdoba, Argentina.

Lipid dispersion of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Cholesterol (Chol) and
Palmitoyl ceramide (P-Cer) in a wide range of relative composition were prepared by the traditional
method of extrusion and compared with lipid dispersions formed by the Rapid Solvent Exchange
method (RSE) [1], which allows lipid vesicles to adopt its spontaneous curvature, without external
influences such as filtration or sonication. Thus, spontaneous curvatures of the ternary dispersions were
evaluated and related to the different phase states characterized in these mixtures [2]. The data shows
that, as we move from a fluid phase to the fluid and gel phases coexistence regions, the vesicles showed
a higher polydispersity in diameter, which appears to depend on the composition of the fluid phase that, in
turn, depends on the relative cholesterol/ceramide ratio.

This work was supported by: FONCyT, CONICET and SECyT-UNC

1- J. T.Buboltz, G. W.Feigenson. Biochi.Biophys. Acta 1417 (1999) 232.

2- B. M. Castro, L. C. Silva, A. Fedorov, R.F.M. de Almeida, and M. Prieto. J.Biol.Chem. Vol 284,NO.34
pp.22978.


BLM10_A simple model to predict the number of unilamellar liposomes in a suspension
Montanari, J, and Alonso, S. del V.
Laboratorio de Biomembranas, GBEyB (IMBICE-CONICET), Universidad Nacional de Quilmes, Roque
Saenz Pea 352, (1876) Bernal, Buenos Aires, Argentina. E-mail: jmontanari@unq.edu.ar
In particulate systems such as liposomes, concentration units are not enough to describe the drug
distribution in suspensions, as they are not homogeneous. In certain in vitro assays, exposure to different
number of particles (containing the same amounts of active in the same global volume) introduces an
extra variable regarding to contact phenomena. Volume and area of liposomes decrease upon extrusion,
while their quantity grows, as new vesicles are formed. Aiming to a rapid estimation of the number of
unilamellar liposomes present in a suspension, a mathematical method was developed, based on the
area and molecular weight of lipids and the mean size of the liposomes. Unilamellar liposomes loaded
with a probe were prepared by extrusion and counted by fluorescence microscopy and Tunable Resistive
Pulse Sensing (Q-Nano). Size was determined by Dynamic Light Scattering. There was about a 90%
coincidence between the theoretical results and the number of unilamellar liposomes counted for two
liposomes populations of different mean size. This model could be a useful complement for interpretation
of in vitro experiments in which results could depend on the distribution of actives into different quantities
of liposomes.


BLM SAB 2013
34

BLM11_A new method for the accurate determination of refractive index on Langmuir
Monolayers by Reflectometry
Pusterla, J.M. and Oliveira, R.G

CIQUIBIC (CONICET) and Departamento de Qumica Biolgica, Facultad de Ciencias Qumicas,
Universidad Nacional de Crdoba, Haya de la Torre y Medina Allende, X5000HUA- Crdoba, Argentina.
Monolayers at the air/water interface have been widely used as biomembrane models under controlled
intermolecular organization (lateral molecular area). Surface pressure, surface potential, reflectivity (R)
and other magnitudes can be precisely determined on these planar monomolecular films. However, some
physical parameters such as the refractive index (n
m
) still remain elusive. This is important because its
precise quantification allows for the determination of the thickness of the film by R, for instance on a
Brewster Angle Microscope (BAM)
1
. The uncertainties of n
m
determine important errors in the calculation
of monolayers thickness that propagates non-linearly.
Here we present an analytical method for the experimental determination of n
m
in monolayers based on
the principle of refractive index matching as used in bulk suspensions of liposomes
2
. By using a BAM
setup and monolayers spread over subphases with different and known refractive index (n
s
), a minimum
in R is search as a function of n
s
. In these conditions, the n
m
is equal to the known n
s
. The results shown
correspond to monolayers of isolated bovine myelin and two lipids with n
m
previously reported, DMPC and
cerebrosides. The n
m
values remain approximately constant, slightly increasing in a range of 0.005 to
0.01 upon compression. The obtained values allows for determination of thickness.
Acknowledgements: We thank funding from CONICET, SECyT-UNC and FONCYT.
1- Hnon, Sylvie. Review of Scientific Instruments. 1991. 936.
2- Ardhammar, Malin. PNAS. 2002. 15313.

BLM12_Interfacial behavior of a novel non-ionic azobenzene amphiphile. Towards a new
membrane photoswitch
Benedini, L
a
; Sequeira; M. A.
a
; Fanani;L.
b
, Maggio;B
b
; Dodero, V.I
a
a. INQUISUR-CONICET, Baha Blanca, Buenos Aires.b. CIQUIBIC-CONICET, Crdoba, Crdoba.

The comprehension of the stimulus-response behavior in biological is a great challenge [1]. In this
context, azobenzenes amphiphiles are versatile compounds which could be used as molecular switches
triggered by light [2]. We have obtained a new non-ionic amphiphile with photomodulated capacities that
allow using it as a membrane photoswitch. The film properties at different temperatures of the pure
derivative in both conformations were evaluated in Langmuir films at the air-water interface. The
capability of interaction and modification of a membrane model was evaluated by polarized optical
microscopy, electron microscopy and differential scanning calorimetry. In Gibbs monolayers, penetration
experiments showed that both isomers were able to interact differentially with the lipid monolayer. The (Z)
isomers showed the highest surface pressure cut off probably related to the increased the polarity of the
molecule.
Acknowledgements: Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.

1. Alberts, B. ; Bray, D. ; Lewis, J. ; Raff, M. ; Roberts, K. ; Watson, J.D. Molecular Biology of the cell,
5rd ed, Garland Publishing: New York, 2008.
2. Sebai, S. C.; Cribier, S.; Karimi, A.; Massotte, D.; Tribet, C. Langmuir. 2010. 14135


SAB 2013 BLM
35

BLM13_Surface behaviour of sphingomyelines with very long chains (C28-C32) PUFAs
and their interaction with premixed or enzymatically generated ceramides

Pealva DA
1
, Wilke N
2
, Maggio B
2
, Aveldao MI
1
, Fanani ML
2

1
INIBIBB, CONICET-UNS, Baha Blanca,
2
CIQUIBIC, UNC-CONICET, Crdoba, Argentina.
E-mail: lfanani@fcq.unc.edu.ar
Molecular species of sphingomyelin (SM) with nonhydroxy (n) and 2-hydroxy (h) very long-chain
polyunsaturated fatty acids (n- and h- 28:4, 30:5 and 32:5) abound in rat spermatogenic cells and
spermatozoa. These SMs are exclusively located on the sperm head, where they are converted into the
corresponding Cer by sphingomyelinase after completion of the acrosomal reaction. The aim of this study
was to gain some insight into the surface properties of this unique type of sphingolipids and how such
properties change by the SM Cer conversion. SM and Cer species were isolated by HPLC [1] and
organized in Langmuir films, alone and in SM/Cer mixtures. Compression isotherms for all six SMs under
study were compatible with a liquid-expanded state and showed large mean molecular areas. Only the
longest SMs (n- and h-32:5 SM) underwent phase transition upon cooling. h-28:4 SM show typical
general properties whereas h-28:4 Cer exhibited an easily compressible liquid condensed phase [2]
which may results from its higher conformational freedom in such phase. In premixed and enzymatically
generated h-28:4 SM / h-28:4 Cer films, Cer-rich domains with a high incorporation of SM were formed. In
conclusion, while the SMs from sperm behave in a regular way, the corresponding Cers show atypical
properties that may be relevant for the structural rearrangement occurring in the acrosome-reacted sperm
membrane.
This work was supported by FONCyT, CONICET, SECyT-UNC and SECyT-UNS.
1- D.A. Pealva, et al, J. Lipid Res. (2013) 54:2225-35.
2- D.A. Pealva, et al, Biochim Biophys Acta (2013), accepted.


BLM14_Study of the effect of steric interaction in lamellar phases

Barbara B. Gerbelli, Rafael L. Rubim, Emerson R. Silva, Frdric Nallet, Laurence Navailles, Cristiano L.
P. Oliveira, Elisabeth A. de Oliveira.

Some biological process such as membrane fusion and organization of membranes in stacks are
governed by interactions between membranes, which are formed by a mixture of lipids organized in
bilayers. Different structures like sponge phase, vesicles, or stacks can emerge, depending on the lipid
fraction with respect to the solvent, and on the packing of lipids in the bilayers. In this work we investigate
the behavior of multilamellar phases composed of lecithin and a commercial co-surfactant (Simusol),
which is a mixture of ethoxylated fatty acids. Composition of membrane was varied from 100% of lecithin
to 100% of Simulsol. Using X ray scattering and a new procedure to fit the data, relevant parameters
characterizing the lamellar structure were determined as a function of membrane composition. Scattering
data illustrating the swelling of the lamellae for different amounts of co-surfactant are presented with the
respective behavior of the Caill parameter. The incorporation of co-surfactant to lecithin membrane
shifted the equilibrium bilayer separation

Acknowledgements: To CNPQ, FAPESP, CAPES, INCT /CNPQ, Egide. Soleil synchrotron kindly
allocated beam time on the SWING station, proposal number 20101084.


BLM SAB 2013
36

BLM15_Surface properties of liposomes mixtures of phosphatidylcholines and
phosphatidylethanolamines.
Sierra,M.B
1
., Pedroni,V
1
., Morini,M
1
., Disalvo,E.A
2
., Alarcon,L
1
., Appignanesi,G
1
.
1- Laboratorio de Fisicoqumica Dpto. de Qumica. INQUISUR Universidad Nacional del Sur
2 -Laboratorio de Biointerfases. Centro de Investigacin y Transferencia, de Santiago del Estero
Phase properties studies of mixtures of DMPC/DPPC and DMPC/DMPE performed by means of
differential calorimetry and fluorescence microscopy, determined the existence of phase segregation as a
function of temperature and composition. It is inferred from these studies that the phases in contact
produce areas of contact, which affect surface properties of the membranes in contact with the aqueous
medium. However, there are few systematic studies about the surface potential that arise from the
presence of segregated phases. Thus, in this paper the zeta potential of multilamellar liposomes of
DMPC/DPPC and DMPC/DMPE mixtures was determined at 50C and 60C respectively, which are the
phase transition of the lipids, in a 1mM KCl medium. For the DMPC/DPPC zeta potential discontinuities
are observed as mole fraction of DPPC increases. Two mole fractions show particularities, which coincide
with what was reported for these mixtures in gel state [1] and with bigger sized liposomes. Since these
lipids are miscible in the whole range of molar fractions, the results could be explained as a consequence
of the organization of polar headgroups at the lipidic interphase. This conclusion is also supported by the
behaviour of unilamellar and multilamellar mixtures of DMPC/DMPE where the polar headgroup plays an
important role in the stabilization due to steric factors being this influence more noticeable than the one
related to the change of length of the hydrocarbon chain, as shown by the DMPC/DPPC mixtures.
Results are in agreement with Makino [2] proposal for the placement of PC headgroups on the liposome
interphase according to its phase state.
Acknowledgements: With funds from CONICET and UNS.
1- M. del C. Luzardo,G. Peltzer, and E. A. Disalvo. Langmuir. 1998. 5858.
2- K. Makino, T. Yamada, M Kimura. T Oka, H. Ohshima and T. Kondo. Biophys.Chem.1991. 175.

BLM16_Interaction of amphiphilic cyclodextrins with phospholipids in Langmuir
monolayers
Pinzn Barrantes, J.
a
,Hoyos de Rossi, R.
a
, Maggio, B
b
., Vico, R.
a
a
INFIQC-CONICET, Departamento de Qumica Orgnica,
b
CIQUIBIC-CONICET, Departamento de
Qumica Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba.

Cyclodextrins (CDs) are water-soluble molecular cages with a hydrophobic interior that allows the
inclusion and hence solubilization of various molecular species. These biocompatible cyclic
oligosaccharides, with low toxicities and no immune response are currently used as hydrophobic drug
carriers. CD-containing organized supramolecular structures can be obtained through assembly of
amphiphilic CDs
1
or by their insertion into integrated systems such as lipid membranes.
2
In previous work
we study the capability of amphiphilic CD to form Langmuir monolayer as well as the properties of these
films.
1,2
Here we present the properties of mixed films formed by an amphiphilic CD with palmitoyl oleyl
phosphatidyl choline (POPC) at different mole fractions. The miscibility and interactions of the
components have been characterized by A isotherms and the excess of free energy of mixing (G
exc
).
The results indicate non ideal mixing. Additionally, the more stable composition is dependent, al low
the more stable film is enriched in POPC, while at high there are two minima in G
exc
at X
POPC
=0.20 and
X
POPC
=0.8.
1
Vico, R., de Rossi, R.H., Maggio, B. Langmuir 2010. 8407.
2
Roux, M., Sternin, E., Bonnet, V., Fajolles,C., Djedani-Pilard, F. Langmuir 2013. 3677.

SAB 2013 BLM
37

BLM17_Folic acid: pro-oxidant action and characterization of a system based in SPC
liposomes
Marsanasco M.
a
, Piotrkowski B.
b
, Calabr V.
b
, Chiaramoni N.S.
a.
and Alonso S. del V
a

a Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
b Fisicoqumica, Facultad de Farmacia y Bioqumica, UBA e Instituto de Bioqumica y Medicina
Molecular-CONICET

The aim of this work was to study the antioxidant action of folic acid (FA) and characterize liposomes
encapsulating FA, before and after pasteurization. Soy phosphatidylcholine (SPC) liposomes were
obtained by the Bangham method with FA and/or in combination with stearic acid (SA) or calcium
stearate (CAS), with vitamin C (VC) and in combination with vitamin E (VE). Oxidative stability was
studied with TBA and ORAC methods. Characterization was performed by means of surface charge (Z
potential), membrane packing (probes MC540 and laurdan), size distribution by dynamic light scattering
(DLS) and rheological behavior.
The pro-oxidant action of FA was observed via TBA method, even after pasteurization. The VE resists the
pro-oxidant effect of the FA. With the ORAC method, no effect was observed with FA alone or in
combination with VE. The three formulations with FA had similar size distributions and Z potential values.
FA favors Merocyanine 540 (MC540) interaction with SPC and SPC:SA and increased laurdan GP when
combined with VE, in all of the formulations studied. Although the heat treatment decreased the viscosity
of SPC and SPC:SA and increased the viscosity of SPC:CaS; all systems showed Newtonian behavior,
which made these systems attractive for industrial applications.
Acknowledgements Universidad Nacional de Quilmes, Universidad de Buenos Aires and CONICET.


BLM18_Effect of hormone thyroids on ripple gel phase membranes
Marcelo C. Sosa Morales
a
, Ana C. Juarez
b
, Doly M. Chemes
b
, Rosa Maria S. lvarez
a

a
INSIBIO (CONICET-UNT), Instituto Superior de Investigaciones Biolgicas, Chacabuco 461, San M. de
Tucumn, Tucumn, T4000ILI, Argentina

b
Instituto de Qumica Fsica, Universidad Nacional de Tucumn, San Lorenzo 456, San. M. de Tucumn,
Tucumn, T4000ILI, Argentina


Since some time ago, our interest was focused on the study, from a microscopic point of view, of the
action mechanisms of certain biomolecules on cell membranes. The elucidation of the structural
characteristics of the molecules involved in these processes is carried out primarily by Raman
spectroscopy. This technique is a powerful tool to understand the interactions between phospholipid
membranes and biologically important molecules such as thyroid hormones (TH) (1). As yet, the
experimental evidence indicated that the membrane in gel phase hinders access of HT to the
hydrophobic region of the bilayer, in such a way that a significant portion of the hormones remains
anchored in the polar region. This fact might lead to interactions between PO2 and CO lipid groups with
the alanine moiety of the HT. Comparison between the DMPC/HT and DMPG/HT systems allow us to
better understanding of the nature of the interactions that take place in the hydrophilic region of the
membrane, while helping us to predict the degree of insertion of hormones in the hydrophobic region
according to the structural characteristics of the bilayers.
1- A.A.Petruk, M.C.Sosa Morales, R.M.S.lvarez, Spectrochim Acta Part A, (2013) 112, 403




BLM SAB 2013
38

BLM19_Interaction of nisin with anionic membranes. Purification and Raman study
Marcelo C. Sosa Morales
a
, Augusto Bellomio
a
, Ana C. Juarez
b
, Rosa Maria S. lvarez
a

a
INSIBIO (CONICET-UNT), Instituto Superior de Investigaciones Biolgicas, Chacabuco 461, San M. de
Tucumn, Tucumn, T4000ILI, Argentina
b
Instituto de Qumica Fsica, Universidad Nacional de Tucumn, San Lorenzo 456, San. M. de Tucumn,
Tucumn, T4000ILI, Argentina

Nisin is an antibiotic peptide ribosomally sintetized by Lactococcus lactis that inhibits the growth of a wide
range of gram-positive bacteria. It has a net positive charge (+5) and its weight is approximately 3kDa.
Commercially, it is available as a lyophilized powder containing 2.5% (w/w) nisin.
Purification was carried out using chromatographic methods (1). The bactericidal activity of fractions
obtained was visualized on agar plates. In addition, Tricine SDS-Page and Mass Spectrometry were
performed to confirm the purity of nisin.
Raman Spectroscopy was used to study the interaction of this small peptide with anionic models
membranes. We evaluated and interpretated signal spectral changes considered specific markers of the
lipid packaging, the number of conformers gauche in the hydrocarbon chains and the degree of hydration
of the polar heads. For this purpose, dipalmitoylphosphatidylglycerol (DPPG) and
dilauroylphosphatidylglycerol (DLPG) were utilized. Measurements were made at room temperature.
Analysis of DPPG/Nisin and DLPG/Nisin systems will allow us to know if lipid phase state impact on the
interaction and hence to have a better understanding about the action mechanism of nisin.

1- Abts, Andr, International Journal of Peptides, (2011), Volume 2011, Article ID 175145

BLM20_Interactions and Elastic Properties Of Lipid Membranes: The Role of A
Cosurfactant
Rubim, R L
1
; Bougis, K
2
; Gerbelli, B B
1
; Silva, E R
1
; Oliveira, C L P
1
; Navailles, L
2
; Nallet, F
2
; Oliveira, E
A
1
.

1
Complex Fluids Group, Experimental Physics Department, Institute of Physics, University of So Paulo,
So Paulo, Brazil
2
Research Centre Paul Pascal, Pessac, France
In this work we investigate interactions between lipid membranes and their elastic properties, with the
incorporation of ethoxylated fatty acids, commercially known as Simulsol. Small Angle X-ray Scattering
(SAXS) experiments were carried out in lamellar phases in order to obtain structural information and
using a model to the scattered radiation, we can obtain the thickness of the membrane and the Caill
parameter, which is related to the flexibility of the membrane. Applying an osmotic pressure to the
membranes we can access information about the interactions between the bilayers and evaluate the
effect of changing their flexibility and the interface. Combining the information obtained from both
methods it is possible to characterize the elastic parameters of the membrane. We observe that the
insertion of the cosurfactant increases the flexibility of the membranes, as well as the diluted and
repulsive interactions. This study brings a new method to evaluate interactions between membranes and
characterize its elastic properties.
Acknowledgements: This work was supported by FAPESP, process number 2011/16149-8, and INCT-
FCx.
1- DOI: 10.1021/la402962c
2- C. Oliveira et al. J. Appl. Cryst.. 2012. 1278.

SAB 2013 BLM
39

BLM21_Insertion of Glyphosate into a lipid bilayer: Study by molecular dynamics
Frigini, E.; Martin, O. A. and Porasso, R. D.
IMASL-CONICET, Departamento de Fsica, Universidad Nacional de San Luis, Italia 1556, 5700-San
Luis, Argentina
Herbicides are very useful for agriculture, allowing the elimination of weeds for the optimum development
of crops. In the last decades, glyphosate has been the most widely used herbicide in the world. Currently,
it is used without an accurate knowledge of its adverse effects on human health. Independent studies
have shown that the glyphosate present some toxicity in laboratory animals
(1-3)
. In order to understand
how the glyphosate interacts with a cell, in a first approximation, we model the process of insertion of this
herbicide molecule into a lipid bilayer, using the molecular dynamics technique. The main goal is to
determine the precise location of the glyphosate within the membrane, determining the free energy
profiles of this process
(4,5)
.
Acknowledgements This work was supported by PIP-112-2011-0100030 from CONICET and P-328402
from the UNSL, Argentina

1- E. A. Smith and F. W. Oehme. Vet Hum Toxicol. 1992. 531
2- C. Cox. Glyphosate. J. Pesticides Reform, 1995. 14.
3- A. Paganelli et al, Cem. Res. Toxicol, 2010,1586
4- G.M. Torrie and J.P. Valleau. J. of Computational Physics. 1977. 187.
5- Porasso, R, et al. J. Phys. Chem. B. 2009. 9988


BLM22_To Be Or Not To Be In Membrane Domains: Transbilayer Asymmetry And
Sphingomyelin-Dependent Preferential Partitioning Of The Acetylcholine Receptor

Perillo V.L
a
; Pealva, D.A.
a
; Vitale, A.J.
b
, Aveldao, M.I.
a
; Barrantes F.J
c
; Antollini S.S.
a

a
Instituto de Investigaciones Bioqumicas de Baha Blanca / Universidad Nacional del Sur, Argentina.
b

Instituto Argentino de Oceanografa / Universidad Nacional del Sur, Argentina.
c
Lab. Molec. Neurobiol.
Biomed. Res. Institute, UCA-CONICET, Argentina.
The preferential partitioning of the nicotinic acetylcholine receptor (AChR) in liquid-ordered (Lo) domains,
heterogeneous membrane domains commonly known as rafts,is thought to be a part of its clustering
mechanism. Previous studies from our group have shown that AChR lacks preference for Lo domains
when reconstituted in sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) model systems. Here we
study the effect on the possible Lo-preferential partitioning of purified AChR reconstituted in two model
systems (POPC:Chol, 1:1 and POPC:Chol:SM, 1:1:1) under: a) induced transbilayer asymmetry, by
addition of brain sphingomyelin (bSM) to the external hemilayer; and b) the presence of different pure SM
species in the model membrane (bSM, 16:0-SM, 18:0-SM or 24:1-SM). AChR distribution was evaluated
by fluorescence resonance energy transfer efficiency between the AChR intrinsic fluorescence and
Laurdan or dehydroergosterol fluorescence, and also by determining the presence of AChR in detergent-
resistant and detergent-soluble domains (1% Triton X-100, 4C). Both studies show that the induction of
transbilayer asymmetry or the presence of 16:0-SM or 18:0-SM, as opposed to bSM or 24:1-SM, leads to
a preferential partitioning of AChR in Lo domains. Thus, the localization of AChR in Lo domains strongly
depends on the characteristics of the host lipid membrane.

BLM SAB 2013
40

BLM23_Changes in biophysical properties of membranes containing sphingomyelin with
very long chain PUFA induced by its hydrolysis
Pealva DA
1
, Fanani ML
2
, Maggio B
2
, Aveldao MI
1
, Antollini SS
1

1
INIBIBB, CONICET-UNS, Baha Blanca, and
2
CIQUIBIC, UNC-CONICET, Crdoba, Argentina.
E-mail: dpenalva@criba.edu.ar

Very long-chain (C24 to C36) polyunsaturated fatty acids (VLCPUFA) are important acyl groups of
sphingomyelin (SM) and ceramide (Cer) of mammalian spermatozoa. In rat sperm, SM species containing
PUFA with 28-32 carbon atoms are exclusively located on the heads. After inducing the acrosomal
reaction almost complete hydrolysis such SMs occurs, leading to gametes considerably enriched in the
corresponding Cer species. The aim of this study was to evaluate the effects of the sphingomyelinase-
induced conversion VLCPUFA-SM VLCPUFA-Cer on the biophysical properties of a binary model
system (POPC/SM). The VLCPUFA-containing molecular species of SM were isolated from rat testes by
a combination of chromatographic techniques. Egg-SM, whose properties are widely known, was used for
comparison. Unilamellar liposomes (LUVs) were prepared and Dynamic Light Scattering was used to
evaluate their structures/sizes before and after enzymatic hydrolysis. The potential increase in the
interaction between different populations of liposomes (fission/fussion) after the SM Cer hydrolysis was
evaluated by FRET assays using NBD-PE as donor and Rh-PE as acceptor, and the lateral segregation
of phases after Cer generation was followed by anisotropy of different fluorescence probes (laurdan, DPH
and NBD-PE). In all biophysical properties measured, the SM species containing VLCPUFA contrasted
with those of the egg-derived saturated SM. The longer and bulkier acyl chains of VLCPUFA-Cer may
play a role in favouring the fusion/fission events that occur in the head of spermatozoa during the
acrosomal reaction, a process that requires topological lipid intermediates with negative curvature.

BLM24_Surface activity of L- ascorbic acid derivatives
Mottola, M.
1, 2
, Villanueva, M.
2
, Fanani, M.L.
1
, Vico, R
2
.
1
CIQUIBIC CONICET, UNC.
2
INFIQC - CONICET, Departamento de Qumica Orgnica, UNC.
E-mail: mili_m26@hotmail.com
L-ascorbic acid derivatives are molecules of potential pharmacological interest (as well as alimentary and
cosmetic) due to its antioxidant properties and amphiphilic nature.
These vitamin C derivatives (ASC
n
n= 10, 12, 14) were synthesized according to a modified method of
the main procedure already reported in literature [1] and were compared with the commercial derivative
ASC
16
. These molecules were characterized through
1
H
13
C NMR and IR, showing stability during three
weeks.
We found that, contrary to ASC
10
and ASC
12
, ASC
n
with n= 14 and 16 form stable Langmuir monolayers
at room temperature. ASC
16
films show phase transition from a liquid-expanded (LE) to a liquid-
condensed (LC) or crystalline phase, whereas the other derivatives presents only a LE phase.
All these compounds have a complex surface behavior and display a favorable penetration into


phospholipid monolayers with strong interaction among them.
This work was supported by: SECyT-UNC, FONCyT and CONICET. MLF and RV are Carrer Researchers
of CONICET, MM and MV are undergraduate students of UNC.

1- Capuzzi, G.; Nostro, P. Lo; Kulkarni, K.; Fernandez, J. E. Langmuir 11 (1996) pp. 3957
2- M. Mottola, N. Wilke, L. Benedini, R.G. Oliveira, M.L. Fanani. Biochimica et Biophysica Acta 1828
(2013) pp. 2496.

SAB 2013 BLM
41

BLM25_Characterization of the interaction of polyphenols with model membranes.
Effects upon viscosity and protection against oxidative agents.

de Athayde Moncorvo Collado A, Morero RD, Minahk CJ
Instituto Superior de Investigaciones Biolgicas (INSIBIO) CONICET-UNT. Chacabuco 461, San Miguel
de Tucumn.
Polyphenols are secondary metabolites commonly found in a wide variety of plants and vegetable-origin
aliments, characterized by the presence of two or more phenolic groups. These substances are subject of
great interest in the last years, due to their potential multiple bio-active features, including their well-
known antioxidant capacity. Importantly, a widely accepted characteristic about polyphenols is their ability
to interact with biological membranes.
For the present study, four polyphenols from different chemical families (resveratrol, naringenin,
epigallocatechin gallate and enterodiol) were tested for their effect on membrane viscosity by
fluorescence techniques. For that purpose, liposomes of dipalmitoyl phosphatidylcholine (DPPC) alone or
in combination with 40% cholesterol were prepared by extrusion and labeled with either laurdan, DPH or
TMA-DPH. Anisotropy was measured at different temperatures. On the other hand, protection against
oxidative agents such as hydrogen peroxide and Cu II was spectrophotometrically studied following the
increase of conjugated dienes. Fluorescence studies showed that the presence of polyphenols induced a
marked increase in the viscosity with a concomitant alteration in the order of the phospholipids, which
was polyphenol-dependent. On the other hand, all compounds tested were able to decreased the
oxidation levels induced by the Fenton reaction. However, little correlation was found with the membrane
interaction inferred from anisotropy measurements.
Results presented in this work indicate that polyphenols are able to closely interact with biological
membranes. Therefore, further studies in this area would result in a better understanding of the potential
uses beyond their anti-oxidant properties.

BLM26_Effect of pressure in assembly and disassembly of surfactant aggregates
Espinosa Y.R
1
, Grigera J.R
1,2


1
Centro de Qumica Inorgnica, CEQUINOR (UNLP-CONICET).
2
Departamento de Ciencias Biolgicas,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata. Argentina.
Hydrophobic interaction manifests itself in numerous phenomena, including the processes of self-
assembling, formation of micelles and protein folding

[1]. We studied the effects of high pressure,
temperature and solvent on hydrophobic interactions, responsible for micellization of amphiphilic
surfactants, using Molecular Dynamic simulations in a system of sodium dodecyl sulphate (SDS) in water,
heavy water and a pure non polar solute (Lennard-Jones) Preliminary results indicate that (i) At
pressures around 1-1,5 kbar causes disassembly of the formed micelles. (ii) In the range of the pressure
higher than 1,5 kbar, the pressure effects is completely reversed, the number aggregation increases
causing a redistribution of surfactant molecules changing the geometry of the aggregates, in agreement
with experimental observation. (iii) In Lennard-Jones solvent reverse micelles are formed, due to the lack
of hydrophobic interaction. (iv) Thermal analysis indicated than at low temperature the SDS molecules
are aligned in parallel, similar to lamellar phase diminishing the solvent accessible surface area; when
temperature is increased; the structural transition change producing predominantly micellar configurations
with a high aggregation number and high solvent accessible surface area.
1- Meyer, E.E.; Rosenberg, K.J.; Israelachvili, J. PNAS 2006, 15739.

BLM SAB 2013
42

BLM27_Characterization of Vesicle-Micelle transitions by Fluorescence Correlation
Spectroscopy (FCS) and Isothermal Titration Calorimetry (ITC)

Dodes Traian MM
1,2
, Pignataro MF
1
, Levi V
2
, Gonzlez Flecha FL
1

1
IQUIFIB-CONICET, Dpto. de Qumica Biolgica, FFyB, Universidad de Buenos Aires
2
IQUIBICEN-CONICET, Dpto. de Qumica Biolgica, FCEyN, Universidad de Buenos Aires

Structural and kinetic studies of membrane proteins require their reconstitution in amphiphilic systems to
ensure their proper folding and stability in aqueous media. The structure of these systems ranges from
micelles to vesicles depending on the relative concentrations of phospholipids and detergent. To
characterize the behaviour of these systems and the transitions among them we used Fluorescence
Correlation Spectroscopy (FCS) and Isothermal Titration Calorimetry (ITC). We used FCS to measure the
diffusion coefficient of the particles and thus this technique allowed us to gather information about the
heterogeneity of the studied systems. ITC allows detecting membrane solubilisation by measuring the
heat exchanged upon the addition of detergents. Working with a system similar to that used in membrane
activity protocols we could observe the abrupt change in size characteristic of a micelle-vesicle transition
at a phospholipid / detergent mole fraction around 0.75 and by ITC around 0.8 mole fraction the heat
signature changes from endothermic to exothermic. These experiments contribute to our understanding
of the correlation between the physical properties of the amphipilic systems and the function of
membrane proteins inserted in them.
With grants from ANPCyT and UBA.

BLM28_Biophysical and Biochemical characterization of Proteoliposomes harboring
Annexin V and Alkaline Phosphatase
M. Bolean
1
, A.M.S. Simo
1
, Kiffer-Moreira, T.
2
, M.F. Hoylaerts
3
, J.L. Milln
2
and P. Ciancaglini
1

1
Departamento de Qumica, FFCLRP-USP, Ribeiro Preto, SP, Brazil,

2
Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA,
USA,

3
Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
In this work, we standardized the proteoliposomes harboring Annexin V (AnxA5) and alkaline
phosphatase (TNAP) in order to obtain the best lipid composition to optimize the incorporation and
function of proteins. The kinetic parameters for the hydrolysis of the substrates by TNAP, in the absence
and presence of AnxA5, were determined at physiological pH. The proteins were incorporated into
liposomes constituted by DPPC and DPPC:DPPS (5, 10 and 15%). The best catalytic efficiency was
obtained for the system containing DPPS 10%. DSC studies show that with the DPPS concentration
increased in the DPPC liposomes is observed a progressive broadening of the transition peaks. The
presence of AnxA5 causes a decrease in H values and when TNAP is present in the proteoliposomes,
that effect is even greater. AnxA5 was able to mediate Ca
2+
influx into the DPPC and DPPC:DPPS 10%-
vesicles. The presence of TNAP in the proteoliposomes did not affect AnxA5-mediated Ca
+2
uptake.
However, the presence of AnxA5 affected significantly the hydrolysis of the substrates by TNAP. Studies
with Giant Unilamellar Vesicles (GUVs) confirmed the functional reconstitution of AnxA5 in the mimetic
system. Acknowledgements: CNPq, CAPES, FAPESP.
SAB 2013 BLM
43

BLM29_Structural properties of CHAPS micelles, studied by molecular dynamics
simulations.
Herrera, Fernando E.
a
; Garay, A. Sergio
a
; Rodrigues, Daniel E.
a,b
a
Departamento de Fsica, Facultad de Bioqumica y Ciencias Biolgicas, Universidad Nacional del Litoral
(UNL), Ciudad Universitaria, 3000 Santa Fe, Argentina.
b
INTEC (CONICET-UNL), Gemes 3450, 3000
Santa Fe, Argentina.
Detergents are essential tools to study biological membranes. They are frequently used to solubilize lipids
and integral membrane proteins, and to evaluate the strength of interaction of the membrane
components. The nondenaturing zwiterionic detergent usually named CHAPS was designed for
membrane biochemistry and integrates the effects of sulfobetaine-type detergents and bile salts. Despite
of the available experimental data little is know about the molecular structure of its micelles.
We performed Molecular Dynamics simulations to study the aggregation in micelles of several numbers of
CHAPS (1-16) starting from an homogeneous water dilution. We developed and validated the forcefield
parameters to describe the interactions of the molecule. After 50ns of simulation all the systems result in
the formation of stable micelles. We characterize the molecular shape (gyrate radii, volume, surface) of
the micelles. We analyzed the molecular structure (RDF, salt bridges, H-bonds, SAS) and found that the
main interactions that lead to the stability of the micelles are the electrostatic ones among the polar
groups of the tails and the OH's from the rings moiety. At variant with micelles of other compounds,
CHAPS- show a grain-like heterogeneity with hydrophobic micro-pockets. Our results agree with avalaible
information from NMR and X-ray. The rotational diffusion of the micelles is also characterized to compare
with EPR spectroscopy results.

BLM30_Design, Synthesis and Characterization of a novel azoniosome.
Sequeira; M. A.; Mari, I.; Benedini, L.; Dodero, V. I
Depto de Qumica- INQUISUR, Universidad Nacional del Sur-CONICET, Baha Blanca (AR).

Niosomes are promising new systems for Drug Delivery. They possess a vesicular lamellar structure
similar to liposomes; however they are constituted by mixtures of water insoluble ionic surfactants and
cholesterol [1]. Various groups have attempted to use this event to design vesicular systems based on
azoamphiphiles using either ionic or non-ionic head. However, the selection of the polar group head and
its relationship with the tail of the amphiphile was not adequate to achieve the formation of vesicular
aggregates and/or the photomodulation in water [2, 3].
Through the design and chemical synthesis, we obtained a new non-ionic azoamphiphile whose relative
head / linker/ tail is optimal for the formation of photoswitchable vesicles. This system has potential use
as a phototriggered drug delivery system or azoniosome.

Acknowledgements: Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.

1. Gupta S. et.al. Polymer 2012, 53, 3053.
2. Kordel C.; Popeney C.; Haag R. Chem. Commun. 2011, 47, 6584.
3. Orihara Y. et. al. Langmuir 2001, 17, 6072.





BLM SAB 2013
44

BLM31_Compressibility modulus and states of water in lipid monolayers.
Fras, M.A.,Salcedo, C.L.,Cutro, A.C. and Disalvo, E.A..
Laboratorio de Biointerfases y Sistemas Centro de Investigacin y Transferencia, de Santiago del Estero
(CITSE)
The lipid monolayer properties have been studied by compressing lipids spread in a large surface up to
its collapse. In this methodology, the lipids spread at huge areas are considered as being in a gas phase
and are compressed at a temperature below the critical one (the transition temperature) until
condensation. The thermodynamic treatment is compared with a real gas in which the condensation is
analyzed as a consequence of the manifestation of the intermolecular forces between the lipids. In other
words, lipid interacts between them as gas particles in vaccum. A more realistic approach to account on
monolayer behavior is that lipids, even at large areas, are in contact with the water phase. Upon
compression, the energy input is not merely used to make work of compression but to overcome the
friction of hydrated lipid molecules with the water (stationary) phase. Thus, the lipids drag water during its
compression, force its reorganization and/or a displacement work can occur. In order to clarify the
hydration role in the compression/expansion isotherms we have compared and evaluated the
compresibility of monolayers compressed from the very diluted state (gas phase) with that obtained after
the stabilization of the lipids in a force-free state. This study allows to comprehend the role of water
confined at the interphase processes on compressibility.
Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
1- K. Ariga, Y. Okahata, Langmuir 10 (7) (1994) 2272



BLM32_Enhanced antiradical activity of gallic acid included in lipid interphases.
Cutr, A.C.
1
, Salcedo, C.L.
1,2
, Fras, M.A., Nazareno, M.A.
2
, Disalvo, E.A.
1

Laboratorio de Biointerfases y Sistemas Biomimticos
1
y Laboratorio de Antioxidantes y Procesos
Oxidativos
2
, Centro de Investigacin y Transferencia, de Santiago del Estero (CITSE)
Polyphenols are well known by its effects as antioxidant agents and by its effects on the hydration layers
of lipid interphases. In this regard, it is known that gallic acid and its family are able to decrease the dipole
potential and to act in water as a strong antioxidant. In this work we have studied both effects on lipid
interphases in monolayers and bilayers of phosphatidylcholines. The results show that the antiradical
activity of gallic acid adsorbed to DMPC membranes increases five-fold with respect to that found in
water. In parallel, the gallic acid increases the negative surface charges of LUVs and decreases the
dipole potential. As a result, the positively charged radical species such as ABTS
+
are able to penetrate
more easily the membrane, enhancing the antiradical activity. The role of interfacial water in the
antiradical activity and the possible association of gallic acid with the phospholipid molecules were
studied by means of surface pressure/area curves, dipole potential measures and FTIR spectroscopy.
These results allow discussing the effect of interfacial water on the antioxidant reaction catalysed by
lipids.
Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
1- Huh NW, Porter NA, McIntosh TJ, Simon SA. Biophys J, 1996. 71, 3261.
2- Salcedo C.L., Lpez de Mishima B.A., Nazareno M.A. Food Res. Int., 2010. 43, 1187.



SAB 2013 BLM
45

BLM33_FTIR interaction between glycine and DPPC in hydrated state.
Norma M. Ale
1
, Aida Ben Altabef
1,2
, Andrea Gmez Zavaglia
3
and Sonia B. Daz
1
1
Instituto de Qumica Fsica. Facultad de Bioqumica, Qumica y Farmacia. U.N.T. San Lorenzo 456.
T4000CAN Tucumn, Argentina. e-mail:sobedi63@yahoo.com.ar.
2
Instituto de Qumica del Noroeste (INQUINOA)-CONICET-Tucumn, Argentina.
3
(CIDCA)-CONICET- Universidad de La Plata, Argentina.

This glycine`s family (betaine, carnitine, dimethylglycine or glycine) takes part in a variety of biochemical
reactions. The main function of a protector is to regulate loss and exchange of water in the biological
structures. The protective capacity of these compounds on the membranes has been studied on
numerous occasions. The existing information is fundamentally empirical, and do not know the
mechanisms of interaction of these compounds with the membrane at molecular level.
The aim of this work is analyze the interaction of the main functional groups of dipalmitoylphosphatidyl
choline (DPPC) with glycine (gly) by infrared spectroscopy Fourier Transform (FTIR) in hydrated state.
Our results revealed intermolecular interactions between the headgroups of DPPC and gly. These
interactions would indicate that the phosphate group of the lipid membrane forms hydrogen bonds with
gly in replacement of structured water.
1. Gmez Zavaglia, A.; Tymczyszyn, E.; De Antoni, G.; A. Disalvo J. Appl. Microbiol., 95(6): 1315-1320
(2003).
2. Daz, S. B.; Biondi de Lpez, A. C. and Disalvo, E. A. Chemistry and Physics of Lipids 122:153-157
(2003).
3 Crowe, L.M.; Mouradian, R.; Crowe, J.H.; Jackson, S.A.; Womersley, C.; Biochim. Biophys. Acta,
769:141150 (1984).


BLM34_Raman and FTIR interaction between l-cysteine methyl ester and DPPC in
anhydrous state
J. M. Arias
2
, M. E. Tuttolomondo
1
, S. B. Daz
1
and A. Ben Altabef
1,2

1
Instituto de Qumica Fsica. Facultad de Bioqumica, Qumica y Farmacia. Universidad Nacional de
Tucumn San Lorenzo 456. T4000CAN Tucumn.
2
INQUINOA-CONICET.

This work is the contribution of a number of studies about the cysteine family (L-cysteine, L-cysteine ethyl
ester and L-cysteine methyl ester) to understand lipid membrane interaction. The cysteine family has a
thiol group that takes part in a variety of biochemical reactions. The possible formation of weak hydrogen
bonds at receptor sites is of considerable interest, as it might contribute to a biological response.
The objective this work is understand and analyze the interaction of the complex of L-cysteine methyl
ester.HCl (CME) with lyophilized liposomes of dipalmitoylphosphatidylcholine (DPPC) by Raman and
infrared (FTIR) spectroscopies.
Our results revealed intermolecular interactions between the headgroups of DPPC and CME. These
interactions would indicate that the phosphate group of the lipid membrane forms hydrogen bonds
probably with S-H groups of CME in replacement of structured water.
The Raman analysis shows that the CME has little interaction with the hydrophobic region of DPPC. The
intensity ratio analyses show that the density of lateral packing of the acyl chain (I
2881
/I
2846
) and the
intensity ratio relevant to Gauche-trans rotamers (I
1096
/I
1062
) do not have significant changes. The I
2933
/I
2846
intensity ratios show an increase in movement and rotational disorder without an increase of the number
of Gauche-trans rotamers.
1- J.L.R. Arrondo, F.M. Goi, Chem. Phys. Lipids 96 (1998) 5368.
2- C. B. Fox, R. H. Uibel, J. M. Harris, J. Phys Chem. 2007, 111, 1142811436.
BLM SAB 2013
46

BLM35_Surface topography of lipid-peptide mixtures at the air-water interface using
BAM: role of physical state of the lipid.
Caruso, B, Ambroggio EE, Wilke, N, Fidelio GD.
CIQUIBIC,Depto.QumicaBiolgica, Fac. Cs.Qumicas, CONICET, UNC.

Previous studies in our laboratory showed that the interaction of peptides with membrane lipids depend
on the secondary structure of the former and the physical state of the latter (1, 2). Here, we contribute to
this subject by imaging of Langmuir monolayers using the Brewster Angle Microscopy technique (BAM).
Monolayer isotherms of the well-known Melittin did not present compressional hysteresis, in keeping with
a prevalence of -helix structure. Melittin form homogeneous monolayers at BAM, but when mixed with a
lipid that forms Liquid Expanded (LE) monolayers (T
c,dmPC
21C) it induced domain formation (phase
segregation) at lateral pressures that dependent on film composition. Although pH affects the isotherm of
pure Melittin, this did not affect the phase diagram of Melittin/DMPC. On the contrary, when mixed with
the more liquid POPC (T
c,poPC
-2C), the domains were not observed. On the other hand, the -sheet
A1-42 peptide exhibits a different behaviour when mixed with either DMPC or POPC. In A1-42/DMPC
mixtures, although phase segregation did not occur at a defined lateral pressure, a fibrillar domain pattern
became apparent upon compression. In A1-42/POPC mixtures no domains were observed. We
conclude that the physical state of lipid phase is not only important for lipid-peptide miscibility but also for
lateral topography.

Acknowledgements: Supported by grants from SeCYT-UNC, CONICET and FONCYT (Argentina).
1- Fidelio et al. BBA.1986. 49.
2- Ambroggio et al. BiophysicalJournal. 2005. 2706.


BLM36_Lipid bilayer fluid phases are not always fluid: a simulation experiment.
Pinto, O.A (1), Fras, M.A.(2), Disalvo, E. A.(2)
(1) Laboratorio de Sistemas Nanoestructurados y Electroqumica., (2) Laboratorio de Biointerfases y
Sistemas Biomimticos. Centro de Investigacin y Transferencia, de Santiago del Estero (CITSE).

FTIR measurements showed that the macroscopic disordered state of the lipid phase has, at the
molecular level, different arrangements of the methylene groups according to the lipid composition. Thus,
fluid membranes are not always fluid because membrane has, according to the acyl chain composition,
different ratios of connected and isolated methylene populations. In this work, we reproduced the typical
curves of changes in the asymmetric stretching modes of CH
2
of phosphatidylcholines with temperature
by modelizing the membrane system as a Lattice Gas in which each element (the CH
2
residues of the
acyl chains) is coordinated with six neighbors. The free energy change of each element state is given by
modulated by a cooperativity parameter accounting for the interaction with the first neighbors. The
Montecarlo simulation was run using the dynamic of Glauber or single spin flip and the algorithm of
Metropolis. The results show that the introduction of water blocks the membrane elements frustrating
some of the elements to contribute to the transition. In consequence, the freezing/frustration of some
elements changes the number of isolated elements as found by FTIR spectroscopy. It is concluded that
the state of the cooperative units in the so-called fluid state are not similar due to the chemical
composition of the acyl change and the water amount buried in it.
1- E.A.Disalvo, A.M. Bouchet, M.A. Fras, Biochim. Biophys. Acta 2013, 1828, 1683.
2- Thermal Biophysics of Membranes, Thomas Heimburg, Published Online: 20 SEP 2007 Print ISBN:
9783527404711, Online ISBN: 9783527611591 DOI: 10.1002/9783527611591
SAB 2013 BLM
47

BLM37_Liposomes and L-cysteine in the treatment of respiratory diseases
Perrota, R., Fernandez Ruocco, M.J., Siri, M., Chiaramoni, N.S., Alonso, S. del V.

Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
Respiratory diseases are very common and affect a large proportion of the population worldwide. For
therapeutic agents to reach the target cells is essential to bypass the barriers of the lung architecture.
With the main goal to overpass these barriers and destabilize them, we developed lipid transporters that
can encapsulate L-cysteine This amino acid, with sulfur in its structure may break the disulfide bonds of
the epithelial barrier and disrupt mucus network [1].
These lipid transporters were formulated with polymerizable lipid DC8,9PC mixed with DMPC . When
irradiated with UV light DC8, 9PC has the ability to form conjugated double bonds increasing stability [2,
3]. Transporters were formulated with two amino acid concentrations: 10 and 50 mol % (relative to lipid).
In order to characterize these systems we studied the polymerization efficiency and the hydrophobic
defects in the bilayer surface. Interactions were analyzed by FTIR. We found that increasing amino acid
concentration, polymerization efficiency decreases presumably due to increased hydrophobic surface
defects. FTIR data obtained confirmed that the L-cysteine interacts strongly with the liposome surface.
The design formulation should be considered as a strong candidate to overcome the mucus barrier. To
confirm this efficiency in vivo testing is going to be carried out in the Laboratory of Dr. Marcelo Morales
(UFRJ, Brazil).
1- Poelma, F.G.J., et al., Int. J. Pharm. 1990, 64: 161-169.
2- Chiaramoni, N.S., et al., J. Liposome Res. 2010, 20(3): 191-201.
3- Temprana, C.F., et al., Chem. Phys. Lipids. 2012, 165(5): 589-600.

BLM38_Molecular properties involved in the effects of steroids on membrane biophysical
state
Wenz J.J.
Instituto de Investigaciones Bioqumicas de Baha Blanca, Universidad Nacional del Sur, Camino La
Carrindanga Km 7, B8000FWB Baha Blanca, Argentina. Tel +54(291)4861201. jwenz@criba.edu.ar.

In order to learn about the factors that govern the effects of steroids on the physical state of membranes,
a library of 82 steroids was studied regarding their ordering, rigidifying, condensing and/or raft promoting
activity on membranes, concurrently with several molecular properties.
Steroids were classified according to their reported activity on membranes into one of two categories by
defining a dichotomous variable (0s and 1s). Each molecular structure was subjected to geometry
optimization by means of a semi-empirical procedure (AM1) and several molecular descriptors were next
computed for the optimized low energy conformations. The obtained data were analyzed by means of
logistic regression and mean contrasting.
Comparing steroids displaying ordering, rigidifying, condensing and/or raft promoting activity with their
counterparts having the opposite effect, substantial differences were found in the partition coefficients
(octanol-water), surface area, volume, number of rotatable bonds (mostly present in the carbon alkyl side-
chain), molar refractivity and molecular weight. Despite the intrinsic correlation between the molecular
properties, the results collectively point toward a positive correlation between steroids lipophylicity and
their ordering, rigidifying, condensing and/or raft promoting effects on bilayers.


BLM SAB 2013
48

BLM39_Surface and hysteresis properties of lipid interphases composed by head group
substituted phosphatidylethanolamines.
Salcedo, C.L.
1,2
, Bouchet, A.M.
1
, Nazareno, M.A.
2
, Disalvo, E.A.
2
, Frias, M.A.
1

Laboratorio de Biointerfases y Sistemas Biomimticos
1
y Laboratorio de Antioxidantes y Procesos
Oxidativos
2
, Centro de Investigacin y Transferencia de Santiago del Estero (CITSE, UNSE-CONICET)
This work analyzes the surface properties of PE-containing membranes modified at the head group
region by the addition of methyl and ethyl residues at or near the amine group. These residues alter the
lipid-lipid and lipid-water interactions by changes in the hydrogen bonding capability and the charge
density of the amine group thus affecting the electrostatic interaction. The results obtained by measuring
the dipole potential, the zeta potential, the area per lipid and the compressibility properties allow to
conclude that the H-bonding capability prevails in the lipid-lipid interaction. The non-polar groups attached
to the C
2
-carbon of the ethanolamine chain introduce a steric hindrance against compression and
increases the dipole potential. The analysis of areas suggests that lipids with methylated head groups
have a much larger compressibility at expense of the elimination of hydration water, which is congruent
with the broader extent of the hysteresis loop.
Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
1- Salcedo, C.L., Bouchet, A.M., Nazareno, M.A., Disalvo, E.A.Frias, M.A. Colloid & Surfaces, B
Biointerfaces, 2014. 113, 243.
2- BouchetA.M., FrasM.A., AlmaleckH., LairionF., MartiniF., DisalvoE.A., GordilloG., Biochim.
Biophys. Acta, 2009. 1788,918.


BLM40_Vitamin C: antioxidant and pro-oxidant action in SPC liposomes
Marsanasco M.
a
, Piotrkowski B.
b
, Calabr V.
b
, Alonso S. del V
a
. and Chiaramoni N.S.
a

a Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
b Fisicoqumica, Facultad de Farmacia y Bioqumica, UBA e Instituto de Bioqumica y Medicina
Molecular-CONICET.
The aim of this work was to study and characterize liposomes with vitamin C (VC) before and after
pasteurization. Liposomes were obtained according to Bangham method with soy phosphatidylcholine
(SPC) and SPC in combination with stearic acid (SA) or calcium stearate (CAS). Then VC or VC
combined with Vitamin E (VE) was added. Oxidative stability was studied with TBA and ORAC methods.
They were characterized by surface charge (Z potential) and membrane packing (MC540 and Laurdan),
size distribution (DLS) and also its rheological behavior. Results showed that the TBA method has an
influence per se on the antioxidant action of VC that maintains its activity even after pasteurization,
probably due to the protective effect of liposomes on the thermolbil VC [1]. VC changes Z potential from
negative to positive, shift that induces liposomal aggregation. The VC favors the entry of merocyanine
540 in SPC and SPC:SA liposomes and increament of laurdan GP in SPC:CaS. Similar trends were
obtained when combining both vitamins: VC and VE. Rheometric studies showed Newtonian behavior
and only SPC had increased viscosity post pasteurization. In summary, these results demonstrates that
SPC based liposomes have a potential application in food industry encapsulating both vitamins (VC and
VE).
Acknowledgements Universidad Nacional de Quilmes, Universidad de Buenos Aires, CONICET .
1- Marsanasco, M., Marquez, A.L., Wagner, J., Alonso, S. del V and Chiaramoni, N.S. (2011). Food Res
Int 44: 30393046.
SAB 2013 BLM
49

BLM41_Discussing the use of light scattering in the characterization of polidisperse
colloidal systems
Nomura, D. A. , Enoki, T. A., Silveira, N. P. and Lamy, M. T.
Grupo de Biofsica, Instituto de Fsica, Universidade de So Paulo, SP, Brazil
Dynamic (DLS) and Static (SLS) light scattering are widely used techniques in the study of colloidal
aqueous systems, like polymers, proteins or lipid aggregates dispersions. In SLS, the intensity of the
scattered light is measured, and the particle radius of gyration calculated. DLS measures time fluctuation
of the scattered light intensity, what allows the calculation of the particle diffusion coefficient, and, with the
use of the Stokes-Einstein equation, an effective particle radius. With aqueous dispersions of polystyrene
nanospheres of discrete sizes ((21.0 1.5) nm, (46.0 2.0) nm and (92.0 3.7) nm) we use DLS and
SLS techniques, at different scattering angles, and discuss the sensitivity of each technique to
characterize monodisperse systems, and the dimensions of particles in a multicomponent dispersion. For
monodisperse systems, SLS was not sensitive to particles of 21 nm, being more limited than DLS in the
characterization of small particles. In multicomponent dispersions, we show that it is very important to
have in mind that both SLS and DLS techniques give Z-average diameter values, that are weighted by
(radius)6, what means that larger particles contribute much more to the final result. Moreover, with DLS,
the size distribution analysis through double exponential or Contin methodologies can be rather
misleading. Also found unreliable is a polidispersity coefficient obtained from the method of Cummulants.
Discussion presented here is fundamental to the use of SLS or DSL in the characterization of unknown
colloidal dispersions.
Financial Support: USP, FAPESP and CNPq.

BLM42_Gold Nanoparticles Functionalized with Cell Penetrating Peptides -Synthesis,
Characterization and Interaction with Biomembranes-
Berberin M.V.
1,2
, Mayorga L.S.
2
, Ciocco Aloia F.
1
and Del Popolo M.G
1

Instituto de Ciencias Bsicas (ICB)
1
e Instituto de Histologa y Embriologa Mendoza (IHEM)
2
,
Universidad Nacional de Cuyo, Mendoza, Argentina

Over the past decades nanoparticles (NP) have prompted a large amount of research in pharmaceutical
sciences, as they offer novel alternatives in the design of drug delivery systems. It is noticeable, however,
that despite the large body of bibliography in this area there is no consensus yet on the way NP are
internalized and distributed within living cells. Size, shape and chemical composition of the surface are
among the factors that control internalization rates. Experiments show that NP intake can occur through
diffusion across the cell membrane, either mediated by the formation of a pore or the invagination of the
lipid bilayer, or follow an endocytic pathway.
In the present work we investigate the interaction of NP with the plasma membrane of living cells, and
explore a possible non-endocytic internalization mechanism. We use human sperms as model cells. Gold
NPs (10100 nm) are synthesized by the reduction of HAuCl
4
in sodium citrate, and the particle surface is
subsequently covered with two different cell-penetrating peptides (CPP). The interaction of the
functionalized NP with the sperms membranes is monitored by electron microscopy. Our results indicate
that, depending on the particle size, concentration, and incubation time, the NP show large affinity for the
cell surface and are able to penetrate the plasma membrane, reaching the acrosome and the cell
nucleus. In order to rationalize the interaction between the CPP-covered nanoparticles and the lipids of
the cell surface, we have investigated by Molecular Dynamics simulations the adsorption and penetration
of a standard CPP molecule (Arg
9
) into a model lipid bilayer (DPPC). Simulations predict a strong a
binding energy to the lipid surface, while the penetration of the CPP into the membrane core is
accompanied by the formation of a water channel.
BLM SAB 2013
50

BLM43_Monitoring CHAPS self-assembly by EPR spectroscopy and Factor Analysis
Rodi, Pablo
1
; Bocco Gianello, M.D.
1
; Gennaro, A.M.
1,2

1
Departamento de Fsica, Facultad de Bioqumica y Ciencias Biolgicas, UNL, Santa Fe
2
IFIS Litoral (CONICET-UNL), Santa Fe

CHAPS is a zwitterionic synthetic detergent derivative from bile salts. It is a facial detergent, and due to
its peculiar characteristics, CHAPS self assembly has been subject of several works in the last years, and
there are open discussions regarding the characteristic of the resulting micelles. In this work we obtain
EPR spectra of the spin label 12-SASL in CHAPS solutions of a wide range of concentrations. The
spectra show strong changes with concentration. Using Principal Factor Analysis we demonstrate that all
the spectra can be reproduced as linear combinations of only three fundamental spectra, and obtained
their relative weights at each detergent concentration. One of them corresponds to 12-SASL free in water,
and the other two correspond to the label in motionally restricted environments. One of the motionally
restricted components appears at a concentration coincident with the experimentally reported cmc of
CHAPS, and it was assigned to 12-SASL in a Type 1 micelle. The relative weight of this component
gradually decreases at increasing concentrations, with the rise of the component assigned to a Type 2
micelle. Rotational correlation times of the spin labels for each of the micelles were obtained by fitting the
spectra with slow motion conditions, and compared with the micelle rotational diffusion times calculated
by MD (poster Herrera et al.). This comparison allows us to make estimations about the aggregation
number.



BLM44_Oncoproteins induce multilamellar structure formation in DMPC vesicles
Borioli G, Gaggiotti MC and Del Boca M
CIQUIBICCONICET, Dep. de Qumica Biolgica, Fac. de Ciencias Qumicas, UN Crdoba.
gborioli@mail.fcq.unc.edu.ar

The Immediate Early oncoproteins c-Fos, c-Jun and Fra-1 are intrinsically unstructured amphitropic
proteins that interact with lipid membranes. In this work, we explored the effects of c-Fos, c-Jun and Fra-1
on membrane remodeling. We have used two complementary techniques, Small Angle X Ray Scattering
(SAXS) and Transmission Electron Microscopy (TEM), to evaluate a system of pure dimiristoyl
phosphatidyl choline (DMPC) or binary (DMPC/PIP
2
, phosphatidyl inositol diphosphate) 100 nm
liposomes , incubated with one of the oncoproteins. SAXS reveals that the three proteins induce the
formation of highly correlated multilamellar structures over the phospholipid phase transition [1], while
TEM shows two coexisting populations of membranes, one of aggregated vesicles and the other of
multilamellar structures, similar to the so-called myelin figures [2]. The spacing differs between SAXS and
TEM measurements, which could be accounted for by the difference in the hydration degree of the
samples. On the other hand, the spacing also differs between the c-Fos and Fra-1 induced multilamellae.
This is a very interesting contribution which may be important to the biological role of oncoproteins.

Acknowledgements: Brazilian SynchrotronLight Laboratory (LNLS),SECyT-UNC, CONICET, FONCyT.
1- Interactions of the transcription factors c-Fos and c-Jun with phospholipid vesicles. Torriani. I.; Borioli
G.A.; del Boca M. Activity Report, Brazilian Synchrotron Light Laboratory 2006
2- Gaggiotti, M.C. Tesis doctoral 2011. Fac. de Ciencias Qumicas, Univ. Nac. de Crdoba



SAB 2013 BLM
51

BLM45_Thermotropic and structural study of sphingolipid mixtures
Dupuy, F; Oliveira, R; Maggio, B.

Centro de Investigaciones en Qumica Biolgica de Crdoba CIQUIBIC-CONICET/UNC, Haya de la Torre
S/N, Ciudad Universitaria, Crdoba, Argentina. Email: fdupuy@fbqf.unt.edu.ar
The hydrocarbon chain of the lipids is a major determinant of the phase behavior of these molecules: long
and saturated acyl chains induce gel/solid ordered phases with a high degree of intermolecular
association. However, lipid phase states can be dramatically altered in multicomponent systems by non-
ideal mixing. In Langmuir monolayers, we demonstrated the formation of an azeotropic mixture between
the short chain ceramide C10:0 Cer and the sphingomyelin N-acylated with palmitic acid C16:0 SM, in
which a condensed phase is obtained at surface pressure values where both components are in
expanded state. In this work, we studied the thermotropism of the sphingolipids C10:0 Cer, C16:0 SM and
their binary mixtures by high sensitivity differential scanning calorimetry. Also, structural information of the
different lipid dispersions was obtained from small angle synchrotron X-ray diffraction. The results indicate
lipid mixing in the gel state but phase separation upon melting. Also, bilayer mesomorphism was
stabilized in the mixed state, although pure C10:0 Cer formed an unusually ordered inverted phase.
Acknowledgements: We thank to the Brazilian Synchrotron Light Laboratory LNS (CNPEM/MCT) for X-
ray beamtime at SAXS2 beamline under project D11A SAXS1 10716.




BLM46_Interfacial stabilization of the antitumoral drug Paclitaxel in monolayers of
ganglioside GM1
Heredia,V.
a
, Dupuy, F.
b
, Maggio, B.
c
, Beltramo, D.
a

a
Unidad de Biotecnologa,CEPROCOR- Min.CyT Pcia. de Crdoba;
b
INSIBIO-CONICET/UNT;
c
CIQUIBIC-CONICET/UNC, Depto. Qca. Biolgica, Facultad de Ciencias Qumicas, UNC
Paclitaxel (Ptx) is an antitumoral drug that inhibits microtubule depolymerization. However, its low water
solubility (less than 1 g.ml

1
) is a drawback in treatment of cancer patients. Recently, we demonstrated
that paclitaxel (Ptx) can be loaded into GM1 ganglioside micelles, to render a stable water soluble
formulation that increases the drug solubility up to 6.3 mg.ml
-1
. In order to get insights on the membrane
interaction of Ptx, we studied the interfacial behavior of the drug in the absence and in the presence of
GM1 by means of the monolayer technique at the air-water interface. Gibbs adsorption experiments and
compression isotherms were carried out in a Langmuir balance, equipped with surface potential detection
and amenable to Brewster angle microscopy. This allows to evaluate the affinity of Ptx towards GM1, the
lateral packing of the mixtures and the surface topography of the molecules in the film. The results
indicate that Ptx becomes stabilized at the interface in the presence of the ganglioside, leading to an
enhanced stability of the mixed GM1/Ptx, film at higher surface pressures compared to pure PTx.


Biofsica de protenas y cidos nuclicos (Biophysics of proteins and nucleic acids ) SAB 2013
52

BPA1_Protein-DNA interactions in extremophiles.

Ariel Aptekmann, Alejandro Nadra
IQUIBICEN-UBA

Protein-DNA interactions are central to cell activity regulation including transcription initiation. The TATA
box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP
is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable
and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs),
from below 0 C to more than 100 C. Thus, the archaeal TBP family is ideally suited to study the
evolutionary adaptation of proteins to an extremely wide range of temperatures (1).
We expect TBP and TATA box to coevolve responding to a number of factors, adaptation to temperature,
pressure, salinity, and other extreme physico chemical conditions.
We characterized the TATA box for different archeal phylum in terms of length, base composition,
information content, and analized its relation with OGT, which is predicted to have an effect in those traits
by Schneider's molecular information theory (2).

1 - Kopitz A, Soppa, J Spectrochimica Acta Part A : Molecular and Biomolecular Spectroscopy, 2009 ,
page 799
2 - Schneider. J. Mol. Biol. 1986, page 415




BPA2_Biochemical Characterization of Human Receptor-Type Protein Tyrosine
Phosphatase ICA512 Ectodomain expressed in Pichia pastoris.
Samus, I.
1
, Noguera, M. E.
1
, Ferreyra, R. G.
1,2
, Ermcora, M. R.
1,2

1
Departamento de Ciencia y Tecnologa, Universidad Nacional de Quilmes.
2
IMBICE-CONICET.
Human Receptor-Type Protein-Tyrosine Phosphatase ICA512 (or IA-2) is a transmembrane protein
located in secretory granules of neuroendocrine cells. Identified as one of the main antigens of
autoimmune diabetes, it is associated with protein secretion processes
1
. During insulin secretion, the
cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the
transcription of the insulin gene. The structures of the intracellular pseudocatalytic and extracellular
mature domains are known, but the transmembrane domain and several other parts of the receptor are
poorly characterized. Previously, we solved the structure of the mature ectodomain (ME ICA512, residues
449-575) and identified potential dimerization interfaces involved in the regulation of the secretion
2
.
Furthermore, ICA512 is a glycosylated protein and molecular modeling studies suggest that the
positioning of the sugar residues imposes steric restraints on the type of dimerization interface. To
address this question, we express ME ICA512 in Pichia pastoris. The protein secreted to the culture
medium was purified and characterized by mass spectrometry and size-exclusion chromatography. The
implications of these findings for biological activity will be discussed.
Acknowledgements: CONICET, UNQ and ANPCyT.
1- Hermel et al. Eur J Neurosci. 1999. 8:2609.
2- Primo et al. J Biol Chem. 2008. 283:4674.

SAB 2013 BPA
53

BPA3_Defining the role of the Sco proteins in the assembly of the Cu
A
site
Morgada MN
a
, Abriata LA
b
, Vila AJ
a

a
Instituto de Biologa Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioqumicas y
Farmacuticas, Universidad Nacional de Rosario-Conicet, Rosario, Argentina
b
Swiss Federal Institute of Technology, cole polytechnique fdrale de Lausanne (EPFL), Lausanne,
Switzerland
The dinuclear copper center Cu
A
is the electron entry point of the cytochrome c oxidase (COX). It funnels
the electrons from reduced cytochrome c to the Cu
B
center at subunit I of COX where O
2
is reduced to
water molecules. The correct assembly of this metal center is essential for the function of the complex
and thus for the survival of the cell.
Metallochaperones are proteins capable of binding copper ions and transfer them to their target proteins
that use them as a cofactor, and at the same time reduce the toxic effect. In the case of the assembly of
the Cu
A
site in humans, two proteins of the Sco family have been found as essential for its formation:
Sco1 and Sco2.
1

Both proteins binds copper ions through a CxxxCx
n
H motif, being able not only to transfer copper but also
able to transfer the electrons to maintain the Cys from the Cu
A
protein in its reduced state for the binding
of the copper ions.
2

Using a model of the eukaryotic oxidase, we followed by NMR the interactions between these two
putative metallochaperones finding that Sco1 is able to transfer to copper while Sco2 is important to
maintain the Cys ligands in the reduced state.
1- Robinson, N.J. and D.R. Winge, Annu Rev Biochem, 2010. p. 537-62.
2- Abriata, L.A., et al., Nat Chem Biol, 2008. p. 599.


BPA4_Rheological properties of regular insulin and aspart insulin Langmuir monolayers
at the air/water interface: condensing effect of Zn
2+
in the subphase.
Grasso, E.J., Oliveria, R.G., Maggio B.
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC-CONICET), Dpto. de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Pabelln Argentina, Ciudad
Universitaria, X5000HUA, Crdoba, Argentina.
The interfacial behavior of regular insulin (Reg-insulin) and aspart insulin (Asp-insulin) was critically
affected by the presence of Zn
2+
in the subphase. This cation induced a condensed-like behavior in the
compression isotherms, especially apparent for Reg-insulin films when observed by Brewster angle
microscopy. Immediately after spreading, Reg-insulin, but not Asp-insulin, showed bright patches that
moved in a gaseous-like state. Moreover, Zn
2+
caused marked variations of the surface electrostatics of
both insulin monolayers and considerable hysteresis of their molecular organization. By oscillatory
compression-expansion cycles, we observed in all cases the development of a dilatational response to
the surface perturbation, and both monolayers exhibited well-defined shear moduli in the presence of
Zn
2+
, which was higher for Reg-insulin. Development of a shear modulus indicates behavior resembling a
nominal solid, more apparent for Reg-insulin than for Asp-insulin, suggesting the presence of viscoelastic
networks at the surface.
This work was supported by: CONICET, FONCyT and SECyT-UNC.

BPA SAB 2013
54

BPA5_Topological study of the -lactam-sensor proteins from Methicillin resistant
Staphylococcus aureus (MRSA).
Peris,D.; Belluzo,B.S.; Llarrull, L.I.
Instituto de Biologa Molecular y Celular de Rosario (IBR), Ocampo y Esmeralda, Predio CONICET -
(2000) Rosario
The membrane proteins BlaR1 and MecR1 play key roles in resistance to -lactam antibiotics in MRSA
strains. It is proposed that they sense and transduce the signal to unleash the transcription of the
resistance genes.
1
However, the topology of these proteins and the molecular events that are involved in
the transmission of the signal remain controversial. Here we report a topology study of the proteins
expressed in E. coli (where we have previously shown that BlaR1 is active) based in a strategy that
involves the construction of C-terminal fusions to EGFP.
2
This reporter does not fold correctly when
translocated to the periplasmic space through the Sec System and, therefore, it does not fluoresce when
fused to a periplasmic loop. In order to determine the number of membrane-spanning domains and the
localization of the different domains, we characterized the different fusion proteins by fluorescence
spectroscopy in whole cells and by fluorescence microscopy, and we normalized the expression of the
different constructions in E.coli by Western blot analysis.
Acknowledgements
The PEW Charitable Trusts, ANPCyT, CONICET

1- Llarrull, L.I., Fisher, J.F., Mobashery, S. Antimicrob. Agents and Chemother. 2009. 4051.
2- McKenzie, N.L., Nodwell, J.R. Microbiology. 2009. 1812.


BPA6_The structure of plant pri-miR172 and its complex with HYL1.
Suarez, IP
1
; Hbarnter, C
2
; Rasia, RM
1

1 Instutute of Molecular and Cellular Biology of Rosario (CONICET-UNR). Ocampo y Esmeralda, predio
CONICET, 2000, Rosario, Argentina.
2 Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Gttingen, Germany.
MicroRNAs are essential gene regulators in multicellular organisms. Plant miRNA precursors (pri-
miRNAs) are processed in the nucleus by a protein complex formed by DICER-LIKE1 (DCL1), HYL1 and
SERRATE. HYL1 is a double stranded RNA binding protein, with no catalytic activity, that ensures the
accuracy of the processing reaction. The mechanism by which itguides the RNAse DCL1 to the correct
location within the precursor is unknown. pri-miRNAs display highly variable secondary structure features
that could be essential to define the location of the miRNAs within their sequences. In this work we
characterized the secondary structure of Arabidopsis thaliana pri-miR172 and a variant that hinders
processing in vivo by means of enzymatic probing. The mutant shows an alteration in the secondary
structure distribution of the upper stem. We mapped the location of HYL1 within the precursor by RNAse
and DMS protection assays. The protein binds to the lower stem of the precursor, and the size of the
protected region suggests that both RNA binding domains bind in line on the precursor. Binding of HYL1
induces conformational changes on the upper stem region. Remarkably, the first nucleotide of the miRNA
sequence becomes particularly sensitive to DMS modification in the complex with HYL1. Altogether our
results show that HYL1 recognizes specifically the pri-miRNA structure and induces conformational
changes on distant parts of the precursor that contribute to the specificity of the processing reaction.
Acknowledgements: ANPCyT, CONICET, DAAD
SAB 2013 BPA
55

BPA7_Overexpression of the BlaR1 membrane protein implicated in resistance to -
lactams in Staphylococcus aureus
Belluzo, BS, Llarrull, LI
Instituto de Biologa Molecular y Celular de Rosario (IBR), Ocampo y Esmeralda, Predio CONICET -
(2000) Rosario
BlaR1 is a membrane receptor implicated in -lactam antibiotic resistance in Staphylococcus aureus. This
protein is composed of various transmembrana helixes, followed by a cytoplasmic domain and an
extracellular sensor domain capable of detecting de presence of antibiotic (1). The predicted cytoplasmic
domain contains Zn(II)-dependent protease activity and it has been proposed that it is activated in
response to binding of the antibiotic to the sensor domain (2). Activated BlaR1 degrades the gene
repressor BlaI, which triggers the expression of the BlaZ-lactamase. We are interested in the
spectroscopic study of the signal transduction mediated by BlaR1. In order to overexpress this protein, we
usedactive natural variants lacking the first three transmembrane helixes. We evaluated the expression
as fusions to different soluble tags that were shown to help in the overexpression of several membrane
proteins, and we are now developing a protocol of purification from inclusion bodies, denaturation and
refolding.
Acknowledgements
The PEW Charitable Trusts, ANPCyT, CONICET

1- H. Z. Zhang, C. J. Hackbarth, K. M. Chansky, H. F. Chambers, Science 291, 1962 (2001).
2- L. I. Llarrull, S. Mobashery, Biochemistry 51, 4642 (2012).


BPA8_Expression and purification of a novel flavohemoblobin from Mycobaterium
tuberculosis.
Piccinni, F.E
1,3
. Bonamore A.
3
, Marti M. A.
1,5
, Nadra A.D.
1,2
, Boffi, A.
3
and Estrin, Daro A.
4,5

1
Depto. Qumica Biolgica Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aire,
2
IQUIBICEN-CONICET,
3
Laboratory for advanced technology in the production of pharmaceuticals,
Department of Biochemical Sciences, Sapienza University, Roma, Italia.
4
Depto Qumica Inorgnica,
Analtica y Qumica Fsica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Argentina
5
INQUIMAE-CONICET

Flavohemoglobins, widely found in bacteria and yeast, have both a globin and a ferrodoxin reductase
domain (FAD-binding domain). The flavohemoglobin from E. coli has been thoroughly characterized as
an important feature against nitrosative stress. A novel flavohemoglobin studied in M. tuberculosis shows
some structural similarity to the latter one from E. coli. However, it is thought to have a different role in
protection against oxidative damage that does not include NO scavenging. The cell membrane is
protected by its cycling between the ferrous and ferric state due to a D-lactate dehydrogenase activity.
The globin domain can be easily obtained in BL21 DE3 Star E. coli strain. Since it is a rather soluble
protein, its purification consists of a two-steps column protocol and results in a high purity product.
Therefore, several characteristics can be studied simply by UV-visible spectrophotometry. The globin
domain shows a high affinity for oxygen and appears to be able to bind fatty acids in the ferric and ferrous
states. It can be oxidized with dithionite or ferricianide, which will allow further studies on the nature of the
binding (i.e.: by gas mass chromatography). The understanding of this protein might help unravel the
pathogenic pathways of M. tuberculosis.
1- Alessandra Bonamore and Alberto Boffi. IUBMB Life. 2008. p. 19.
2- Kanak L. Dikshit and col. The Journal of Biological Chemistry. 2012. p. 16435.
BPA SAB 2013
56

BPA9_Coagulation of casein micelles induced by bacterial enzymes: effect of cations
Lombardi, J
1,4
; Folmer, AP
2
, Brandelli, A
2
; Risso, P
1,3,4
; Boeris, V
1,4

1
UNR.
2
Universidade Federal do Rio Grande do Sul.
3
IFIR.
4
CONICET.
The aim of this study was to determine the effect of Ca
2+
, Na
+
, Zn
2+
and Mg
2+
on the milk coagulation
process induced by the enzymatic pool P7 (EPP7), secreted by Bacillus sp. P7. The significance of the
concentration of each cation on the time of coagulation (t
c
) and fractal dimension of clots obtained (D
f
)
was analized. The turbidity () of casein micelle (CM) suspensions in the range of 450-650 nm were
recorded. parameter, which is related to the particle size, was obtained from the slopes of the linear
graphs of log vs. log time (t). D
f
, which estimate the degree of compactness of the aggregates formed, is
the maximum value achieved and t
c
was determined as the time at which stabilizes after the sharply
rising. The effect of these four cations on the EPP7 enzymatic activity and on the initial size (initial ) of
the particles were also studied. The presence of Ca
2+
, Na
+
or Zn
2+
increased initial values, which
corresponds to the formation of larger CM. This makes sense since the shielding of the negative casein
charges facilitate their aggregation. The variables t
c
and D
f
proved to be also dependent of Ca
2+
, Na
+
and
Zn
2+
. On the other hand, Mg
2+
has not a significant effect on these variables (p>0.05). As the
concentration of Ca
2+
increased (up to 15 mM) the D
f
decreased; however, the increase in the
concentration of Zn
2+
(up to 0.25 mM) the D
f
increased. The concentration of Na
+
(up to 100 mM)
changes the effect of Zn
2+
on the D
f
: at higher NaCl concentration, less is the effect of Zn
2+
(the ionic
strength is probably the responsible of this change, not the Na
+
specifically). These three cations caused
a decrease of t
c
. This finding confirms that the t
c
is not directly related to the enzymatic activity of EPP7,
since Ca
2+
and Mg
2+
had a stimulatory effect on EPP7 activity, while Na
+
and Zn
2+
caused a decrease on
its activity. In conclusion, the cations evaluated have a differential effect on the CM aggregation process.


BPA10_Auto catalytic ROS dependent proteolysis of ICA512
Toledo P., Ros A., LLovera R., Primo E., Ermcora M.
Laboratorio de Expresin y Plegado de Protenas, Univ. Nac. de Quilmes
In previous works of our group several structural features of the membrane proximal ectodomain of
ICA512 (MPE ICA512; residues 469-557) were clarified and two main different association modes were
described; namely 4-4 and 2-2. These two possible interfaces of dimerization have similar
crystallographic properties but differ in critical aspects. While 4-4 comprises a region with high degree
of sequence conservation within proteins of the same family, 2-2 involves the surface responsible for
the dimers found in solution. To gain further insight in this topic an extended version of MPE ICA512
including residues 469 to 576 was prepared and characterized. The added residues link the ectodomain
to the transmembrane domain (residues 576-600). The new construction allowed to test hypothesis
regarding the topology or the ectodomain and its orientation on the membrane as well as to assess the
mechanism and participation of the added residues in the autocatalytic, ROS dependent, proteolysis of
the receptor reported in previous works by our group.
1- Primo ME, Klinke S, Sica MP, Goldbaum FA, Jakoncic J, Poskus E, Ermcora MR. J Biol Chem.
2008. 283:4674.
2- Primo ME, Jakoncic J, Noguera ME, Risso VA, Sosa L, Sica MP, Solimena M, Poskus E, Ermcora
MR. PLoS One. 2011. 6:e24191.

SAB 2013 BPA
57

BPA11_DSC and RMN approach to study the complex unfolding mechanism of a pdz
domain
Torchio, G
1,2
; Burgos, I
3,2
; Fidelio, G
3,2
; Arn, M
4,2
; Gallo, M
4,2
; Ermcora, M
1,2
; Sica, M
5,2
.
1.Laboratorio de Plegado y Expresin de Protenas, Univ. Nac. de Quilmes-IMBICE-CIC, Argentina
2.CONICET, Argentina. 3.Centro de Investigaciones de Qumica Biolgica,Univ. Nac. de Crdoba,
Argentina. 4.Fundacin Instituto Leloir, Argentina. 5.Centro Atmico Bariloche, Argentina

Many PDZ domains have been extensively studied from a biophysical perspective since they are
important and ubiquitous modules of protein-protein interactions, with particular characteristics of folding
and binding. Our model of study is the PDZ domain of the 2-syntrophin protein (2S-PDZ), which is
directly involved in the regulation of insulin secretion. We have shown that thermal unfolding curves of
2S-PDZ are not as expected for a protein that follows a two state nor a three state model of folding (1),
as reported for other PDZ domains. Moreover, the unfolding curves of 2S-PDZ are more reminiscent of
a downhill regime, or multiple state mechanism of folding (2-3).
Here we present additional evidence that supports the hypothesis of a complex folding mechanisms for
2S-PDZ. Differential scanning calorimetry (DSC) experiments discard two- and three-state models,
showing a more complex scenario. DSC experiments also rule out oligomerization during thermal
unfolding, at least at concentrations up to 2 mg/ml. Although some aggregation is detected at high
temperatures, this only seems to be associated to a minor population of 2S-PDZ and thermal
denaturation probed to be highly reversible, as a second and a third scan of DSC are completely
superimposable. Also NMR experiments suggest the existence of a minor population of 2S-PDZ that
could populate an alternative conformation.
(1) Torchio, G; Ermcora M; Sica, M. Biophysical J. 2012. p2835
(2) Oliva, F; Muoz, V. J. Am. Chem. Soc. 2004. p8596(3) Naganathan AN; Perez-Jimenez R; Sanchez-
Ruiz JM; Muoz V. Biochemistry. 2005. p7435

BPA12_Isolation and characterization of calreticulin oligomers
Moreschi A. C., Durand E. S., Montich G.G., Decca M.B.
Departamento de Qumica Biolgica-CIQUIBIC, Facultad de Ciencias Qumicas, Universidad Nacional de
Crdoba. Haya de la Torre y Medina Allende,5000, Crdoba, Argentina.
Calreticulin (CRT) oligomerization occurs in living cells as a response to stress, and leads to an
enhancement in chaperon and polypeptide binding activities of this protein. Recruitment of CRT
oligomers seems to be critical for the scaffolding of stress granules in cytoplasm, a mechanism that
regulates mRNA translation as a response to stress. Extended CRT oligomerization has been also
proposed to act as a large platform for binding of different substrates. Despite the importance of CRT
oligomers in expanding CRT functions, no structural characterization of CRT oligomers has yet been
performed.
Here we describe a method for isolation of CRT oligomers as well as its biophysical characterization.
Circular dichroism spectroscopy was used to determine structural properties of CRT oligomers in
comparison with monomeric CRT. We found that the global secondary structure of calreticulin is retained
in oligomers whereas tertiary structure is partially lost. Differential scanning calorimetry showed no
cooperative transition in the thermal denaturation of CRT oligomers, and a decreased thermal stability in
comparison with CRT monomers.
Acknowledgements: This work was supported by grants from the Agencia Nacional de Promocin
Cientfica y Tecnolgica, CONICET, SECyT Universidad Nacional de Crdoba.

BPA SAB 2013
58

BPA13_Towards regulation of peptide-DNA interaction using an allosteric molecule
Quirolo, Z. B.
a
;Mascareas, J.L.
b
;Dodero, V. I
a
a- Depto de Qumica- INQUISUR, Universidad Nacional del Sur-CONICET, Baha Blanca (AR). b-
CIQUS, Universidad de Santiago de Compostela (ES).

Cells receive a wide variety of cellular and environmental signals which must be processed to generate
specific and timely genetic responses. The first step in the expression of genetic information, namely the
synthesis of RNA from DNA template, is tightly regulated by DNA-binding proteins called Transcription
factors (TFs). One of them, is GNC4, a TFs found in yeast which recognizes it cognate DNA binding
domain as a helical dimer. The structural motif is composed by two regions: one responsible of
recognition named the basic region and the other is the leucine zipper domain responsible of
dimerization. Previously, we have used this model peptide to prove spatial and temporal control [1, 2].
Now, we envisaged the use of a truncated GNC4 peptide which possesses only the basic region
chemically modified at the N-terminal in order to promote dimerization and DNA recognition in the
presence of an allosteric molecule. This molecule possesses two -Cyclodextrins to promote peptide
dimerization and a linker based on an azobenzene moiety which is able to change its conformation upon
light irradiation. The chemical synthesis, structural characterization and the initial binding experiments are
presented.

Acknowledgements:Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Blanco J. B.; Dodero V. I.;Vzquez M. E.; et.alAngew. Chem. Int. Ed. 2006, 118, 8390.
2- Mosquera, J.; Jimnez-Balsa, A.; Dodero, V. I.; Vzquez, M. E.; Mascareas, J. L. Nature Comm,
2013, 4, 1874.


BPA14_Evaluation of 33-mer gliadin assemblies. Towards understanding its
immunomodulator role in gliadin related diseases.
Herrera, M. G.
a
; Lonez, C.
b
;Celej, S.
c
;Ritacco, H.
d
, Ruysschaert, J-M.
b
; Dodero, V. I
a
a
INQUISUR- UNS-CONICET, Baha Blanca (AR).
b
CBSB-Universit Libre de Bruxelles (BE).
c
Dto.
Qumica Biolgica, CIQUIBIC,CONICET, Fac. Ciencias Qumicas, UNC Crdoba (AR).
d
INFISUR-UNS-
CONICET, Baha Blanca (AR).

Gliadin, a protein present in wheat, rye and barley undergoes incomplete enzymatic degradation during
digestion, producing several peptides. One of them is a 33-mer peptide, LQLQPF(PQPQLPY)
3
PQPQPF,
a proline-rich fragment which elicits an immune response in susceptible individuals and it is associated
with gluten sensitivity and celiac disease [1]. Recently, our group has demonstrated that the 33-mer
gliadin peptide is able to self- assemble into nanospheres which align into fractal linear arrays above a
certain concentration under physiological conditions [2]. Herein, the evaluation of 33-mer self-assembly
system by fluorescence spectroscopy techniques, ATR-FT-IR and DLS is presented. Interestingly, 33-mer
assemblies are able to induce NFk, a transcription factor related to inflammatory reactions, in a dose
dependent manner. Our results demonstrate a relation between 33-mer assemblies and inflammation,
which can be relevant in the early steps of gliadin intolerance disorders.

Acknowledgements:Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Sapone, A. et al. BMC Medicine. 2012, 10:13.
2- Herrera, M. G., Zamarreo, F., Costabel, M., Ritacco, H., Htten, A., Sewald, N., Dodero, V. I.
Biopolymers. 2013, in press.


SAB 2013 BPA
59

BPA15_Catalytic activity of beta galactosidase entrapped in a silicate matrix. Effect of
the substrate type, porous structure and aging time.
Burgos, M.I.
a
, Carrer, D.
b
, Perillo, M.A.
a

a: Instituto de Investigaciones Biolgicas y Tecnolgicas (IIBYT, CONICET-Universidad Nacional de
Crdoba). b: Instituto de Investigacin Mdica Mercedes y Martn Ferreyra.
Protein encapsulation in solid matrixes is of interest for biotechnological purposes and it also serves as a
model of molecular crowding. We have successfully entrapped the enzyme -galactosidase (-gal) in
silicate gels via a sol-gel reaction. The catalytic activity of encapsulated -gal (E-gal) was compared with
the activity of the soluble form (S-gal) with two different substrates (ONPG and PNPG) and at different
aging times. Data were analyzed with a Michaellis-Menten model. With the substrate ONPG, we found a
Vmax value higher for E-gal than for S-gal and this difference increased at longer aging times. On the
contrary, with PNPG, Vmax was not affected neither by the enzyme condition (S-gal and E-gal) nor by
the aging time. The porous structure of the silicate matrix was also studied as a function of aging time of
the gels. Thus, fluorescence correlation spectroscopy (FCS) was applied through the analysis of
diffusional properties of fluorescent probes of different sizes, entrapped in the gel matrix. We observed an
anomalous diffusion in some conditions, suggesting a self-similar pore structure, and aging time
dependent changes in diffusion coefficients. Taken together our result supports the hypothesis that there
is a relationship between the catalytic mechanism proposed for -gal and water availability in the silicate
gel since the ratio between bulk and structured water depends on the pore size.
Acknowledgements: Authors thank CONICET, foncyt and Secyt-UNC for the financial support. MIB is a
postdoctoral fellow and MAP and DC are career members of CONICET.


BPA16_The Thermal Unfolding of 2-Glycoprotein
Soledad Bazn, Mariana Paolorossi and Guillermo Montich
Departamento de Qumica Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba,
CIQUIBIC (CONICET)

2- Glycoprotein I (B2GPI) is an abundant glycoprotein of human plasma. It participates in blood
coagulation processes and the clearance of phosphatidylserine exposing- apoptotic cells. B2GPI is also
the major antigen involved in the aberrant immune response that characterizes the antiphos-pholipid
syndrome.
B2GPI consists of a single chain of 326 aminoacids arranged in five domains. Four of them are the so
called sushi or short consensus repeat (SCR) domain with its conserved beta sandwich- arrangement.
The fifth domain is larger and it has a lysine rich region that was shown to participate in the interaction of
B2GPI with anionic lipids. B2GPI has four N-glycosilation sites with complex bi and triantennary complex
glycans containing sialic acid that comprise ~20% of its total weight.
To study the folding behavior of this multiple domain glycoprotein we performed differential scanning
calorimetry at various pH and salts conditions. We found that at pH 7 the protein unfolding proceeds with
a pre-transition at lower temperatures. This effect is more pronounced in the absence of salt. In order to
characterize this earlier intermediate we perfomed circular dichroism studies of the thermal unfolding of
B2GPI in the far and near UV region. The far UV signal shows a two state unfolding transition centered at
the same temperature of the calorimetric one. In contrast, the near UV signal shows a more complex
behavior. These results would indicate that the folding intermediate has a different tertiary structure
protein while conserving its secondary one.
Acknowledgement: S.B. wants to thank FONCyT for it postdoctoral fellowship. We also wish to
acknowledge the following institutions for its support: CONICET, SECyT- UNC and FONCyT.
BPA SAB 2013
60

BPA17_Lactose binding kinetics of the -galatosil binding protein Galectin-1
Romero J. M.
1
, Estrin D. A.
2
, Rabinovich G. A.
3
, Di Lella S.
1,3
1
INQUIBICEN-CONICET, FCEN-UBA;
2
INQUIMAE-CONICET, DQIAyQF, FCEN-UBA,
3
Laboratorio de
Inmunopatologa, IBYME-CONICET. juanm.romero18@gmail.com
Galectin-1 is a -Galatoside binding protein involved in cell comunication and diferentiation processes.
1
It
consists of a 134-aminoacid domain which has been proved to form a homodimer at physiological
conditions.
2
In a previous work, we have studied the thermodynamics of lactose binding and the simmetry
of the homodimer applying computational simulation techniques
3
. Now we employ guided molecular
dynamics to characterise the binding and unbinding process, paying special attention to the molecular
determinants of the kinetic barrier, which turns out to be mainly the result of hydrogen bonds breaking and
forming. Furthermore, we measure the association and dissociation rate constants by stopped flow
experiments. We find both constants to be 206 M
-1
.s
-1
and 0,98 s
-1
, respectively, and correlate them with
the calculated energy barriers, while discussing results with previous reported kinetics experiments.
4
Acknowledgements: Conicet, ANPCyT, UBA, Fundacin Sales, Consejo Interuniversitario Nacional, and
Dr. Madia Trujillo
1- Rabinovich GA and Toscano MA, Nat. Rev. Immunol, 2009.
2- Lpez-Lucendo et al, J Mol Biol, 2004.
3- Romero JM and Di Lella S, Unpublished results, 2012
4- Ghler et al, Biophysical J., 2010



BPA18_Tinkering with coordination in globins

Boron, I.
1,2
, Chisari, L.
1
, Wetzler, D. E.
1
, Capece, L
2
, Marti, M
1,2
, Estrin, D
2
, Nadra, A
1

1
Departamento de Qumica Biolgica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Buenos Aires, Argentina, C1428EGA
2
Departamento de Qumica Inorgnica, Analtica y Qumica Fsica, INQUIMAE-CONICET, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, C1428EGA
Background. Human Myoglobin (Mb) and hemoglobin are relevant examples of pentacoordinated (5c)
heme proteins and many of their biological functions involve the coordination of exogenous ligands in the
sixth position of the heme group. On the other hand, in several globins direct coordination of HisE7 to the
Fe atom leads to the formation of a hexacoordinated (6c) globin, as noted in neuroglobin (Ngb) and
cytoglobin, among others. It has been suggested that this bond and the equilibrium between the 6c and
the 5c state regulate ligand affinity. Recent work by Nadra et al. has shed light on the key determinants
for globin hexacoordination, engineering Mb and Ngb by swapping their CD region. A partially 6c
myoglobin and a subtly 5c Ngb shown that each chimeric protein shifted its coordination state according
to its CD region. Goal. With the aim of further displacing the coordination equilibrium, we designed point
mutations to enhance the observed behaviour. Results. By exploring the contacts stabilizing the CD
region we have found a contact between a lysine and a glutamic acid which may account for Mb's
preference for the 5c state. Molecular dynamics simulations, equilibrium spectroscopy and ligand binding
results for a chimeric Ngb punctual mutant suggest differences in agreement with the theoretical
predictions.

SAB 2013 BPA
61

BPA19_Effect of molecular crowding on the conformation and thermal stability of -Gal
Nolan, MV and Perillo, MA
IIByT (CONICET-UNC), ICTA and Ctedra de Qumica Biolgica, Dpto. de Qumica, Facultad de
Ciencias Exactas, Fsicas y Naturales(UNC). e-mail: vnolan@efn.uncor.edu
In previous works we studied the effect of molecular crowding on beta galactosidase (-Gal) enzymatic
activity. The results showed that V
max
was just slightly affected, while the affinity of the enzyme (K
M
)
suffered a significant decrease at growing molecular crowding levels. In that opportunity we also found,
as a first approach to the study of the enzyme conformation in crowded systems that -Gal fluorescence
spectrum suffered a red shift when the crowding agent concentration was increased from 0 to 35 % W/V.
In the present work, studies on the conformation and thermal stability were carried out using both infrared
(IR) and fluorescence spectroscopy were done to investigate possible changes on the enzyme structure
due to overcrowding using polyethylene glycol molecular weight 6000 (PEG
6000
) was used as crowding
agent. The -Gal IR spectrum showed an important diminution in the main -structure band (at around
1620 cm
-1
) when the molecular crowding agent concentration was increased up to 35 % W/V. At the
same time, typical bands corresponding to disordered structures appeared. On the other hand, the effect
of crowded systems on -Gal thermal stability was noticeable, prevented the typical protein aggregation
that occurs in proteins denatured by temperature and denaturation occurred in a less cooperative way.
Taken together our results showed that in crowded conditions -Gal is thermodynamically trapped in a
conformation more open, flexible and stable than in dilute solutions.
Acknowledgements: we thank Dr Montich for the access to the IR equipment. Work financed with grants
from CONICET, Foncyt, SeCyT-UNC. MVN and MAP are career members of CONICET.


BPA20_Thermal inactivation of the NA
+
,K
+
-ATPase, ATP and Mg
2+
effects.
Placenti, M.A., Kaufman, S.B. Gonzlez Flecha, F.L. And Gonzlez-Lebrero, R.M.
IQUIFIB-Departamento de Qumica Biolgica, Facultad de Farmacia y Bioqumica, Universidad de
Buenos Aires, Junn 956, 1113, C.A.B.A., Argentina. E-mail: aguedaplacenti@gmail.com
Folding and stability are key factors for the proper biological function of proteins, therefore the importance
of its study. Na
+
,K
+
-ATPase is an integral membrane protein which couples ATP hydrolysis to the
transport of three Na
+
out and two K
+
into the cell. During this catalytic cycle, the enzyme interconvert
between two conformers, E
1
and E
2
.The aim of this work is to characterize the effect of natural ligands
(ATP and Mg
2+
) on the thermal inactivation of Na
+
,K
+
-ATPase. In a typical thermal inactivation
experiment, the enzyme is incubated for different time periods at several temperatures in the presence or
absence of the ligand. The functional or structural integrity of the protein is followed by measurement of
some property, in our case, ATPase activity, Trp fluorescence and circular dichroism spectroscopy. We
observed that thermal inactivation in all conditions tested followed a first-order kinetic. The decrease of
ATPase activity is concomitant with the conformational change detected by Trp fluorescence.
Additionally, circular dichroism experiments showed a slight change in the secondary structure when the
ATPase was fully inactivated. A clear stabilization of the enzyme was observed when ATP or Mg
2+
was
present in the incubation media. Even though Na or ATP are known to displace the equilibrium of the
enzyme towards E
1
conformer, our results showed that those ligands have opposite effects in terms of
thermal stability of the Na
+
,K
+
-ATPase.
With grants from: UBACyT, CONICET and ANPCyT.
BPA SAB 2013
62

BPA21_Analysis of the interaction between dengue virus NS3 helicase and ssRNA under
equilibrium conditions
Incicco JJ
1
, Gebhard LG
2
, Gonzlez-Lebrero RM
1
, Gamarnik AV
2
, Kaufman SB
1
1
Instituto de Qumica y Fisicoqumica Biolgicas y Departamento de Qumica Biolgica, Facultad de
Farmacia y Bioqumica, Universidad de Buenos Aires, Ciudad de Buenos Aires, Argentina.
2
Fundacin
Instituto Leloir-Consejo Nacional de Investigaciones Cientficas y Tcnicas, Ciudad de Buenos Aires,
Argentina.
Dengue virus nonstructural protein 3 (NS3) is a molecular motor protein that unwinds RNA duplexes
driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This RNA-helicase was
shown to bind preferentially to single stranded (ss) RNA along which it would move with 3' 5'
directionality. Here we report results from the study of the interaction between dengue virus NS3 helicase
and ssRNA with a quantitative fluorescence titration technique.
Binding experiments were performed with a series of fluorescein-labeled ssRNA oligomers of different
lengths. Model-independent analysis of the titration curves provided estimates for the stoichiometry and
the macroscopic equilibrium constants. Experimental results were well described by a statistical
thermodynamic model that takes into account the overlap of potential binding sites and cooperative
interactions between adjacent NS3 molecules. Global fitting of this model to all titration curves gave a
binding site size of 9.1 0.1 bases per NS3h; an intrinsic association constant of 0.0048 0.0001 nM
-1

and a cooperativity factor of 3.4 0.7.
(With grants from Universidad de Buenos Aires, from CONICET and from ANPCyT, Argentina).

BPA22_GOSPEL enhances in vitro nitric oxide-induced aggregation of GAPDH
Gonzlez, M.C., Ingaramo, M.C., Romero, J.M., Curtino, J.A. and Carrizo, M.E.
CIQUIBIC-CONICET, Depto. Qumica Biolgica, Facultad de Ciencias Qumicas., U. N. de Crdoba. E-
mail: ecarrizo@fcq.unc.edu.ar
Among its many functions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a proapoptotic role
associated with oxidative and nitrosative stress. Nitric oxide stress leads to reversible S-nitrosylation of
the active site cysteine of GAPDH. When S-nitrosylated GAPDH binds to the E3-ubiquitin-ligase Siah1
which possesses a nuclear localization signal that promotes the translocation of the complex to the
nucleus, where both proteins have cytotoxic effects. This cascade is regulated by S-nitrosylated GOSPEL
(GAPDH's competitor Of Siah Protein Enhances Life) whose association with GAPDH prevents the
cytotoxic interaction GAPDH-Siah1 (1).
Oxidative stress, produced either by hydrogen peroxide or nitric oxide, can also promote the formation of
amyloid-like aggregates of GAPDH through disulfide bonds involving its active site cysteine (2). It is worth
mentioning that GAPDH has also been found deposited as insoluble aggregates in some
neurodegenerative diseases.
Here we present the analysis of the effect of GOSPEL on the in vitro aggregation of GAPDH induced by
nitric oxide. Our results indicate that, under these conditions, both proteins co-aggregate and that the
aggregation of GAPDH was enhanced in the presence of GOSPEL. Our work also includes the
characterization of the aggregates obtained and the study of the nature of the interaction between the
proteins therein.
1. Sen, N. et al. (2009) Neuron 63, 81-91.
2. Nakajima, H. et al.(2007) J. Biol. Chem. 282, 2656226574.
SAB 2013 BPA
63

BPA23_Spying into the supramolecular arrangement of -synuclein amyloid oligomers

Gallea JI and Celej MS
Dto. de Qumica Biolgica,CIQUIBIC,CONICET, Facultad de Ciencias Qumicas, Universidad Nacional
de Crdoba.

Amyloid oligomers are the most toxic species in many devastating neurodegenerative disorders, which
are characterized by the misfolding and pathological aggregation of a particular polypeptide chain. In
Parkinsons disease the protagonist is the 140-aa presynaptic protein -synuclein (AS). Very little is
known about the structure of AS oligomers and their mechanisms of toxicity.
We showed that AS oligomers adopt a distinctive antiparallel -sheet structure. In this work, we use site-
directed fluorescence-labeling to define a map of cooperatively folded units and intermolecular proximities
in AS oligomers.
Spectral analysis of Acrylodan-labeled AS oligomers showed that all the labeled positions along the
sequence are shielded from the solvent, in contrast to reported results. Complementary GdmCl
denaturation experiments revealed two distinguishable cooperatively folded units. Excimer formation in
Pyrene-labeled AS oligomers revealed close proximity of positions 9 and 76 in adjacent molecules. Our
results will contribute to model the supramolecular structure of AS oligomers, and therefore, to the
understandig of the structure-based toxicity of such species.

Acknowledgements: Drs. Bertoncini and Jovin for proving us the several Cys-AS variants. SECyT-UNC,
FONCyT, CONICET and ISN-CAEN for financial support.
1- Celej, MS et al.,.Biochem J. 2012. 719.
2- Van Rooijen, BD. J Mol Biol. 2009. 826.

BPA24_The Supramolecular structure of gliadin protein is modulated by pH: toward
understanding the molecular triggers of gliadin related pathologies.
Herrera, M.
.
G.; Veuthey, T. V.; Dodero, V.I

a
INQUISUR- UNS-CONICET, Baha Blanca (AR).

Gliadin has been described as the most toxic protein fraction of gluten related with different human
diseases, such as celiac disease, durhing disease, ataxia and gliadin sensibility and allergy, among
others [1, 2]. It has been hypothesized that gliadin can reach the intestinal lumen inducing an increase in
intestinal permeability that seem to be a critical and early event in the pathogenesis of these diseases [3].
Although it is known the ability of gliadin to form high molecular weight aggregates [4], it has not been
performed a complete characterization of those aggregates. Considering the relevance of pH in the
digestion process, we decided to carry out a supramolecular evaluation of gliadin at different pHs by a
physicochemical approach. The aim will be directed to shed light on the molecular self-organization
depending on environmental stimulus. Herein, the evaluation of gliadin aggregates by a combination of
UV-Vis, steady state fluorescence, dynamic light scattering, optical and electron microscopy is presented.

Acknowledgements:Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.

1- Rubio-Tapia, A.; Murray, J. A. Curr opin gastroenterol. 2010, 116.
2- Sapone A, Bai J.C, Ciacci C, et al. BMC Med.2012, 10, 13.
3- Hadjivassiliou, M.; Williamson, C. A.; Woodroofe, N. Trends in immunol.2004, 25, 578.
4- Kasarda D.D, Bernardin J.E, Thomas R.S. Science. 1967, 203.
BPA SAB 2013
64

BPA25_Growth Hormone Releasing Hexapeptide is able to form Nanotubes: a
complementary study by Small Angle X-Ray Scattering, Transmission Electron
Microscopy and Molecular Dynamic Simulations
Barbosa, LRS
1
, Santana, H
2
, Avila, CL
3
, Cabrera, I
4,5
, Pez, R
2
, Falcn, V
2
, Pessoa, Jr. A
6
, Ventosa, N
4,5
,
Veciana, J
4,5
, Itri, R
1

1
Inst. de Fsica, USP, Brazil.
2
Dept. of Pharmaceutical Technology, CGEB, Cuba.
3
INSIBIO, CONICET-
UNT, Argentina.
4
Dept of Molecular Nanoscience and Organic Materials (ICMAB-CSIC), Spain.
5
CIBER
de Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Spain.
6
Dept of Biochemical and
Pharmaceutical Sciences, School of Pharmaceutical Sciences, USP, Brazil.
Growth hormone releasing peptide belongs to a class of synthetic growth hormone secretagogues, which
stimulate growth hormone secretion from somatotrophs in several species including humans. Besides, it
was recently used in vivo in the treatment of amyotrophic lateral sclerosis (ALS) disease. In the present
study, we investigated the self-assembling properties of GHRP-6, using small angle X-ray scattering,
transmission electronic microscopy (TEM) as well as molecular dynamics simulation. The combined
results demonstrated that GHRP-6 self-assembles into very long nanotubes, with inner and outer cross-
sections of 7(1) and 13(1) nm, respectively, at 20 mg/mLin phosphate buffer solution at pH 7.4. At
concentrations > 30 mg/ml, both SAXS and cryo-TEM results indicate that the shape of the long
aggregates remains the same and a bundle of nanotubes are disposed in a hexagonal arrangement in
solution, with a very well defined center-to-center distance of circa15 nm, according to SAXS data
analysis These results indicates that GHRP-6 self-assembles like dimers in the cylinder wall. Molecular
dynamics simulations also reflect the capability of the peptides to self-assemble like amphiphilic
molecules in aqueous solutions, giving additional details about the arrangement of the peptides within the
nanotube.
BPA26_Structural characterization of heparin-induced GAPDH protofibrils preventing -
synucleinoligomeric species toxicity
Avila, CL
1
; Torres-Bugeau, CM
1
; Barbosa, LRS
4
, Morand Sales, E
4
; Ouidja, O
2.3
, Socas B
2,3
; Raisman-
Vozari, R
2
, Papy-Garcia, D
3
, Itri R
4
, Chehn R
1

1 INSIBIO and Inst.deQca Biolgica Dr Bernab Bloj (CONICET-UNT). 2 INSERM. Thrapeutique
Exprimentale de la neurodgnrescence. 3 Laboratoire CRRET, Universit Paris EstCrteil, 4 Instituto
de Fsica da Universidade de So Paulo
Oligomeric arrangement of -synuclein has been associated to neuronal cell loss in neurological
diseases. Although -SNis mainly found in the cytosol, the presence of misfolded or aggregated -
SNoutside the cell suggests that it might play a role also at the extracellular level. Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and glycosaminoglycans (GAGs) have been found associated to -
SN amyloid aggregates in PD. However, it remained to be determined whether the interplay between
GAGs, GAPDH and -SN may exert a protective or a deleterious role on the development or evolution of
PD. We demonstrate that -synuclein oligomers toxicity on cultured human dopaminergic neurons can be
prevented by a newGAPDH specie formed in the presence of heparin. Structural characterization,
performed by SAXS shows a cylinder-like specie with average size of 22 x 12 nmcompatible with a
protofibril. Using computational modelling we obtained the first all-atom model of the GAPDH protofibril
capable to satisfy all the experimental restrictions derived from SAXS and mass spectrometry. Moreover,
we propose a pathway for GAPDH aggregation involving the loss of the tetrameric state of the enzyme
with a concomitant appearance of native-like dimeric species, which quickly assemble to protofibrils. It
seems plausible that heparansulphates from brain extracellular matrix can effectively interact with
GAPDH and induce the formation of protofibrils able to scavenge -SN displaying an important role in
cellular proteostasis.
SAB 2013 BPA
65

BPA27_Functional and structural comparison between protofibrils of rabbit muscle and
human GAPDH
Defonsi, E, Vera, C, Chaves, S, Torres-Bugeau, CM, Minahk, CJ, Bellomio, A, Avila, CL,Chehin, R.
Instituto Superior de Investigaciones Biolgicas (INSIBIO), CCT-Tucumn and Instituto de Qumica
Biolgica Dr Bernab Bloj (CONICET-UNT) Chacabuco 461 (T4000ILI) Tucumn, Argentina
GAPDH is an ubiquitous enzyme that in addition to its central role in energy production presents highly
diverse non-glycolitic functions in the intra or extracellular space. Several independent studies suggest
that it might also be related to neurodegenerative diseases. We recently reported that highly sulfated
GAGs like heparin and heparin sulphates, are able to trigger GAPDH amyloid aggregation. The GAPDH
protofibrils formed during the early stages of the aggregation process are able to efficiently accelerate -
Sinuclena (AS) aggregation. We proposed that these protofibrils could play an important role
sequestering the AS oligomeric species involved in amyloid disease spreading in the extracellular space.
Still, all the experiments were performed using commercially available GAPDH from rabbit muscle. In this
way the study of the ability of protofibrillar species from human GAPDH to neutralize AS toxic species
acquire relevance. In the present work we have expressed and purified human GAPDH and compared
the aggregation kinetics of human GAPDH vs. rabbit muscle GAPDH. Structural comparison of these
proteins as well as their aggregation intermediates and fibrils were performed by spectroscopic
techniques. The ability of the different protofibrils to neutralize the membrane perturbation capability of AS
oligomers was also compared. The relevance of the different mutation of human and rabbit GAPDH in
heparin binding and aggregation was assessed using bioinformatic tools. This study represents a
milestone in the assessment of the feasibility of using heparin-induced-GAPDH protofibrilsas a
therapeutic agent in the treatment of PD.

BPA28_Loop insertions as a tool to study folding and stability of human frataxin
Noguera, M.E., Roman, E.A., Faraj S.E. and Santos, J
Instituto de Qumica y Fisicoqumica Biolgicas (CONICET-UBA), Junn 956, Ciudad Autnoma de
Buenos Aires, Argentina.
A complete description of the protein structure requires, in addition to the average localization of all of its
atoms, the changes in time of these positions, i.e. its dynamic behavior, and ultimately the
characterization of the complete native ensemble, including low-populated conformations. The study of
protein dynamics is crucial to understand biological function. Throughout the structural study of the
human frataxin, we found that there is a relationship between the dynamics of the C-terminal region
(CTR) and loop-1, the segment linking the helix-1 with the strand-1 of this single domain protein:
sequence mutations that affect the dynamics of CTR, also alter the dynamics of loop-1.
In this work, we explored these relationships through the manipulation of protein sequence: introducing
mutations in the loop-1 region to alter its local dynamics, and assessing its effects in the dynamics of
other regions of the protein.First of all, we investigated whether the loop-1 is tolerant to the sequence
changes, without severe alterations in the stability of the native state. For this purpose, we designed two
variants: in one of them, the only two residues which establish tertiary contacts with the rest of the protein
were mutated to alanine, whereas in the second variant, substitutions and insertions were performed
resulting in a loop extension with six new glycine residues. Both variants were characterized by
spectroscopic and hydrodynamic methods, and both were shown to be well folded, stable, and
monomeric. These results will be presented along a preliminary assessment of the influence of these
mutations in conformational dynamics.
BPA SAB 2013
66

BPA29_Stability of the ATP binding domain of a hiperthermophilic Cu(I) transport
ATPase against sodium dodecyl sulfate

Alvaro A. Recoulat Angelini and F. Luis Gonzlez Flecha

Laboratorio de Biofisica Molecular. IQUIFIB Departamento de Qumica Biolgica, Universidad de
Buenos Aires - CONICET, Argentina.

In this work, we characterize the stability against the detergent sodium dodecyl sulfate (SDS) of the ATP
binding domain (ATPBD) of the Cu(I) transport ATPase from Archaeoglobus fulgidus CopA, a
hiperthermophilic -helical membrane protein that is involved in transporting Cu+ throughout biological
membranes. To accomplish this, we carry out experiments registering the intrinsic fluorescence of the
unique tryptophan resildue as a function of the detergent concentration. A decrease in the fluorescence
was observed in the range of 0.03 1.6 mM of SDS with 3 different phases in the downfall. This decrease
was proving to be reversible by dialysis and even by dilution of the sample, which recover the original
fluorescence of the protein. We also observed that intrinsic anisotropy increased in the range 0.2 - 1 mM
SDS, indicating that the SDS-denatured protein acquire a rigid structure. Finally, we explore the
interaction of CopA-ATPBD with the fluorescent probe 1-anilino-naphthalene-8-sulfonate (ANS),
observing an initial increase of the ANS fluorescence until 0.3 mM SDS followed by decrease up to a
constant value at 2-3 mM SDS. We can hypothesize that SDS-induced unfolding of CopA-ATPBD
proceeds through an intermediate state that acquire an expanded -molten globule like- globular structure,
holding the ANS in a more hydrophobic environment.

With grants from UBACyT and ANPCyT

BPA30_Quantitative analysis of local dynamics of the C-terminal region of human
frataxin
Faraj, SE;
1
Aran, M;
2
Gallo, M;
3
Santos, J.
1

(1) Instituto de Qumica y Fsicoqumica Biolgicas (UBA-CONICET). (2) Fundacin Instituto Leloir. (3)
Dipartimento di Scienze e Tecnologie Chimiche, Universit di Roma Tor Vergata, Italy.
Friedreich's ataxia is a progressive neurological hereditary disease, caused by the deficiency in the
activity of frataxin (FXN), a mitochondrial iron chaperone that regulates the transference of metal ions to
other proteins. Previous studies from our laboratory found that the C-terminal region (CTR) is a crucial
element in the stabilization of FXN. The local unfolding of the CTR may enable the protein to smoothly
modulate its dynamics and stability, acting as a conformational lock.

In an attempt to quantify the stability of the interaction between the CTR and the rest of the domain, we
replaced, one at a time, three Leu residues from the CTR which establish hydrophobic interactions with
core residues by Cys residues. Cys mutants are useful tools to analyze local dynamics through the
reactivity of their free thiol, allowing us to infer the dynamics at particular positions (in which Cys are
placed). We followed the reaction of Cys modification by DTNB (Ellmans reagent), and found that
different positions possess varying rates of modification. Our results indicate that the difference in free
energy of local unfolding of the CTR is half the global stabilization energy. Besides, molecular dynamics
simulations results are consistent with our experimental observations, and allow us to estimate average
accessibility of Cys residues for each variant, to assess the effective contribution to observed reactivity
from local unfolding events. Altogether, these results were analyzed side by side with wild-type protein
and L198R mutant NMR relaxation dispersion experiments.
Acknowledgements: ANPCyT, UBACyT and CONICET.
SAB 2013 BPA
67

BPA31_Cool adaptation: An electrostatic switch defines the signaling state of
thermosensor DesK
Inda, Mara Eugenia
1
, Michel Vandenbranden
2
, Diego de Mendoza
1
, Jean-Marie Ruysschaert
2
, Larisa
Cybulski*
1
.
1- Instituto de Biologa Molecular y Celular de Rosario (IBR)- CONICET and Departamento de
Microbiologa, Facultad de Ciencias Bioqumicas y Farmacuticas, UNR, Rosario, Argentina.
2- Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and
Bioinformatics, Universit Libre de Bruxelles, Brussels, Belgium.
DesK is a multipass transmembrane protein that allows the bacterium Bacillus subtilis to adjust the levels
of unsaturated fatty acids required to optimize membrane lipid fluidity after a cold stress. This is achieve
by its bifunctional cytoplasmic catalytic domain that alternates between kinase/phosphatase activity.
Temperature sensing, however, involves a built-in instability caused by a group of hydrophilic residues
located near the N-terminus of the first transmembrane (TM) segment. These residues detect changes in
membrane thickness that occur as a consequence of temperature variations, promoting the required
conformational change
1
. Nevertheless, the core question, still remains: how is the information sensed by
the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain that allows
switching between kinase and phosphatase states? At the inner interphase, there is a linker region that
connects the TM sensing domain with the cytoplasmic catalytic domain. A helix/random coil
conformational duality is mechanistically exploited to transmit temperature-dependent conformational
changes from the transmembrane to the intracellular region. By combining genetic, spectroscopic and
biochemical techniques the role of the linker in DesK signal transduction is studied. We also assess the
role of lipids charges variations as well as ionic force in the interaction of the linker with the membrane
inner phase as additional factors that modulate kinase to phosphatase activity ratio.
1. Cybulski, Martin, Mansilla, Fernandez, de Mendoza. Curr. Biology. 2010. 1539

BPA32_Quaternary Structure of HPRT Trypanosoma cruzi: A role for the C-terminal
region?
Valsecchi, WM
1
; Santos, J
1
; Delfino, JM
1

1
Departamento de Qumica Biolgica, IQUIFIB (UBA-CONICET). FFyB, UBA.
Hypoxanthine/guanine phosphoribosyl transferase (HPRT), a globular / protein of 221 amino acid
residues, catalyzes the recovery of hypoxanthine and guanine, and is essential for the survival of
trypanosomatids. It was described in literature that the asymmetric unit of the highest resolution crystal
contains an HPRT dimer where subunits are closely associated. However, dynamic light scattering (DLS)
experiments performed in our laboratory indicate that HPRT behaves as tetrameric structures in solution.
In this work, we introduce our first structure determined at 2.65 that, interestingly, shows that HPRT
crystallized as a tetramer. Remarkably, the experimental model obtained lacks the C-terminal region (CR)
of the protein (residues 198-229), pointing to the high mobility of this stretch of residues. Based on the 3D
structure, preliminary MDS results and controlled proteolysis experiments, we hypothesized that CR
interactions may participate in the stabilization of the quaternary structure of HPRT. In addition, we
studied the conformation by CD and multimerization state (DLS) of mutant F164W. Enzymatic activity
assays were also performed. In this mutant, a Phe side-chain located in the active site was replaced by
Trp providing a local sensor: the fluorescence quantum yield of a unique Trp residue in the protein.
Altogether these results indicate that, as well as wild-type, F164W behaves a tetramer, is active, and well-
folded. To further understand the molecular basis of the catalytic process, our main goal will be to analyze
the effect of different ligands on the stability and the activity of HPRT.
Acknowledgements: ANPCyT, UBACyT and CONICET.
BPA SAB 2013
68

BPA33_Solvent-mediated assembly of new chemically modified Phe-Phe-Cys peptides
Sequeira, M. A.; Learte, S.; Dodero, V. I

Depto de Qumica- INQUISUR, Universidad Nacional del Sur-CONICET, Baha Blanca (AR).

Self-assembly is a natural process that spontaneously and reversibly organizes molecular units into
ordered structures through non-covalent interactions. Among all biological molecules that can self-
assemble, peptides, specifically dipeptides and tripeptides, offer numerous advantages such as chemical
variation and biocompatibility therefore they can be used in various biological and non-biological
applications [1, 2]. Previous work have defined dipeptide system composed by Phe-Phe as a central motif
for the formation of ordered self-assembled tubular, spherical and two-dimensional structures at the nano-
scale [3]. Herein, we present the synthesis and supramolecular characterization of new Phe-Phe-Cys
derivatives which undergo solvent-mediated assembly. In order to establish a relation between structure
and morphology of the aggregates, it has been incorporated different functionalities at the N-terminal.
Additionally, the cysteine located at the C-terminal allows the chance to modulate aggregation based on
external stimulus, due to cysteine oxidationreduction capabilities promoted by pH and additives
changes.

Acknowledgements: Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Ozin, G. A.; Hou, K.; Lotsch, B. V.; Cademartiri, L.; Puzzo, D. P.; Scotognella, F.; Ghadimi, A.;
Thomson, J. Mater. Today2009, 12, 12.
2- Demirel, G.; Malvadkar, N.; Demirel, M, Langmuir Letter2010, 26(3), 1460.
3- Reches, M.; Gazit, E. Phys. Biol. 2006, 3, S10.



BPA34_Design and characterization of specific p38 peptide inhibitors
Bucci, H.A.
1
, Lopez, E.D.
2
, Turjanski, A.G.
2
, Wetzler, D.E.
1

1
Departamento de Qumica Biolgica, IQUIBICEN, FCEN UBA
2
INQUIMAE, FCEN UBA
Mitogen activated protein kinases (MAPKs) are serine/threonine kinases that play an important role in
regulating various cellular processes including cell growth, differentiation, inflammation and apoptosis.
The development of inhibitors of MAPKs is an important research area for various diseases such as
cancer, diabetes, arthritis and inflammatory diseases. The kinase domain is highly abundant in the human
genome, therefore it is very important to develop specific inhibitors for each particular MAPK. The study of
the specificity of peptide binding to MAPKs is relevant not only for the design of specific inhibitors but also
for understanding the mechanisms of protein-protein recognition in this signaling network. This work
consists on designing specific p38 peptide inhibitors by computational methods to be tested in vitro
using biophysical techniques. Based on the crystal structure of p38 complexed to the docking site of the
transcription factor MEF2A, we calculated the binding energy of wild-type and point mutation peptides.
We propose four potential inhibitors with higher binding energy than the wt peptide. We obtained highly
purified recombinant p38. We present here in silico calculations and a detailed biophysical
characterization of the protein that will be used to test the proposed inhibitors that we are currently
synthesizing.




SAB 2013 BPA
69

BPA35_Studying structure and aggregation of amyloid proteins using small organic
compounds as molecular probes
Valiente Gabioud A.A.
1
, Bertoncini C.
1,2
, Outeiro T.F.
3
,Griesinger C.
4
, Fernndez C.O.
1,2

1
Instituto de Biologa Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina,
2
Laboratorio
Max Planck de Biologa Estructural, Qumica y Biofsica Molecular de Rosario (LMPbioR), UNR.
3
Dept.of
Neurodegeneration and Restaurative Research, University of Gttingen, Gttingen, Germany.
4
Dept. of
NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Gttingen, Germany

The misfolding of proteins into a toxic conformation is proposed to be at the molecular foundation of a
number of neurodegenerative disorders including Alzheimer and Parkinsons disease. One common and
defining feature of protein misfolding diseases is the formation and deposition of amyloid-like fibrils.
Currently, no preventive therapy is available for Parkinson and Alzheimer diseases. Identification of
therapeutic drugs is not only complicated by a lack of understanding of many of the key aspects of the
pathogenesis of these diseases but also by the multifactorial etiology of them. The aggregation pathway
of the proteins linked to these disorders represents then an obvious target for therapeutic intervention.
The detailed understanding of the phenomenon of amyloid fibrillation and its inhibition is therefore highly
clinically important. Our work paves the road for the high resolution characterization of the modulatory
effect of small compounds on amyloidogenic intrinsically disordered proteins, which is critical for the
rational design of efficient anti-amyloid agents to treat Parkinson and Alzheimers disease.

Acknowledgements: ANPCyT, CONICET, Fundacin Bunge y Born, Max Planck Society, Alexander von
Humboldt Foundation.


BPA36_28-mer peptide from enterocin CRL35 displays bacericidal activity
E. Masias
1
, P.R da Silva Sanches
3
, L. Saavedra
2
, A. Bellomio
1
, E. Cilli
3
, C. Minahk
1
1
Instituto Superior de Investigaciones Biolgicas, Chacabuco 461. San Miguel de Tucumn, Argentina.
2
Centro de Referencia para Lactobacilos, Chacabuco 145. San Miguel de Tucumn, Argentina.
3
Instituto
de Qumica de Araraquara UNESP. Rua Prof. Francisco Degni, 55 Araraquara - So Paulo. Brasil.
Email: emilmas@hotmail.com

Enterocin CRL35 is a pediocin-like bacteriocin produced by Enterococcus mundtii CRL35. This
antimicrobial peptide is active against food-borne pathogen Listeria monocytogenes. In this work we used
the following synthetic peptides: the full-length bacteriocin (43- mer) and short derived peptides from the
C-terminus and N-terminus of enterocin CRL35 (28-mer and 15-mer respectively). Peptide binding to
target cells was analyzed by fluorescence polarization. The Ka values calculated from experimental data
indicated that the 3 peptides could bind similarly to the sensitive cells. Dissipation of membrane potential
assays showed that although all 3 peptides were able to dissipate the transmembrane electrical potential,
the 15-mer peptide produced a slower and incomplete dissipation. 43-mer has a MIC of 100 nM while 28-
mer and 15-mer have a much higher values of 10 and 20 M, respectively. However, only 43-mer and 28-
mer peptides displayed bactericidal effect against target cells. 15-mer peptide showed a bacteriostatic
effect since no cells were killed by this peptide in HEPES buffer. These results open up the possibility of
designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a
promising approach for combating Listeria.





BPA SAB 2013
70

BPA37_Labatidin interaction study using lipid monolayer
Barbosa, S.C.
1
, Cilli, E.M.
2
, Stabeli, R.G.
3
, Itri,R.
4
and Ciancaglini, P.
1

1
Departamento de Qumica, FFCLRP-USP, Ribeiro Preto, SP, Brasil;
2
Departamento de Bioqumica e
Biotecnologia, IQ-UNESP-Univ. Estadual Paulista, Araraquara, SP, Brasil.
3
Universidade Federal de
Rondnia (UNIR); Fundao Oswaldo Cruz - Fundao Oswaldo Cruz - Rondnia (FIOCRUZ), 76812-
245 Porto Velho, RO, Brasil.
4
Instituto de Fsica, IF-USP, So Paulo, SP, Brasil
Labaditin (VWTVWGTIAG) is a hydrophobic cyclic peptide from Jatropha Multifida and is composed of 10
residues. It has been studied due to its rigid conformation, which is considered significant for biologic
activity. The Labaditin (Lo) peptide and its open chain analogs (L
1
) were synthesized and its adsorption
on the monolayers of dipalmitoylphosphatidylcholine (DPPC), DPPC/cholesterol and DPPC/dipalmitoyl
phosphatidylserine (DPPS) mixtures was studied. The monolayers were obtained by spreading the lipid,
solubilized in chloroform, on the aqueous surphace (20L, 1mM) of a Langmuir system. The
measurements of A isotherms were done using the Wilhelmy (10 mm/min) method, varying the
concentrations of peptide from 0.0117 to 0.0704M. The results indicated that Lo changed the packing
density of the structures in DPPC. The increase of the initial pressure and a more expanded curve led to
the interpretation that the highest peptide interaction was with the DPPC isotherm. The neutral
phosphocholine groups were in direct contact with Lo peptide, thereby increasing peptide adsorption by
hydrophobic interaction. In DPPC/cholesterol, the monolayer was highly expanded. In the presence of the
Lo and L
1
peptide the adsorption was lower than in pure DPPC. This is probably due to highly the packed
DPPC molecules in the presence of cholesterol. Therefore, the peptide adsorption depends highly on the
condensing effect of cholesterol in the DPPC monolayer. In DPPC/DPPS mixtures both peptides showed
similar interaction with this monolayer, but less than pure DPPC, probably due to the anionic membrane.
So, the cyclic peptide showed more interaction with the DPPC monolayer, as already has been observed
by fluorescence spectrometry. This has probably occurred because of hydrophobic interaction. Although
the L
1
peptide has charged terminals, it had a stronger interaction with the anionic membrane.
Financial Supports: CAPES, CNPq and FAPESP.
BPA38_A lectin and phospholipases A
2
from Bothrops diporus venom: identification and
sequencing by mass spectrometry
Yunes Quartino, Pablo J.
a
, Portela, M.
b
, Lima A.
b
, Durn R.
b
and Fidelio G.D.
a

a
CIQUIBIC, CONICET, Universidad Nacional de Crdoba, Argentina.
b
Institut Pasteur de Montevideo,
Uruguay.
We report protein sequences from lipolitic fractions of venom of Bothrops diporus, a viper from Argentina
known as Yarar Chica. We have previously cloned two phospholipases and produce them via
heterologous expression and subsequent renaturation (1). By similarity and identity to public database
sequences, we classified these as type A
2
(PLA
2
). In the present work we used conventional
chromatographic techniques coupled to MALDI-TOF-TOF mass spectrometry analysis to prove the
existence at the protein level of the cloned sequences and portions of new PLA
2
isospecies. Along this
work we also found a lectin dimer for which it is proposed the disulfide intermolecular connectivity.
Acknowledgements
CONICET, FONCYT, SECYT-UNC (Argentina) and Unidad de Protemica y Bioqumica Analticas,
Institut Pasteur de Montevideo (Uruguay).

1- Yunes Quartino P.J., Barra J.L., Fidelio G.D. Biochemical and Biophysical Research
Communications.2012.321.

SAB 2013 BPA
71

BPA39_pH effect on the folding mechanism of frataxin from Psychromonas ingrahamii
Roman EA, Santos J, Craig PO
Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of
Pharmacy and Biochemistry, University of Buenos Aires, Argentina

Frataxin is a protein that participates in iron binding and delivery to other protein partners. Its absence in
humans yields Friedreich's Ataxia. In our laboratory we study the iron binding and folding mechanism of
human and Psychromonas ingrahamii frataxin (pFXN). We previously reported that pFXN stability is
highly modulated by pH in the 6-8 pH range. In this work, we studied the folding reaction of pFXN as a
function of pH by computational techniques. We used a coarse grained structure based model
supplemented with a general electrostatic potential to extensively sample the folding and unfolding
transitions and evaluate pFXN conformational stability. Residues were reduced to 3 atoms over which the
bonded and non bonded forces were projected. In this model only the residues that make contact in the
native state interact favorably whereas all the others interact only repulsively through excluded volume
effect. The model was supplemented with a coulombic electrostatic potential to account for the effect of
pH on the stability of the protein. Preliminary results show that protonation of histidine residues highly
modulate protein stability. The protonation of key residues and their contribution to the overall effect was
also evaluated.
Acknowledgements. We thank Diego Ferreiro and Rodolfo Gonzalez Lebrero for fruitful discussions.





BPA40_Characterization of iron binding to frataxin from Psychromonas ingrahamii
Rigal JB, Noguera M, Santos J, Roman EA.
Instituto de Qumica y Fisicoqumica Biolgicas, Universidad de Buenos Aires, Buenos Aires, Argentina

Frataxin is a protein highly conserved protein among biology kingdoms. In humans its absence yields
Friedreich's ataxia. Its biological function is to bind and deliver iron to other protein partners. In our
laboratory we study iron binding and its effect on stability and dynamics of different frataxin variants. In
this work, we characterized iron binding to frataxin from Psychromonas ingrahamii at different metal and
protein concentrations. The resulting binding isotherm will define the metal affinity and stoichiometry to
this protein. Previous results reported for other frataxin variants suggest that metal binding could move
protein equilibrium towards oligomeric species
1
. This fact will be studied by light scattering and native gel
techniques.
1- Bou-Abdallah, F., S. J. Mol. Biol. 2004 (341):605.





BPA SAB 2013
72

BPA41_Stability and folding kinetics of frataxin from Psychromonas ingrahamii are
highly modulated by pH
Roman EA, Gonzalez-Lebrero RM, Santos J.
Instituto de Qumica y Fisicoqumica Biolgicas, Universidad de Buenos Aires, Buenos Aires, Argentina
Frataxin is a highly conserved protein among biology kingdoms. In humans its absence yields Friedreich's
ataxia. In our laboratory we study iron binding and its effect on stability and dynamics of different frataxin
variants. In this work, we explored folding kinetics of frataxin from Psychromonas ingrahamii (pFXN) at
different pHs. Previous results from our laboratory revealed that pFXN stability is highly modulated by pH
in the 6 to 8 range
1
. Molecular dynamics simulations also suggested that histidine residues could be
participating in this effect. Here we introduce stopped-flow experiments studying pFXN folding and
unfolding reaction by monitoring Trp-fluorescence and circular dichroism at different pHs. Results show
that both signals reproduce well our previous equilibrium measurements by means of equilibrium constant
and total signal change. Moreover, observed rate constants obtained by both kinetic measurements yield
similar results. Our experiments indicate that as the pH becomes more acid, the rate constant of folding
becomes higher and unfolding rate constant lower. Future experiments with histidine point mutations
would shed light in the role of these residues and pH in general in the folding reaction.
Acknowledgements. We thank Diego Ferreiro and Ignacio Sanchez for fruitful discussions.

1- Roman, E., A. Biochimica et Biophysica Acta. 2013 (1834):1168-80.


BPA42_Conformation coalescence of -barrel proteins at sub-aggregating
concentrations of TFE mimics the formation of aggregation prone species.
CR Angelani
1
, JJ Caramelo
2
, JM Delfino
1
, LM Curto
1

1
Instituto de Qumica y Fisico-qumica Biolgicas Prof. Alejandro Paladini, BA, Argentina
2
Instituto de investigaciones Bioqumicas de Buenos Aires, Fundacin Instituto Leloir. BA, Argentina
98 and 78 are two functional all- sheet variants of IFABP (intestinal fatty acid binding protein),
being useful models to study the determinants related to aggregation of -barrel proteins. Albeit
displaying increased conformational plasticity, these variants exhibit a native-like -barrel topology and
are able to support a cooperative folding. Interestingly, while IFABP and 98 are monomers, 78 is
dimeric. These proteins share a common nucleation-elongation mechanism of aggregation, where the
rate-limiting step is the formation of dimeric nuclei. A straightforward correlation between their intrinsic
stability (IFABP78>98) with their aggregation propensity (78>IFABP98) cannot be
established. This observation appears at odds with the established notion that perturbations of the native
fold necessarily favor the population of aggregation-prone species. To understand the early events
leading to the formation of the critical nucleus, the initial conformational changes occurring after the
addition of an aggregating inducing co-solvent were examined (25 % v/v TFE). The spectral changes in
the far and near UV CD were measured immediately after diluting the protein in TFE. Interestingly, the CD
spectra for each of the proteins revealed the coalescence of all three proteins into conformations richer in
content. The same behavior was observed when incubating at sub-aggregating concentrations of TFE
( 10%v/v). In this scenario, low concentrations of co-solvent might produce conformational changes
similar to those leading to the aggregation prone species. This approach might help in understanding the
early conformational changes controlling the onset of aggregation.


SAB 2013 BPA
73

BPA43_What is the information component in sequences of repeat-proteins ?
Espada R*, Mora, T

, Walczak, AM

, Ferreiro DU*
*QB, FCEN, UBA IQUIBICEN, CONICET

Laboratoire de Physique Statistique and

Laboratoire de Physique Theorique, CNRS, Universite P. et M.


Curie, cole ormale Superieure, Paris, France.

The amino acids sequence of natural proteins contains information about the folding and motion of
biomolecules, defining the energy landscape. Due to conflicting signals between folding (physical) and
functional (biological) constraints and the inherent evolutionary noise, it is not trivial to decompose how
these factors contribute to shaping the amino acid sequences. Here we analyze the ankyrin-repeat
protein family, which have copies of a ~33 amino-acid motif and typically fold into elongated objects of
stacked alpha-helices and beta-turns. The apparent simple architecture of repeat proteins suggests that
the folding properties of a complete domain (the stability and the cooperativity of the array of repeats) can
be derived from the microscopic description of the balance of the energetic terms of each element and its
interactions with its near neighbors. By analyzing the correlation structure of alignments of ankyrin
repeats, we quantify the Mutual and the Direct Information between the positions and distinguish
positions with varying information content. We detect that the contributions to the information content are
slightly higher for positions within than between repetitions. We find that most amino acid pair-interactions
can be well predicted by direct information ranking, even when no structural information was used as
input.


BPA44_Grafting a functional iron-binding motif onto a thioredoxin scaffold
Vazquez, DS

*; Agudelo, WA

; Aran, M

; Gallo, M

; Gonzlez, Flecha, FL

; Gonzlez Lebrero, MC

; and
Santos, J

IQUIFIB (UBA-CONICET), Departamento de Qumica Biolgica, Facultad de Farmacia y Bioqumica,


Universidad de Buenos Aires, Argentina.

Fundacin Instituto Leloir and IIBBA-CONICET, Buenos Aires,


Argentina. *Contact e-mail: dsv@qb.ffyb.uba.ar / jsantos@qb.ffyb.uba.ar
Proteins belonging to the CyaY family contain acidic residues clusters arranged in tandem, presumably
involved in iron exchange and principally located in the N-terminal region. We endeavored to obtain an
individual iron-binding motif grafted onto a foreign protein, thioredoxin (TRX) to evaluate the intrinsic
affinity and the role of neighbor residues, bypassing limitations associated with the natural scaffold, such
as the presence of multiple binding sites and cooperativity between them. In this first work, we present
the characterization of internal dynamics and global properties of the apo and holo conformations, carried
out by computational (MD simulations) and equilibrium experiments. As judged by circular dichroism
analysis and its hydrodynamic behavior, the protein conserves wild-type signatures. More importantly, the
global stability is not significantly altered upon mutation but shows a slightly diminished oxidoreductase
activity in absence of iron. In addition, MDS analysis of the secondary structure suggests that the binding
of the metal ion may stiffen the grafted region. Furthermore, we evaluate the iron affinity of an isolated
peptide containing the iron binding motif (K
D
=100.1M, pH4.1) and its conformation, in the presence and
absence of the metal ion. The results suggest that 5-6 residues of the peptide acquired helical structure
upon iron binding. Finally, by the grafting approach, we expect to shed light in the understanding of
protein-metal dynamics and the impact on protein function.
Acknowledgements: for grants from UBACyT, ANPCyT, and CONICET.

Enzimologa (Enzymology) SAB 2013
74

ENZ1_Functional Characterization of Human Glycogen Branching Enzyme obtained in
Sf9 cells
Issoglio FM, Carrizo ME, Curtino JA.
CIQUIBIC-CONICET, Depto.Qca. Biol., Fac. Cs. Qcas., U.N.C. E-mail: fissoglio@fcq.unc.edu.ar
Glucose is the principal source of energy for most cells. In mammals, glucose is stored as glycogen, the
branched polymer formed by linear -1,4-oligoglucan chains linked by -1,6-glucosidic bonds. Three
enzymes are mainly responsible for the de novo biosynthesisof glycogen. First, glycogenin
autoglucosylation produces a protein-bound oligoglucan that serves as primer for the other two enzymes,
glycogen synthase elongating the chains, and the glycogen branching enzyme catalyzing the cleavage of a
linear segment and transferring it to the 6-position of a non-terminal glucosyl unit. Glycogen branching
enzyme (GBE) is the least studied of the three enzymes. It has been isolated and characterized in some
bacteria, rabbit and rat. In this study we report the production of recombinant human GBE in insect cells
using the baculovirus expression system, and the activity analysis of the recombinant enzyme. Using
amylose as a substrate, the branches introduced by GBE are analyzed after degradation with isoamylase.
The new reducing oligosaccharides generated are subjected to fluorophore-assisted carbohydrate
electrophoresis (FACE)
1
, where saccharides are labeled quantitatively with a fluorophore and then
separated by polyacrilamide gel electrophoresis (PAGE). Using this method we could determine the length
preference of the transferred oligosaccharide chain, and measure GBE activity quantifying the specific
reaction product.
1- Starret al. J. Chromatogr. A, 1996. 295.

ENZ2_Modulation of lipoxygenase activity in linoleicacid/ phosphatidylcholine
monolayers and bilayers.
Medina , A.V.
1,2
, Bouchet, A.M.
2
,Nazareno, M.
1
, Disalvo, EA
2
.
(1) Laboratory of Antioxidants and Oxidative Processes, Institute of Chemistry, Faculty of Agriculture and
Agroindustries, (CITSE) (UNSE-CONICET), (2) Laboratory of Biointerphases and Biomimetic Systems,
Centro de Investigacin y Transferencia de Santiago del Estero)
Lipoxygenases (LOX) are widely distributed in nature, found in almost all higher plants, fungi and animals.
Plant LOX are members of non heme iron-containing dioxygenasesclass that catalyze the addition of
molecular oxygen to fatty acids containing a cis, cis-1,4-pentadiene system to give an unsaturated fatty
acid hydroperoxide as a first of several secondary products (1). The hydroperoxides and free radicals
produced by LOX can degrade vitamins and proteins during food storage. Because many products of the
LOX reaction (or derivatives thereof) are aromatic, the presence of LOX activity in many foodstuffs can
affect their properties, particularly during long-term storage, mainly in undesirable ways (2, 3). The
enzyme activity in micelles can be modulated by the ionic nature of surfactants and the presence of water
(4). Thus, it was of interest to study the modulation of LOX activity in lipid mixtures of its substrate, linoleic
acid (LA), in 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) monolayers in order to relate its activity
with the water activity at the lipid surface. Experiments of surface pressure, Langmuir isotherms and zeta
potential were performed and the product of the LOX catalysis was evaluated by FOX methods (5).LOX
induces an increase in surface pressure due to electrostatic or non-specific interactions. At constant
surface pressure, LOX induces an increase in the area per lipid that is correlated with a decrease in the
surface potential. Finally, a relationship between the different amount of products catalysis and initial
surface pressure that can be related to a surface environment that promotes the reaction.
(1) James, N. Ann. Rev. Plant Physiol Plant MolBiol(1991) 42:145148; (2) Axelrod, B ACS
AdvChemSer(1974) 136:324348; (3) Eskin, NAM, Grossman S, Pinsky A Crit Rev Food SciNutr(1977)
9:141; (4) Medina, A et. al. (2013) SAB Meeting; (5) Nourooz-Zadeh, J et. al. Anal.Biochem.(1994),
220:403-409
ENZ SAB 2013
75

ENZ3_Surfactant type influence on the catalytic activity of soybean lipoxygenase
Medina , AV
1,2
. Chaillou, LL
1
. Nazareno, MA
1,2
.
(1) Laboratory of Antioxidants and Oxidative Processes, Institute of Chemistry, Faculty of Agriculture and
Agroindustries Faculty, National University of Santiago del Estero.(2) CITSE-CONICET.
The catalytic activity of enzymes is influenced by many physical and chemical factors. Model systems are
used to study this activity based on the properties of amphiphilic molecules to self-organize in solution,
such as micellar systems [1]. Lipoxygenases (LOX) are dioxygenases which catalyze the initial reaction of
oxidation of polyunsaturated fatty acids, containing a 1,4-cis,cis-pentadiene system yielding primary
products with conjugated dienehydroperoxides [2]. In food, these enzymes play an important role in
oxidative processes, therefore several technological processes have been developed for the purpose of
eliminating or reducing its activity [3-4]. The objectives of this work were to study the influence of different
type of surfactants on the soybean LOX catalytic activity and to determine the kinetic parameters
associated with the oxidation reaction. The results of this work indicated that the LOX activity depends on
the nature of the surfactant and its concentration. Activity values obtained for Brij 35 and Tween 20
micelles were superior to those of SDS and AOT. Maximum activity values were obtained at 2.5; 0.8; 3;
and 1 mM of SDS, Brij 35, Tween 20 and AOT, respectively. The non-ionic surfactants promote the
activity, whereas ionic surfactants decrease or totally inhibit this activity.
[1] Purich, D.L., Enzyme Kinetics: Catalysis & Control, Elsevier, Oxford, UK, 2010; [2] Kuhn, H., Structural
basis for the positional specificity of lipoxygenases, Prostaglandins & other Lipid Mediators, 62, 255270,
2000; [3] Robinson, D.S., W. Zecai,D. Claire & C. Rod, Lipoxygenases and the quality of foods, Food
Chemistry, 54, 33-43, 1995; [4] Porta, H. & M. Rocha-Sosa, Plantlipoxygenases. Physiological and
molecular features, Plant Physiology, 130, 15-21, 2002.

ENZ4_-galactosidase activity againts different substrates and in the presence of lipid
interfaces
Flores, SS, Perillo, MA, Sanchez, JM.
IIByT.UNC-CONICET. ICTA- FCEFyN. UNC. Av. Velez Sarsfield 1611. Ciudad Universitaria - (5016)
Crdoba (Argentina). e-mail: jmsanchez@efn.uncor.edu
Previously we demonstrated that the activity of a soluble wild-type E. coli -galactosidase (-Gal
wt
)
against both lactose (the natural substrate) as ortho-nitrofenilgalactopiransido (ONPG, artificial
substrate) increases in the presence of multilamellar vesicles (MLVs) composed of neutral and charged
phospholipids. The aim of this study was to compare the activity of a recombinant -Gal (-Gal
His6
)
against two different substrates in the presence of MLVs of different lipid composition. -Gal
-His6
was
overexpressed in E.coli, and the six histidine residues (His-tag) fused to the carboxyl terminus facilitated
purification by ion metal affinity chromatography (IMAC). The enzyme activity was measured by visible
spectrophotometry, in the absence or presence of MLVs of pure egg phosphatidylcholine (EPC interface)
or at 80:20 molar ratio with dioleoylphosphatidyl glycerol (EPC
80
/DOPG
20
) negative zwitterionic interface).
Kinetic parameters were determined by fitting the michaelian model to the experimental data using
nonlinear regression. Our results showed that the enzyme activity was more efficient against ONPG
compared to lactose (kcat/K
MONPG
>kcat/K
MLactosa
) as previously described for other beta galactosidase. An
activation of the enzymatic activity but a decrease in the substrate affinity were observed in the presence
of lipid interfaces against both substrates. Those effects were enhanced by the presence of charged
interfaces favored by electrostatic interactions mediated by the presence His residues of the enzyme. The
nature of the substrate not qualitatively affected the kinetics of the reaction catalyzed by -Gal
-His6
in the
presence of lipid interfaces.
Acknowledgements: Financed by Foncyt, Mincyt-Pcia Crdoba, SeCyT-UNC, Conicet
ENZ SAB 2013
76

ENZ5_Nucleotide specificity in the presence of RNA by dengue virus NS3 helicase
Cababie LA
1
, Incicco JJ
1
, Gebhard LG
2
, Gamarnik AV
2
, Gonzlez-Lebrero RM
1
, Kaufman SB
1

1
Instituto de Qumica y Fisicoqumica Biolgicas y Departamento de Qumica Biolgica, Facultad de
Farmacia y Bioqumica, Universidad de Buenos Aires, Ciudad de Buenos Aires, Argentina.
2
Fundacin
Instituto Leloir-Consejo Nacional de Investigaciones Cientficas y Tcnicas, Ciudad de Buenos Aires,
Argentina
The dengue virus genome encodes a polyprotein which is cleaved by peptidases to give three structural
and seven non-structural (NS) proteins. Among these the NS3 protein presents the characteristic features
of helicases.This protein moves along single strands and unwinds double strands of RNA molecules
driven by the binding and hydrolysis of nucleosides triphosphates (NTPs). It is known that this enzyme
catalyzes the hydrolysis of several NTPs and this activity is enhanced by the presence of single stranded
RNA. We have previously studied the specificity of NS3 for nucleotides ATP, GTP, CTP and UTP in the
absence of RNA and now we examine how this specificity is affected by the presence of RNA. For this
purpose we obtained the substrate curves for the four nucleotides mentioned in the presence of poly(A).
In all cases it was observed lower K
M
and higher k
cat
values than in absence of RNA. According to the
value of the specificity parameter (k
cat
/ K
M
) we obtained the following specificity order: ATP> GTP> CTP>
UTP.
With grants from: Universidad de Buenos Aires UBACyT, CONICET and ANPCyT.


ENZ6_Productive induced metastabilities in allosteric modulation of kinase function
Montes de Oca J.M

.,Rodriguez-Fris A

., Appignanesi G.A

., Fernndez A.S

Seccin Fisicoqumica, INQUISUR-UNS-CONICET-Departamento de Qumica, Universidad Nacional del


Sur, Avda. Alem 1253, 8000 Baha Blanca, Argentina

Instituto Argentino de Matemtica Alberto P. Caldern, CONICET (NationalResearch Council),


Saavedra 15 Buenos Aires 1083, Argentina

Allosteric modulators of kinase function are of considerable pharmacological interest as blockers or
agonists of key cell-signaling pathways. They are gaining attention due to their purported higher
selectivity and efficacy relative to ATP-competitive ligands. Upon binding to the target protein, allosteric
inhibitors promote a conformational change that purposely facilitates or hampers ATP binding. However,
allosteric binding remains a matter of contention since the binding site is not fit to a natural ligand (i.e.
ATP or phosporylation substrate) of the protein. In this study, we elucidate the allosteric binding modality
by unraveling a local structural motif that promotes association with the ligand. We specifically show that
allosteric modulators promote a local metastable state that is stabilized upon association. The induced
conformational change generates a local enrichment of the protein in the so-called dehydrons, which are
solvent exposed backbone hydrogen bonds. These structural deficiencies that are inherently sticky are
not present in the apo form and constitute a local metastable state that promotes the association with the
ligand. This productive induced metastability (PIM) is likely to translate into a general molecular design
concept.

ENZ SAB 2013
77

ENZ7_Investigation of the enzymatic activity of the Na,K-ATPase via ITC
Yoneda, J.S and Ciancaglini P.
Departamento de Qumica FFCLRP-USP Ribeiro Preto, SP, Brazil
Isothermal titration microcalorimetry (ITC) is a sensitive and accurate method for assaying the steady-
state enzyme activity of the Na,K-ATPase. Measurement of the kinetics by ITC has advantages over
other widely used techniques. The continuous monitoring of enzymatic activity is possible in contrast to
other assays such as the colorimetric determination. Moreover, this method does not require the addition
of any extrinsic probes or coupled enzymes (1). Firstly, a series of sequential injections with interval of 40
min (to complete the reaction), of 10 L of ATP (4.6mM) was performed intoNa,K-ATPase solubilizedto
determine the value of H
app
(66,8 kJ/mol). The initial protein concentration was 470 nM. In order to
determine the kinetic constants (V
max
, K
m
) a multiple injection experiment of ATP into enzyme was done.
The addition of substrate causes a decrease in power thermal to maintain isothermal conditions of
equipment. The power thermal that remains is due to the enzymatic reaction and it keeps constant while
the reaction proceeds at steady state. The rate of heat generated by the enzyme is equivalent to the
decrease in power applied by the calorimeter (dQ / dt), which is proportion toV
max
.The curve V
max
vs. [ATP]
was plotted and the results were:V
max
= 126 nmol/L s and K
m
= 0.126mM. The values were consistent with
colorimetric method and the analysis of the heat liberation by Na,K-ATPase is important to consider the
role of this protein in maintenance of body temperature.
Acknowledgements:CNPq, FAPESP
1- Noskeetal, Biochimica et Biophysica Acta (2010) 1797(8):1540-5


ENZ8_Elucidation of the reaction mechanism of the Metalo--Lactamase NDM-1
Palacios A. R.,Llarrull L. I., Vila A. J.
Institute of Molecular and Cell Biology of Rosario (IBR), CONICET, University of Rosario, Rosario,
Argentina
Carbapenems comprise the latest generation of -lactam antibiotics and are used as antibiotics of last
resort to treat infections caused by most -lactam-resistant bacteria. -lactamases, the enzymes that
inactivate these antibiotics, can be classified into Serin--lactamases or Metalo--lactamases (MLs) and
this last class could be subdivided into B1, B2 and B3 subclasses. In spite of the fact that carbapenems
have a structure that renders them highly resistant to most -lactamases, an emerging class of -
lactamases can act against them. In particular, NDM-1 is a B1 ML with a broad substrate spectrum and
the carbapenemase activity of NDM-1 is of great clinical concern
(1)
. In previous works, our group identified
a reaction intermediate in the hydrolysis of carbapenems by BcII, another B1 ML
(2)
, and our unpublished
results indicate that this intermediate is common to all three ML subclasses. Here we report the steady
and pre-steady state kinetics of NDM-1 (both native and Co(II)-substituted) and the reaction mechanism
of the hydrolysis of imipenem. We have confirmed the presence of the characterized reaction
intermediate in NDM-1, this information is the cornerstone for the rational design of a common ML
inhibitor.
Acknowledgements: ANPCyT, NIH, CONICET
1- Mckenna M., Nature, 2013, 394.
2- Tioni M. F. et al., J. Am. Chem. Soc., 2008, 15852.


ENZ SAB 2013
78

ENZ9_Kinetic characterization and substrate preference of GmsPLA
2
-XIA-1 and
GmsPLA
2
-XIB-2 in model membrane systems
Mariani, ME.
1
, Madoery, R
2
, Fidelio GD
1
.
1
Departmento de Qumica Biolgica-CIQUIBIC-FCQ-UNC, Crdoba, Argentina.
2
Departamento de
Fundamentacin Biolgica, Facultad de Ciencias Agropecuarias- UNC, Crdoba, Argentina.
This is the first time secretory Phosphalipase A
2
(sPLA
2
) enzymes from soybean seeds (Glycine max),
denoted as GmsPLA
2
-XIA-1 and GmsPLA
2
-XIB-2, were produced by heterologous expression in E.coli,
renatured from inclusion bodies by guanidine treatment and purified by ion exchange chromatography.
Mixed micelles of phospholipid/Triton X-100 were used in a Colorimetric assay in order to obtain the
optimum conditions for catalysis. Both sPLA
2
s showed a maximum enzyme activity at pH 7, a
requirement of Ca
2+
essentially in micromolar concentrations and optimum temperature for catalysis of
60C, similar parameters to those found in animals and plants. These enzymes showed subtle differences
in the preference for phospholipids with different head groups in the presence and absence of NaCl. The
apparent kinetic parameters (V
max

app
and K
m app
) demonstrate that both enzymes have more preference
for phosphatidilcholine than for phosphatidylglycerol in contrast with the results observed for pancreatic
sPLA
2
. Furthermore, the mode of catalysis was studied. The effect of auxins asindole 3-acetic acid and
indole 3-propionic acid were studied and shows to rapidly stimulate the conversion of phosphatidylcholine
to LPC and free fatty acid.
Acknowledgements: This work has been carried out with grants from CONICET, FONCYT and SeCyT-
UNC (Argentina)

ENZ10_Structure/activity relationship of placental alkaline phosphatase (PLAP)
submitted to denaturing conditions like high temperature and high hydrostatic
pressures.
Clop, E.M.
1
; Bonafe, C.F.S.
2
; Perillo, M. A.
1
1
IIByT (CONICET-UNC). Ct. de Q. Biolgica, FCEFyN, UNC. Av. V.Sarsfield 1611,Cba. Argentina.
2
Laboratrio de Termodinmica de Protenas, Depto. de Bioqumica, Instituto de Biologia, UNICAMP,
Rua Monteiro Lobato, 255, Campinas, SP, Brazil. e-mail: mclop@efn.uncor.edu

In a previous work we investigated the correlation between the catalytic activity of PLAP (GPI-anchored
protein) and the organization of the molecular environment in model membranes at different lateral
packings and in presence of interfacial molecular crowded milieu. We showed that PLAP activity was
capable to probe all these different media. To gain further insights into the mechanism of this
environmental tuning we explore the kinetics of PLAP submitted to different denaturing conditions, such
as the effect of high hydrostatic pressure (in presence of urea) and high temperatures. The state of the
protein was followed by tryptophan intrinsic fluorescence. A multiple unfolded intermediates were found
while V
max
showed a maximum at low denaturing pressure conditions and moderate temperatures. Also a
cooperative behavior was found in the catalytic activity at high urea concentrations. These results showed
that partially unfolded PLAP is capable to exhibit catalytic activity, not necessarily diminishing its
efficiency and plausible by changing its kinetic mechanism. The tune of catalysis in heterogeneous media
may reflect not only diffusional/motional restrictions but also PLAP structure stability in these
environments.
Acknowledgements: SeCyT-UNC, Conicet, Foncyt, CAPES-SPU for the financial support.
SAB 2013 Bioenergtica y transferencia electrnica (Bioenergetics and transfer)
79

BTE1_Accurate parametrization of ubiquinone to obtain a Charmm36 compatible model.
Galassi V.V., Vasques Camargo da Silva J.P., Menegon Arantes G.
Departamento de Bioqumica, Instituto de Qumica, Universidade de So Paulo, So Paulo, Brazil.
Cytochrome bc1 complex is an ubiquitous component of electron transfer chains in prokariotic and
eukariotic organisms. It catalyzes redox reactions of ubiquinone/ubiquinol (UQ) in a pathway called Q-
cycle. We are performing computer simulations with molecular mechanical and hybrid quantum
mechanical (QM) potentials in order to understand the energetics of several steps involved in this cycle.
To start with, we have built and equilibrated a realistic model of a bacterial-like inner membrane, a mixture
of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) 3:1, both with palmitoyiloleoyl chains,
using the novel CHARMM36 parameters. We have also parametrized tetraoleoyl cardiolipin (CL) and
performed simulations of PE-PC bilayers with 10% CL. We have built a new potential for UQ by improving
partial charges and torsional parameters from previously available UQ potentials (Schulten and Tietz
2007, Rg 2013). In comparisons to high-level QM torsional and water or methane interaction profiles,
our new UQ potential shows significant improvement over the previous potentials. Thus, we performed
potential of mean force calculations with our new UQ potential to obtain the free energy of
water/membrane partition of UQ with different isoprenil chain lengths and validated our model by
comparison with experimental data (Lenaz 1996). This accurate ubiquinone and bacterial membrane
model, is now in use to study the diffusion pathway of UQ through the membrane into Cytochrome bc1
and within its two UQ binding sites.
Acknowledgements: FAPESP.
BTE2_Action mechanism of activating anions on the mitochondrial reversible
H
+
ATPase.
Roveri, O.A., Rico, M.
rea Biofsica, Facultad de Ciencias Bioqumicas y Farmacuticas, Universidad Nacional de Rosario,
Suipacha 531, S2002LRK Rosario, ARGENTINA. oroveri@fbioyf.unr.edu.ar
Despite it has been known for almost 5 decades that several anions such as HCO
3
-
, HSO
3
-
and SO
4
2-
activate ATP hydrolysis catalyzed by the mitochondrial ATPase [1], their action mechanism is still under
debate. More recently we have shown that they inhibit the reverse reaction: the mitochondrial ATP
synthesis [2-4]. Here we present a unifying hypothesis trying to explain the action of these anions and
discuss the role of some intermediates in the coupling between ATP hydrolysis and proton translocation.
HCO
3
-
and HSO
3
-
bind to the low affinity non-catalytic site: HCO
3
-
induces a catalytic site conformation with
high turnover for ATP hydrolysis but unable to bind ADP; conversely, HSO
3
-
induces a conformation with
high turnover for both ATP hydrolysis and synthesis. HSO
3
-
can also bind with lower affinity to the
catalytic site as well as SO
4
2-
inhibiting ATP synthesis by binding at or near the Pi binding site, stabilizing
the site in a half-open conformation, unable to synthesize ATP but still able to translocate H
+
driven by
ATP hydrolysis.
Acknowledgements: With grants from CONICET, UNR and ANPCyT.
1- Roveri OA, Calcaterra NB.FEBS Lett. 192, 123 (1985).
2- Lodeyro AF, Calcaterra NB, Roveri OA. Biochim.Biophys.Acta 1506, 236 (2001).
3- Lodeyro, AF, Castelli, MV, Roveri, OA.J.Bioenerg.Biomembr. 30, 269 (2008).
4- Rico M, Roveri OA. SAB 2012.

BTE SAB 2013
80

BTE3_Cyanide protects Escherichia coli NADH dehydrogenase activity from MccJ25
inhibition

Galvn, A. E.; Chaln, M.C.; Schurig-Briccio, L.; Minahk, C. J.; Gennis, R. and Bellomio, A.
Instituto Superior de Investigaciones Biolgicas (INSIBIO-CONICET-UNT), Chacabuco 461 (T4000ILI),
Tucumn, Argentina. Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized antibacterial lasso peptide. It is produced
by Escherichia coli, and is active against close-related bacteria including certain human pathogens like
Salmolella enterica and Shigella spp. MccJ25 acts in sensitive bacteria inhibiting the RNA polymerase
and the bacterial membrane respiratory chain. The rate of cell oxygen consumption is lower in the
presence of the peptide. In a previous report, it was shown that MccJ25 was able to inhibit the NADH
dehydrogenase activity in vitro [1]. Interestingly, in the present work we observed that the reaction
inhibition decreased to 10% when 6 mM cyanide was added. E. coli has two terminal oxidases,
cytochrome bd and cytochrome bo3, which are quinol oxidases. Therefore, the antimicrobial activity and
the effect of MccJ25 on NADH dehydrogenase activity from wild type as well as terminal oxidases mutant
membranes were assayed.
1- A. Bellomio, P.A.Vincent, B.F. deArcuri, R.N. Farias, R.D.Morero, J. Bacteriol. 189 (2007) 4180
4186.





BTE4_Absorption properties of Chlorophylls in the complex LH-II of spinach

Bea E. A. (1), Soba A. (2), Andrade X. (3), Rubio A. (3)
(1) Instituto de Fsica de Lquidos y Sistemas Biolgicos (IFLYSIB), CONICET La Plata; (2) Comisin
Nacional de Energa Atmica (CNEA), CAC Buenos Aires; (3) NanoBio Spectroscopy Group and ETSF
Scientic Development Centre, Universidad del Pas Vasco, Donostia-San Sebastian, Espaa
The light harvesting (LH) is the primary process in photosynthesis. In green plants, this function is
developed by a series of pigment-protein complexes in thylakoid membrane of chloroplasts. The LH-II
complex is the most abundant and is involved in regulating and channeling of excitation energy to
reaction centres, with photochemical activity. It is known that the excitation moves through intricate
networks of pigments which constitute the complex "antenna". Chlorophylls are the main pigments and
are associated with proteins and other pigments. Chlorophyll a is involved in the photochemical activity of
the active site, where it becomes radiant energy (light) into chemical energy when it receives a photon
from the antenna. The dynamics of energy transfer is highly efficient, thus it is attractive and motivating to
know precise mechanisms involved and the role of quantum coherence among excitonic states. In this
work, the excited state of an active centre of the protein, essentially a Chlorophyll a, is characterized
theoretically by the spectrum generated under an Ehrenfest TD-DFT dynamics. Active electronic states
are identified.
Acknowledgements: European Theoretical Spectroscopy Facility (ETSF)


SAB 2013 Teora y modelado de sistemas biolgicos (Theory and modeling of biological systems)
81

TMSB1_Stochastic and Algebraic Methods for Modeling the Binding of Transcription
Factor Cohorts to Cis-acting Gene Regulatory Modules
Mauricio Bustos
a
and Fernando Levstein
b

a
Department of Biological Sciences University of Maryland Baltimore County, 1000 Hilltop Circle,
Baltimore, MD 21250, USA;
b
FAMAF, Universidad Nacional de Cordoba, Cordoba, Argentina.
Gene transcription programs that operate in eukaryotic cells are controlled by networks of transcription
factors (proteins) binding to gene regulatory modules (DNA). Correct modeling of such systems is
essential to understand normal development and to develop strategies that can prevent the emergence
and spread of diseased states, such as cancers and age-related metabolic defficiencies. In human cells
(a diploid organism), such protein/DNA interactions always involve just two copies of every DNA site, and
a small number of molecules of each transcription factor (typically fewer than 100 copies). Attempts to
model the behavior of such small-number systems using mass action kinetics fail to account for stochastic
effects that predominate in discrete systems with small numbers of reacting molecules. Discrete modeling
approaches can avoid such problems, but the resulting combinatorics often presents formidable
computational challenges. We will describe new hybrid approaches for calculating the stationary
distribution of many transcription factors binding to many DNA sites, which are based on a combination of
stochastic simulation algorithms with algebraic methods. The usefulness of such modeling techniques will
be demonstrated on a system comprising eight transcription factors that regulate spatial gene expression
in the Drosophila embryo.

TMSB2_ Use of Modeling as a Pedagogical Tool in an Undergraduate Biology Teaching
Laboratory
Karen Whitworth
a
, Christopher Rakes
b
, Sarah Leupen
a
, and Mauricio Bustos
a

Departments of Biological Sciences
a
and Education
b
, University of Maryland Baltimore County, 1000
Hilltop Circle, Baltimore, MD 21250, USA
Biology students tend to have an overly contextualized understanding of learned concepts, having
difficulty applying those concepts to new systems. Further, they have difficulty understanding how
quantitative models apply to biological systems. Our objective was to use rigorous, quantitative computer
simulations as tools to teach biological concepts and improve quantitative literacy. We developed
stochastic modeling laboratory supplements for two enzyme experiments performed in our introductory
experimental biology laboratory class and designed a study to examine the efficacy of the simulation in
enhancing student learning. In the control group, students collected all data through the traditional hands-
on laboratory procedures. In the treatment group, students explored the model outcomes instead of doing
the traditional hands-on laboratory procedures. In Week 1, course sections were randomly assigned to
the treatment or control conditions. In Week 2, treatment and control sections were switched. We found
that the use of computer simulated experiments for either enzyme lab neither enhanced nor detracted
from student understanding of enzyme kinetics. Students who used simulations changed their views of
the use of simulations and modeling in teaching labs, and teaching assistants were more enthusiastic
about the simulations than about the standard wet labs. In conclusion, exposure of students in an
introductory teaching lab to modeling techniques that partially replace hands-on laboratory procedures
increases student and teaching assistant engagement without harming conceptual understanding of
enzyme physiology.
TMSB SAB 2013
82

TMSB3_Activation free energies in partial steps of the sarcoplasmic reticulum Ca-
ATPase cycle during the transient state of the reaction. An In Silico approach
Guillermo L. Alonso y Dbora A. Gonzlez
Ctedra de Biofsica, Facultad de Odontologa, Universidad de Buenos Aires.
The step-by-step free energy change (G) profile of the Sarcoplasmic Reticulum Ca-ATPase cycle during
the transient state of simulated reactions was analyzed previously (1). G was negative at all the steps.
Here we calculated the step-by-step activation energies (G

)

in the same model. Transient species were
introduced as chemical intermediates at each step; they decay to intermediate enzyme species
accordingly with the mass law, at 6.56x10
12
s
-1
. Other rate constants are as in (1). The transient evolution
of the model was followed with a computer program. The evolution of the ligands concentrations agree
with experimental results. Enzymatic species concentrations lies within 10
-10
10
-6
M, transient species
lies within 10
-19
-10
-17
M, at 0,1 s., when the reaction is in a cuasi-steady-state. These large differences
preclude the use of the standard (G
o
) or basic (G
basic
) free energies to describe G fall along the cycle.
Forward and backward G

are shown for 10 and 100 ms. of simulated reactions; both are negative in the
exergonic forward cycles, at any reaction time, in sealed and permeable membranes, and partial sums
equals each partial G. The results show the absence of any activation energy hill previously proposed
(2)- to be surmounted upon the reaction advance. Similar results were obtained for the binding of 2 Ca
ions to the enzyme through (+)cooperative reactions. The results do not disagree with the modern theory
of the transient-state intermediates (3), since we used rate constants obtained from enzyme catalyzed
reactions, already including transition coefficients which reduce partial activation energies.
(1) Alonso and Hecht, J. theor. Biol (1990) 147:161-176. (2)Pickart and Jencks (1984) J. Biol. Chem. 259:
1629-1643. (3)Hammer-Shiffer (2013) Biochemistry 52:2012-2020.

TMSB4_Molecular Dynamics Studies of Trypanosoma cruzi Trans Sialidase and T.
rangeli Sialidase Mutants at the Covalent Intermediate Stage.

Diego J. Alonso de Armio
a
, Guy Lezama
a
, Daro Estrin
b
, R. M. S. Alvarez
a
, Adrian E. Roitberg
c

a. Instituto Superior de Investigaciones Biolgicas, INSIBIO (CONICET-UNT). Chacabuco 461, San
Miguel de Tucumn, Tucumn, Argentina.
b. Instituto de Quimica Fisica de los Materiales, Medioambiente y Energa, INQUIMAE-CONICET,
Ciudad Universitaria, Pab 2, C1428EHA, Buenos Aires, Argentina.
c. Quantum Theory Project and Departament of Chemistry, University of Florida, Gainesville, Florida,
United Status of America.
Trypanosoma cruzi is the causative agent of Chagas' disease. T. cruzi trans-sialidase (TcTS) was shown
to be an important factor in the microorganism's virulence, and has been proposed as a target for drug
design, a goal that requires a thorough understanding of the enzyme's mechanism. Evidence indicates
that the catalytic mechanism involves a long-lived covalent intermediate (CI), which is later attacked by an
acceptor glycoconjugate, completing the sialic acid transfer. A key question is thus, how does TcTS
protect the CI from hydrolysis until the acceptor glycoconjugate can position itself in the active site for the
transfer reaction to take place. Previous works suggested the existence of a new switch mechanism
sensitive to lactose, which deactivates the enzyme in abscence of acceptor ligand at the CI stage. Here
we present new in silico studies in which we discuss the structural and sequence differences between
TcTS and TrSA responsible for it.

SAB 2013 TMSB
83

TMSB5_Study of the interaction of L-cysteine ethyl ester with DPPC membrane
Marcelo Puiatti,

Juan Marcelo Arias,

Mara E. Tuttolomondo,

Sonia B. Daz,

Aida Ben Altabef

and
Adriana B. Pierini

INFIQC-CONICET, Facultad de Qumicas, Universidad Nacional de Crdoba, Crdoba, Argentina.

INQUINOA-CONICET, Facultad de Bioqumica, Qumica y Farmacia, Universidad Nacional de Tucumn,


S. M. de Tucumn, Argentina. email: mpuiatti@fcq.unc.edu.ar
Experimental and theoretical studies on the interaction of L-cysteine ethyl ester (ECYS) with
dipalmitoylphosphatidylcholine (DPPC) bilayer were carried out. IR and Raman SERS spectra were
employed, as previously used for other systems,[1] to follow the changes produced in the DPPC with
increasing concentrations of ECYS in the solution. Both states of the lipid, gel and liquid crystalline, were
studied at different temperatures. These studies were complemented with Molecular Dynamics
simulations that offer a microscopic view of the process [2], monitoring and measuring the importance of
the different interactions produced between the ECYS and the bilayer.
The shifts of the different IR and Raman signals show a strong interaction with the phosphate group of
DPPC, also changes in the region of the C=O groups. The MD reveals that the protonation state of the
amino group affect the strength of the interaction with the phosphate group, and the penetration of the
probe in the membrane that stay in the region of the ester groups of the lipids.
1- Defonsi Lestard, et al.Spectrochim. Acta A Mol. Biomol. Spectrosc., 2012,97, 479-89.
2- Lyubartsev, A. P.; Rabinovich, A. L. Soft Matter, 2011, 25-39.


TMSB6_Modelling the circadian rhythm generation at two levels of biological
organization.
Nieto, PS
1
; Romn, MD
1
; Revelli, JA
1
; Garbarino, E
2
; Guido, ME
2
; Tamarit, FA
1
.
1- GTMC, FaMAF, IFEG-CONICET. Universidad Nacional de Crdoba.
2- Depto. de Qca. Biolgica, CIQUIBIC-CONICET, FCQ, Universidad Nacional de Crdoba.

Most living organisms exhibit physiological and behavioural circadian rhythms with an endogenous period
of about 24 h, which can be synchronized and anticipate to periodic cues. At molecular level, the
circadian timekeeping is driven by a set of genes, called clock genes, which interact in oscillatory
transcriptional networks within cells. In mammals, these clock genes are expressed in cells throughout
the whole body and their activity is orchestrated from within the brain, in a tiny structure in the
hypothalamus known as the suprachiasmatic nuclei (SCN). The SCN consists of about 20,000 neurons
which are diverse and function in a coordinate fashion since they are able to communicate with each
other, sync their activity and hence become -as a whole- a biological clock both precise and robust. In this
work we present two deterministic mathematical models for studying circadian rhythms generation at two
levels. At the cellular level, we have studied how the dynamics of the molecular clock is affected by the
translational regulation of clock genes. We found that translational regulation introduces time-delays
between the mRNA and protein expression which ultimately affects the period of the molecular clock. At
the multicellular level we use a model of circadian oscillators coupled through different network
architectures, in order to simulate the dynamical behavior observed in SCN slices. We propose
quantitative metrics to characterize the emerging dynamical behavior in the model. We show that these
metrics reect different proles having their origin in the underlying network topology. We posit that these
metrics can be applied to experimental time-series from SCN slices in order to characterize their spatio-
temporal organization and potentially dilucidate their functional connectivity.
TMSB SAB 2013
84

TMSB7_A novel conformational switch determines Trypanosoma cruzi trans sialidase
activity at the covalent intermediate stage
Diego J. Alonso de Armio
a
, Daro Estrin
b
, R. M. S. Alvarez
a
, Adrian E. Roitberg
c

a. Instituto Superior de Investigaciones Biolgicas, INSIBIO (CONICET-UNT). Chacabuco 461, San
Miguel de Tucumn, Tucumn, Argentina.
b. Instituto de Quimica Fisica de los Materiales, Medioambiente y Energa, INQUIMAE-CONICET,
Ciudad Universitaria, Pab 2, C1428EHA, Buenos Aires, Argentina.
c. Quantum Theory Project and Departament of Chemistry, University of Florida, Gainesville, Florida,
United Status of America.
Trypanosoma cruzi is the causative agent of Chagas' disease. T. cruzi trans-sialidase (TcTS) was shown
to be an important factor in the microorganism's virulence, and has been proposed as a target for drug
design, a goal that requires a thorough understanding of the enzyme's mechanism. Evidence indicates
that the catalytic mechanism involves a long-lived covalent intermediate (CI), which is later attacked by an
acceptor glycoconjugate, completing the sialic acid transfer. A key question is thus, how does TcTS
protect the CI from hydrolysis until the acceptor glycoconjugate can position itself in the active site for the
transfer reaction to take place. Previous works hypothesized that water accessibility to the anomeric
carbon of sialic acid at the CI stage may be restricted in TcTS compared to TrSA --a structurally and
mechanistically very similar enzyme with strict hydrolase activity. Our results show not only that this
hypothesis is wrong but suggest a novel switch mechanism sensitive to lactose in CI-TcTS, by which it is
deactivated in absence of acceptor ligand and reactivated upon its binding, by virtue of a differential
flexibility of the backbone in the open vs closed conformation of CI-TcTS which is not present in CI-TrSA.

TMSB8_A modified BMW-MARTINI coarse grained model of POPE and POPC is capable
of reproducing the mechanical properties of pure and mixed monolayers.
Miguel V
a
, Villarreal MA
b
, Perillo MA
a

a
IIByT (CONICET-UNC), ICTA and Ct. Qumica Biolgica, Dpto. de Qumica, FCEFyN, UNC.
b
INFIQC (CONICET-UNC). e-mail:virgimiguel@yahoo.com.ar

Biological membranes consist in a lipid bilayer with selective permeability for different chemical species.
Ions have high energetic barrier to be transferred from water into the membrane. However, voltage-
induced lipid channels can allow current transmission trough membranes, in the absence of protein
channels. Such studies are usually done in a bilayer model system prepared in the presence of n-decane
as a lipid solvent. Our aim is to simulate such membranes and later subject them to external electric
fields. Coarse grained (CG) models are highly useful and reliable tools for the characterization of lipid
systems. BMW-MARTINI is a molecular CG model with a water molecule with three charge sites that
reproduces the experimental dipole potential at the membrane-water interface. BMW-martini force-field
failed to reproduce the experimental surface pressure () mean molecular area (A) compression
isotherms of pure and mixed POPE and POPC Langmuir films (LF). However, LF with similar
compositions with or without n-decane, could be successfully simulated by molecular dynamics after
introducing modifications in the force field. The self-assembly process of a LF was analyzed to determine
whether n-decane was excluded from the monolayer to the lipid- water or to the lipid-air interface. Finally,
MD simulations of mixed POPE-POPC bilayers with charge imbalance were performed.

Acknowledgements: Work financed with grants from CONICET, Foncyt, SeCyT-UNC. MAV and MAP are
career members of CONICET.

SAB 2013 TMSB
85

TMSB9_Statistical Models for Modulated Phases in Membranes
Brito T. , Lenilson; R. Germano; V. B. Henriques
Instituto de Fsica, Universidade de So Paulo, CP 66318, 05314-970, So Paulo, SP, Brazil.
Amphiphile molecules in solution spontaneously self-assemble in several supramolecular aggregates,
such as micelles, lamellar and hexagonal phases, and vesicles. Aqueous suspensions of vesicles formed
by zwitterionic (neutral) phospholipids- such as phosphatidylcholine- present a gel-fluid transition, associ-
ated with the disordering of the lipid acyl chains, usuallycalled the main transition [1]. This phase
transition is sharp, with a latent specific heat and discontinuity in the thermo-dynamic properties of the
suspension, being thus classified as a first-order transition. Several experimental techniques, point out
that the main transition in the case of lipids containing polar headgroups that may undergo ionic
dissociation, such as phosphatidylglycerol [2], may be broadened, depending on acyl-chain length, lipid
concentration, pH, and ionic strength. We propose a statistical model to account for the gel-fluid
anomalous phase transitions in bilayer - or lamellae. Starting from the Hamiltonian of a model known in
the literature to treat magnetic systems that displays modulated phases, the model ANNNI(Axial Next-
Nearest Neighbour Ising) with competition over a network, we studied the phase diagram to try to explain
the profile of the disorder of chains, always establishing a relationship qualitatively, between the
experimental results of specific heat.
Acknowledgements: The authors thanks the POSLATAM for financial support.

1- J. F. Nagle. Ann. Rev. Phys. Chem. 31, 157 (1980);
2- M. T. Lamy-Freund. Chem. Phys. Lipids 122,19 (2003).

TMSB10_GPDH isoforms from Triatoma infestans. An MD approach.
Cossy Isasi S.,
Ctedra de Bioqumica y Biologa Molecular, Medicina, Fac. Cs. Mdicas, Universidad Nacional de
Crdoba.

Two glycerol-3-phosphate dehydrogenase (GPDH) isoforms are present in flight muscles of Triatoma
infestans: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. During
development, the expression of GPDH-2 increased with a longer time of intake, which would imply an
increment in lipid biosynthesis. The GPDH-1 transcript predominated with respect to that of GPDH-2 in
the lower nutritional condition. The predominant isoform in nymph thoracic muscles and gonads has less
mobility than the isoform in adult thoracic muscles(1). The isoforms' activities and transcript patterns in
flight muscles, suggest transcriptional adaptation to metabolic requirements originated by alternative
splicing. The different 3 ends of GPDH isoforms inferred for C-terminal amino acid sequences ETPSEE
for GPDH-1 and FFTKKSLKP for GPDH-2 could condition the enzymatic activity and the sub-cellular
localization needed to accomplish their metabolic role (2). The last hypothesis is particularly well suited to
be tested with MD studies. Intial results of homology based structural models interacting with different
environments will be presented. Gromacs, with Gromos53a6 force field with modified topologies or hybrid
parameters was the main software. NAMD and Lammps were also tested

1- Stroppa MM, Am J Trop Med Hyg. 2013; 88(6):1146-51.
2- Stroppa MM, Am J Trop Med Hyg. 2008


TMSB SAB 2013
86

TMSB11_ Computational characterization of non-common iron III binding sites
Agudelo, WA; Vazquez, DS; Gonzlez Flecha FL; Santos, J; and Gonzlez Lebrero, MC
Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of
Pharmacy and Biochemistry, University of Buenos Aires, Argentina.
The motif (E/D)(E/D)xx(E/D)(E/D) is involved in iron exchange in the CyaY protein family and it is present
in other metal ion binding proteins. Based on the C-terminal -helix of E. coli thioredoxin a model peptide
containing the motif was synthesized and studied. This peptide shows iron III binding and induction of -
helical structure*. However the microscopic details of the motif-ion interactions are unknown. In this work
the system was addressed by computational simulation techniques. The main goal was the
characterization of possible structures of metal peptide complex. To obtain suitable peptide-ion
structures, geometrical search was performed by genetic algorithm and the most promising candidates
were filtered by geometric criteria. Afterward, selected complexes were submitted to molecular dynamics
simulations and the binding free energies were computed by thermodynamic Integration and compared
with experimental results from isothermal titration calorimetry*. In addition, the protonation state of each
residue of the motif was explored in the absence of the metal ion to evaluate a possible effect of
clustering negatively charged residues on individual pK
a
values. * See submitted abstract: Vazquez DS et
al., Grafting a functional iron-binding motif onto a thioredoxin scaffold.
Acknowledgements: ANPCyT, UBACyT and CONICET for their support.
Andreini C, Bertini I, Cavallaro G. et al. J. Mol. Biol. 2009. 356.
Seebeck B, Reulecke I, Kmper A. et al. Proteins. 2008. 1237.

TMSB12_A QM/MM study of the autophosphorylation of the catalytic domain of CopA
Sabeckis, ML.; Romn EA; Gonzlez Flecha, L. and Gonzlez Lebrero MC.
Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of
Pharmacy and Biochemistry, University of Buenos Aires, Argentina.
CopA is the thermophilic Cu+-P-type ATPase from Archaeoglobus fulgidus. This protein couples the
energy of ATP hydrolysis to Cu+ translocation across cellular membranes. In the reaction cycle it is
generated a phosphorylated metastable intermediate that gives the name of P-ATPases to this family of
proteins. The ATP hydrolysis takes place within an intracellular domain which is composed by two
subdomains: a nucleotide binding or N-domain and a phosphorylation or P-domain. This domain
hydrolyze ATP and inorganic phosphate esters at high temperatures even when purified without the rest
of CopA. The aim of this work is to study the active site dynamics and the autophosphorylation reaction of
this domain using computational simulation techniques. In particular it is studied the protonation of D424
(the phosphate acceptor), as well as the proton transfer of the K600 to the reactive phosphate, and its
influence in this reaction. For this, classical and hybrid QM/MM simulations techniques were performed
using the AMBER simulation set of programs, and a proper developed code for the calculus and the
quantum subsystem, based on Density Functional Theory (DFT) and the PBE functional. This code was
modified for the employment of Graphics processing units (GPUs) in its most demanding parts.
Acknowledgements: ANPCyT, UBACyT and CONICET for their support.


SAB 2013 TMSB
87

TMSB13_In Silico evaluation of the auto-aggregation process of 33-mer Gliadin peptide.
M.J. Amundarain
(a)
, F. Zamarreo
(a)
, J.F. Viso
(a)
, M.G. Herrera
(b)
, V.I. Dodero
(b)
, M.D. Costabel
(a)

(a)- Grupo de Biofsica, Departamento de Fsica, Universidad Nacional del Sur (UNS), Baha Blanca,
Argentina. mjamundarain@gmail.com
(b)- Grupo de Qumica Biomolecular, Departamento de Qumica-INQUISUR, UNS-CONICET, Baha
Blanca, Argentina.
Molecular modelling methods represent essential tools to undertake the study of biological systems,
providing a better understanding of both their structure and functionality. In this work we apply molecular
dynamics simulations and electrostatic calculations to analyse the initial steps of auto-aggregation of 33-
mer peptide.
The fragment 33-mer is the result of the partial degradation of the protein -gliadin. This protein, along
with others of the same family, is found in gluten of grains which are toxic to people who suffer from
coeliac disease.
This peptide has been proposed to initiate the inflammation process that leads to the damage of epithelial
cells of the small intestine. However, the molecular bases of this process are not well known yet. We are
particularly interested in studying its ability of auto-aggregation into more complex structures, since
different superstructures have been observed in vitro.
Therefore, we have analysed three possible assemblies in water using GROMACS for the MD simulations
and APBS to evaluate electrostatic interactions.
Acknowledgments: We want to thank CIN, CONICET and UNS, Min. Educ. SPU P:17-16-296.


TMSB14_Molecular basis of differential reactivity of peroxiredoxins to different
substrates.
Lichtig, PL*. Zeida, A*. Hugo, M**., Trujillo, M**., Estrn, DA*.
*DQIAyQF, INQUIMAE-CONICET, FCEN-UBA, Ciudad Universitaria, Pab. 2, C1428EHA Buenos Aires,
Argentina. e-mail: pablo.lichtig@gmail.com
** CEINBIO, Facultad de Medicina, Universidad de la Repblica, Montevideo, Uruguay.
Peroxiredoxins (Prxs) are ubiquitous proteins that use an active site Cys residue to reduce peroxides.
This residue is oxidized to its sulfenic form, which may form inter- or intramolecular disulfide bonds so as
to recycle their activity. In spite of having a highly conserved active site, they have surprisingly different
reactivities towards different peroxides
1
. Alkyl hydroperoxide reductase E (AhpE) is a 1-Cys Prx. It is an
essential homo-dimer of Mycobacterium tuberculosis. It has an extraordinary reactivity when reacting with
peroxides derived from fatty acids (k
ox
=10
8
M
-
s
-1
), yet with hydrogen peroxide it is at least 1000 times
less reactive
2
. We are attempting to understand, by using computational studies, the molecular factors
that determine this differential reactivity. It has been postulated that the kinetic differences previously
described might be a consequence of a hydrophobic groove located in the dimer interface, which could
facilitate the interaction with this kind of substrates. To test this hypothesis, we designed a mutant that we
expect will be monomeric but keep catalytic activity, currently in the process of being synthetized. We also
attempted to measure binding free energy with different substrates by four different computational
methods (MOPAC, MM-GBSA, classical Umbrella-Sampling and Thermodynamic Integration).
1- Ferrer-sueta et al. (2011) Chem. Res. Toxicol. 24, 434450.
2- Reyes, A. M. et al (2011) Radical Biology & Medicine 51, 464473.
TMSB SAB 2013
88

TMSB15_Ascaris suums FABP, AS-p18, shows ambiguous behavior in its interaction
with biological membranes.
Zamarreo F
(a)
., Ibez-Shimabukuro M.
(b)
, Crsico B.
(b)
and Costabel M.D.
(a)

(a) Grupo de Biofsica, Dpto. de Fsica, Universidad Nacional del Sur, Baha Blanca, Argentina.
(b) Instituto de Investigaciones Bioqumicas de La Plata, CONICET-UNLP, Facultad de Ciencias
Mdicas, Universidad Nacional de La Plata, Argentina.
Fatty Acid Binding Protein (FABP) belongs to a family of intracelular lipid binding proteins with the general
function of lipid trafficking. In vivo and in silico studies have shown that different FABPs transfer fatty
acids to membranes by two different mechanisms: collisional or difusional.
As-p18 is a FABP produced by larvae of parasitic nematode Ascaris suum. Structural features such as
extended loops have been observed in this protein. In addition, this protein is secreted to the perivitelline
fluid by the third stage larva, showing a unique characteristic for this family.
In this work we show that As-p18 has an ambiguous behavior in its interaction with biological anionic
membranes with a third mechanism that seems to be a merger between collisional and difusional
mechanisms
In order to study protein-membrane interaction we analyzed the electrostatic involved in the system, and
developed molecular dynamics simulations.


TMSB16_Molecular dynamics provide an atomistic insight into hydrogen exchange/mass
spectrometry experiments.
Defelipe, Lucas Alfredo
1,2
, Lopez, Elias Daniel
1,2
and Turjanski, Adrin Gustavo
1,2
.
1
Departamento de Qumica Biolgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Argentina.
2.
INQUIMAE-UBA/CONICET, Buenos Aires, Argentina.
Proteins are flexible entities that in solution switch between conformations to achieve their
function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand
dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein
protein interactions. However, interpretation of the HX/MS data is difficult, and changes are mostly
explained in relation to available X-ray structures, precluding a complete atomic picture of protein
dynamics. In previous work [1], we applied Molecular Dynamics (MD) simulations to provide atomistic
view of dynamic changes occurring upon activation and binding of ATP to ERK2 MAPK. In the present
work, we extend the analysis to 4 different proteins (Alpha Actinin, p38 alpha, PPAR gamma, hGHv)
where HX/MS data is available in order to establish the transferability of the approach. Additionally, we
developed a VMD Plugin to allow other groups to easily perform the same analysis with their own MD
simulations and HX/MS data.
Acknowledgements: LAD is a CONICET Fellow, EDL is a ANPCyT Fellow and AGT is a member of
CONICET.

1- Petruk A, Defelipe, L, Rodriguez LR, Bucci H, Marti MA, Turjanski AG. JCTC in. press. (2012)

SAB 2013 TMSB
89

TMSB17_Molecular dynamics study of 1-indanone thiosemicarbazones/hydroxypropyl--
cyclodextrin complexes formation: evidences of a cooperative effect
Martini, M.F.; Glisoni, R.; Moglioni, A.; Sosnik, A. and Pickholz, M.
Department of Pharmaceutical Technology, Faculty of Biochemistry and Pharmacy, University of Buenos
Aires, C.P. 1113, Buenos Aires, Argentina and CONICET, Buenos Aires, Argentina
Among the thiosemicarbazones (TSCs) have been reported varied activities: antineoplastic, antibacterial,
antifungal, antiprotozoal and antiviral. In particular, 5,6-dimethoxy-1-indanone TSC was more effective
than ribavirin against the virus of the bovine viral diarrhea, the surrogate model of the hepatitis C virus
(HCV) [1]. However, TSCs display extremely low aqueous solubility and a relatively high tendency to self-
aggregate in water [2]. This behavior hampered the reproducible and reliable evaluation of the biological
activity in vitro [1,2].
Cyclodextrins (CDs) are macrocyclic oligosaccharides that combine a hydrophobic nano-sized cavity with
a hydrophilic surface. The cavity enables the partial or total incorporation of lipophilic molecules.
In this molecular study, we have analyzed the stabilization of inclusion complexes formed between
hidroxy-propyl- -cyclodextrin (-HPCD) and two TSC molecules: 1-indanone TSC (1TSC) and 5,6-
dimethoxy-1-indanone TSC (2TSC). Our results show a differential stability of the complexes formed
according the presence or absence of the substitutents in the indanone ring. Moreover, one type of
complex of 2TSC depends of cooperative effects to stabilize and to be formed, which is not the case of
the similar complex of 1TSC.

1- Finkielstein et al Eur J Med Chem. 2008, 43:1767
2- Glisoni et al, New J Chem, 2010, 34:2047

TMSB18_Describing protein-ligand interactions by molecular dynamics simulations
using explicit mixed solvents
Arcn, JP. Gauto, DF. Dumas, VG. Defelipe, LA. Mart, MA. Turjanski, AG.
INQUIMAE y Departamento de Qumica Biolgica Facultad de Ciencias Exactas y Naturales
Universidad de Buenos Aires.

Proteins may interact with drugs and small organic molecules by establishing specific interactions such as
hydrogen bonds, hydrophobic interactions, aromatic interactions and salt bridges. Despite the fact that
there are a lot of protein-drug crystal structures, it is still very difficult to identify pockets in the protein that
are amenable to drug binding and to determine binding affinities. Taking this into account we have
studied protein interaction with different solvents in order to identify hot-spots that are able to establish
specific molecular interactions.
A set of proteins crystallized with different ligands were subjected to molecular dynamics (MD)
simulations, in their apo form, using explicit solvent. In addition to MD runs with water, three different
solvent mixtures were tested: water/ethyl alcohol, water/acetamide and water/methyl ammonium acetate
[1]. The cosolvents were chosen in order to mimic different kinds of molecular interactions a drug might
establish with a protein. Confined regions with high probability of finding a water or cosolvent molecule,
as related to the bulk, were analyzed to detect possible hot-spots. We found that different cosolvents
preferentially occupy distinctive regions in the binding site, reproducing in whole a vast majority of the
protein-ligand interactions detected in the crystallized structures. In addition, a direct relation between
these regions with high water or cosolvent occupation and certain moieties in ligand structure, e.g.
aromatic rings, was observed.

1- Seco J et al. J. Med. Chem. 2009. p 2363.
TMSB SAB 2013
90

TMSB19_Propagation of epidemics in models with multiple reservoirs
Gualtieri, A. F., Hecht, J. P.
Ctedra de Biofsica, Facultad de Odontologa, Universidad de Buenos Aires, M. T. de Alvear 2142,
1122, Ciudad de Buenos Aires, Argentina
e-mail: agualtieri@odon.uba.ar

Theoretical epidemic models are useful to know what factors could be important for the dynamics of
epidemics (see [1], for example). There are important zoonotic diseases that involve several reservoirs,
such as leptospirosis, leishmaniasis and toxoplasmosis. The aim of this work is to design and explore a
general epidemic model with multiple reservoirs. A model based on differential equations which includes
the dynamics of propagation in reservoirs and humans is designed. We raise a traditional SIR dynamics
(Susceptible-Infected-Recovered) for reservoirs and humans. The system is studied by computer
simulation. The explorations performed show that the number of reservoirs, along with rates of infection
and recovery, influence the dynamics of propagation in the whole system and in the human population.
Simulations also allow to observe how the results of the implementation of control measures depend on
the reservoirs affected by these measures and the parameters of infection and recovery defined by the
model. In conclusion, our model shows that the number of reservoirs is a factor that can affect the course
of the epidemic in humans, and should be taken into account for the implementation of health campaigns.

Acknowledgments: Supported by UBACyT 20020110200037BA.

1- Gualtieri, A. F. & Hecht, J. P. Journal of Life Sciences. 2013. 503.


TMSB20_Structural and functional relationship of a key Lys residue in human telomerase
reverse transcriptase and HIV-1 RT proteins: a comparative study.
Herrera, F.E
(1)
. and Sferco, S.J.
(1,2)

(1) Dpto. de Fsica, Facultad de Bioqumica y Ciencias Biolgicas, Universidad Nacional del Litoral, Santa
Fe, Argentina; (2) INTEC (UNL-CONICET), Santa Fe, Argentina.
Reverse Transcriptase (RT) proteins add dNTP to an existing DNA primer having RNA as a template.
HIV-1 and telomerase reverse transcriptase (TERT) proteins are well known examples. Both have a RT
sequence domain, formed by several motifs (1, 2, A ,B, C, D and E) identified from a few conserved key
residues. Among them, three conserved Asp residues (from motifs A and C) are essential for the catalytic
polymerase activity, both in HIV-1 as well as in telomerases. On the other hand, mutations of the other
key residues modify some properties but do not quench the catalytic activity to zero. The exception is the
Lys902 (located in motif D) of human TERT: when mutated to Ala, Gln or Asn, a null catalytic activity is
measured in vitro. While, when the corresponding Lys220 in HIV-1 is mutated to Gln, the catalytic activity
is still significant compared to WT. In this work, a comparative study was done in order to understand why
the mutations of the conserved Lys in motif D, are crucial in hTERT, but not in HIV-1. A good validated
theoretical model for the human TERT was obtained (the experimental structure is not known) and 10 ns
of molecular dynamics simulations at 300K for the WT, and each of the three mutations were performed.
Similarly, the same kind of MD simulations for the WT and mutated HIV-1, from an experimental structure
were also performed. The results suggest that the Lys902 participates directly into the catalytic site of
human TERT and therefore no mutations are tolerated. On the other side, the corresponding Lys in motif
D of HIV-1 is found far away from the catalytic site, suggesting also that an exchange of roles with the
neighbor Lys219 might occur.

SAB 2013 TMSB
91

TMSB21_Molecular Dynamics Simulation of neuroendocrine hormones in different
media
SN. Monachesi
1
, JR: Grigera
2
and MC. Donnamaria
3

1,3 Instituto de Fsica de Lquidos y Sistemas Biolgicos-IFLYSIB (CONICET-UNLP), 59 N 789, 1900 La
Plata, Argentina, donna@iflysib.unlp.edu.ar
2, Centro de Quimica Inorgnica-CEQUINOR- (CONICET-UNLP) ,47y115,1900 La Plata, Argentina
Exendin-4,(EX4),-hormone of 39 aminoacids, first isolated from the Gila monster saliva-displays more than
50 % sequence identity to Glucagon-like peptide-1,(GLP-1)-mammalian hormone of 39 aminoacids-, bind
in mammalians to the same G-protein coupled receptor and unlike GLP-1 has prolonged duration of action.
In the current work by molecular dynamics (MD) simulations (GROMACS package) some structural and
configurational properties of GLP-1 and of its longeracting analog EX4,in water and physiological
solutions are analyzed. The considered neuroendocrine hormones are interesting because of their
glucoregulatory action (diabetes and obesity control). The MD simulation has proved to be a powerful
technique to predict properties of biomolecules in solution. The MD modeling was carried out in 20 ns at
normal conditions in aqueous and in physiological solutions of EX4 and of GLP-1.The SPC/E model for
water is used. In physiological solutions, moreover, monovalent counter ions are placed at substituted
water positions. The root mean square fluctuation (RMSF) of the C atoms give mobility results. The root
mean square deviation (RMSD) was already calculated to check the simulations convergence. Also final
structures are displayed, showing EX4 the most stable structure. The averaged final conformations show
the hormones forming helixes in the media, in agreement with experimental data.
Acknowledgements: SNM is a FCE-UNLP PHD applicant. MCD and JRG are members of Research
Career of Comisin de Investigaciones Cientficas Prov. de Bs AsCICPBA-, and of Consejo Nacional de
Investigaciones Cientficas y Tcnicas CONICET-, respectively. Authors gratefully acknowledge support
to CONICET&UNLP. MCD also thanks a research personal CICPBA subsidy.
TMSB22_Preliminary results in the hidratation of the hydrophobic region of the 5-fold
symmetry axis of TrV.
Viso J.F.
1
, Zamarreo F
2
, Amundarain M.J.
3
,Gurin Aguilar D.M.
4
,Costabel M.D.
5

1 3 5
Departamento de Fsica - Universidad Nacional del Sur
2
Departamento de Qumica - Universidad Nacional del Sur
4
Universidad del Pas Vasco, San Sebastin Espaa

Triatoma Virus (TrV) is an insect virus that belongs to the Dicistroviridae family and infects several
species of triatomine insects which are the vectors for human trypanosomiasis, commonly known as
Chagas disease. Because of this TrV is proposed as a biological control against this kind of insects.
In this work we study the capsid of TrV, which is an icosahedral structure formed by multiple copies of the
proteins (Vp1, Vp2 and Vp3). In particular we are interested in the 5-fold symmetry axis because of the
channel formed along this axis may be responsible of the interaction of the interior of the virus with the
exterior. Previously we have observed that the pore formed in this axis remains closed in the position of a
ring of aminoacids formed by Valines which created an hydrophobic gate.
Using molecular dynamics we have found certain conditions where the axis is completely full of water
molecules, even in the hydrophobic region. The complete hydratation of the channel may lead to the
opening of the channel and the interaction between the interior and the exterior of the capsid.
Acknowledgments: We want to thank CONICET and UNS, Min. Educ. SPU P:17-16-296.


TMSB SAB 2013
92

TMSB23_Concentration Effects of Sumatriptan on the Properties of Model Membranes
Wood, I.
1*
and Pickholz, M.
1,2
1
Departamento de Tecnologa Farmacutica, FFyB, UBA;
2
CONICET *irewood@gmail.com
Triptans are drugs rationally designed based on the neurotransmitter serotonin (5-HT) and used for the
treatment of migraine [1], acting as receptor selective agonists 5-HT1B/1D/1F [2]. In this work, we report a
Molecular Dynamics (MD) simulations study of the first designed triptan, Sumatriptan, at protonated state
(pSMT), in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC). The
simulations were carried out at three different drug/lipid stoichiometries: 1:75, 1:10 and 1:3, under NPT
conditions. The goal of this study is to evaluate the effects of different concentrations pSMT on the lipid
bilayer properties. Our results show partition of pSMT between the lipid head-water interphase and water
phase. The orientation of PN vector was altered by the presence of pSMT at lipid-water interphase,
showing concentration dependence. The main interactions that stabilized the systems were hydrogen
bonds, salt bridges and cation-. Besides, pSMT molecules have no access to the hydrophobic region of
the bilayer at the studied concentrations. From an atomistic point of view this work could contribute to the
discussion of the drug-membranes interactions regarding the limitation of Sumatriptan to cross the blood-
brain barrier, that could be associated with both the efficacy and adverse effects.
1- Humphrey PPA. Ann NY Acad Sci. 1990. 587
2- Rapoport AL. J Headache Pain. 2001. S87


TMSB24_Validation of GP41-based Chimerical HIV-1 Epitopes by De Novo Design
Methods
Cunha, KC; Rusu, VH and Lins, RD
Department of Fundamental Chemistry, Federal University of Pernambuco, Recife, PE, Brazil,
keila.cunha@ufpe.br and roberto.lins@ufpe.br
We have previously identified immunogenic amino acid sequences from the HIV-1 virus and grafted the
epitope sequences into an artificially-designed highly-stable scaffold protein called Top7. In addition to its
remarkable stability, this protein displays structural elements similar to the identified epitopes. The
structural and dynamical stability of the computationally designed epitopes were previously assessed by
molecular dynamics simulations (1), and CD spectroscopy (2). The stable scaffolds were capable of
presenting a structural motif that can be immunologically recognized (1). However, the devised protocol
comprised of several modeling steps, which prevents new predictions in an efficient fashion. In this work
we have used de novo design techniques to score our engineered chimeras. The Rosetta energy scoring
function within the pmut module was able to qualitatively discriminate the structurally stable and
immunologically active proteins from the unstable chimeras. From six Top7 helical-based systems, the
Rosetta scoring function suggests that stable epitopes could present relative energies higher by a few
units of kcalmol
-1
.
This work was developed with financial support from FACEPE, NanoBiotec-BR/CAPES, CNPq, INCT-
INAMI, nBioNet and the Oswaldo Cruz Foundation

1- Viana, IFT et al. RSC Advances. 2013,3: 11790
2- Viana, IFT et al, LNBI. 2013, 8313: 94



SAB 2013 TMSB
93

TMSB25_De Novo Design of Top7- and GB1-based 4E10 Epitopes
Rusu, VH and Lins, RD
Department of Fundamental Chemistry, Federal University of Pernambuco, Recife, PE, Brazil,
victor.rusu@ufpe.br and roberto.lins@ufpe.br
Broadly neutralizing monoclonal antibodies to HIV-1 are essential for vaccine design. 4E10 is the
broadest known neutralizing antibody (1). It recognizes the contiguous and highly conserved epitope
sequence NWFNIT in the membrane-proximal region of gp41. Residues W672, F673, I675 and T676
have been identified to interact with monoclonal antibody in a helical conformation. Here, we have applied
de novo design techniques to generate Top7- and GB1-based 4E10 epitopes in a helical motif. Top7 and
GB1 were chosen as scaffolds due to their ability to accommodate replacements in their sequence (2-3).
The structural stability of the proposed candidates was subsequently evaluated by molecular dynamics
simulations. The most promising chimeras are discussed based on a combination of energetic criterion
and solvent exposure of the W672, F673, I675 and T676 residues.
This work was developed with financial support from FACEPE, NanoBiotec-BR/CAPES, CNPq, INCT-
INAMI, nBioNet.

1- Cardoso, RM et al. Immunity. 2005, 22: 163
2- Viana, IFT et al. RSC Advances. 2013, 3: 11790
3- Gronenborn AM et al. Febbs Letters. 1996, 398: 312


TMSB26_A simple model to simulate protein-membrane interactions

Villarreal
1
MA, Emperador A
2,3
, Sfriso P
2,3
, Leiva EPM
1
, Orozco M
2,3,4,5
.
1- INFIQC-Depto Matemtica y Fsica. CONICET-Universidad Nacional de Crdoba. Argentina.
2- Institute for Research in Biomedicine, Barcelona, Spain.
3- Joint IRB-BSC Program in Computational Biology, Barcelona, Spain.
4- Barcelona Supercomputing Center, Barcelona, Spain.
5- Department of Biochemistry and Molecular Biology, University of Barcelona, Spain.

We present a minimalistic model to simulate protein-membrane interactions. The protein is represented
with a Go model using only the alpha carbons of the backbone. The lipid bilayer is represented as five
slabs, which account for the water phase, the lipid headgroup, and the membrane interior. The interaction
of the protein with the membrane is based on lipid-water partition coefficients and the exposed surface
area of each amino acid. The interaction potential is modeled with discrete changes in energy, and
therefore can be used in discrete molecular dynamics simulations (DMD). Here we present the
calibration of the model against experimental results of binding constants, and preliminary applications of
the refolding of a transmembrane protein.



Transportadores, receptores y canales (Transporters, receptors and channels) SAB 2013
94

TRC1_Interaction of an ATP analogue with the Plasma Membrane Calcium Pump
Saffioti, N A., Ferreira-Gomes, M., Rossi, JPFC. and Mangialavori, IC.
IQUIFIB- Dr. Paladini -Departamento de Qumica Biolgica, Universidad de Buenos Aires

The Plasma Membrane Calcium Pump (PMCA) is a highly regulated protein present in all eukaryotic
cells. It belongs to the P-type family, a group that shares the characteristic of ion transport coupled to the
ATP hydrolysis involving phosphorylated intermediates during its reaction cycle. In order to study the
conformational changes in the nucleotide binding domain, we assay the 2',3'-O-(2,4,6-
Trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) an ATP analogue with fluorescence properties with
purified PMCA. We have previously shown that TNP-ATP binds to the protein increasing its quantum yield
allowing the detection of conformational changes among intermediates of the reaction cycle
1
. In this work
we characterize the interaction between TNP-ATP and PMCA purified from human red cells in equilibrium
and in steady-state conditions. To this purpose, Ca
2+
-ATPase activity and TNP-ATP fluorescent emission
were measured in the presence or absence of calmodulina or phosphatidylserine. Our results show that
PMCA do not hydrolyze TNP-ATP. This ATP analogue behaves as a pure competitive inhibitor of the
enzyme. Its apparent K
i
is not significantly modified when the pump is incubated in the presence of
activators. Results show that (1) TNP-ATP binds to the same site of ATP and (2) the increase of PMCA
activity by calmodulin or phosphatidylserine do not modify the binding site of the nucleotide.

With grants of ANPCYT, CONICET, UBACYT y NIH
1- Mangialavori IC, Ferreira-Gomes MS, Saffioti NA, Gonzalez-Lebrero RM, Rossi RC, Rossi JP. J Biol
Chem 2013. 2013. In the Press

TRC2_Divalent metal ions transport by Plasma Membrane Calcium ATPase. Effect on
regulation and selectivity.
Ontiveros, MQ., Mangialavori, IC., Martiarena, J., Rossi, JPFC. and Ferreira-Gomes, MS.

IQUIFIB- Dr. Paladini -Departamento de Qumica Biolgica, Universidad de Buenos Aires

The plasma membrane calcium pump

(PMCA) transports Ca
2+
actively to the extracellular medium
coupled to the ATP hydrolysis maintaining the cellular homeostasis. PMCA is activated by interaction with
Ca
2+
-calmodulin complex or by acidic phospholipids, increasing its affinity for Ca
2+
and its maximum
velocity [1, 2]. The aim of this work was to study the transport of different divalent cations that could be
transported by PMCA, the effect on regulation and the selectivity of the pump. We assay the alkaline
metals Be
2+
, Ca
2+
, Sr
2+
and Ba
2+
, and Co
2+
, Cd
2+
and

Pb
2+
from the fourth, fifth and sixth periods. The
experiments were performed with both IOVs and the purified enzyme isolated from membranes of human
erythrocytes. Results show that: (a) PMCA is able to transport Ca
2+
, Sr
2+
, Ba
2+
, Co
2+
and

Pb
2+
with
different capacity and affinity while Be
2+
and Cd
2+
are not transported; (b) PMCA activated by calmodulin
shows an increased affinity for those cations; (c) there is a relation between the ionic radii of cations and
the transport capability of the pump.
These results suggest that the mechanism of expulsion of Ca
2+
mediated by PMCA would also transport
other divalent cations and that the size of such cation must be optimal for the interaction with the binding
site of the enzyme.
With grants of ANPCYT, CONICET and UBACYT

1- Mangialavori y col. J Biol Chem. 2009. 4823
2- Filomatoriy col. J Biol Chem. 2003. 22265

SAB 2013 TRC
95

TRC3_Alumminium inhibits the plasma membrane and the sarcoplasmic reticulum
calcium pumps by different mechanisms
De Sautu, M., Ferreira-Gomes, M., Takara, D., Rossi, JPFC. and Mangialavori, IC.
IQUIFIB- Dr. Paladini -Departamento de Qumica Biolgica, Universidad de Buenos Aires

Aluminium (Al
3+
) is involved with the pathophysiology of neurodegenerative disorders, such as
Parkinsonism dementia and Alzheimers disease. The mechanisms that have been proposed to explain
the action of Al
3+
toxicity are linked to changes in the cellular calcium homeostasis, placing the
transporting calcium pumps as potential targets.
The aim of this work was to study the molecular inhibitory mechanism of Al
3+
on Ca
2+
-ATPases like the
plasma membrane (PMCA) and the sarcoplasmic reticulum (SERCA). These P-ATPases transport
actively Ca
2+
from the cytoplasm towards the extracellular medium and to the sarcoplasmic reticulum,
respectively. For this purpose, we performed enzymatic measurements of the effect of Al
3+
on purified
preparations of PMCA and SERCA. Our results show that: (1) Al
3+
inhibits Ca
2+
-ATPase activity of both
enzymes with similar apparent affinity; (2) in the presence of Al
3+
, the apparent affinity for Ca
2+
of SERCA
decreased, leaving unmodified its affinity for PMCA; (3) Al
3+
increase the apparent affinity for Mg
2+
of both
pump;, (4) Al
3+
increases the phosphorylated intermediate (EP) of PMCA while it has not effect on
SERCA; (5) ATP does not change the apparent inhibitory affinity for Al
3+
; (6) pH does not modify
significantly the apparent inhibitory affinity for Al
3+
for both PMCA and SERCA. This work show for the
first time a different inhibitory mechanism of action for Al
3+
that involves intermediates of the ATP
hydrolysis by these two Ca
2+
-transport ATPases.

With grants of ANPCYT, CONICET, UBACYT y NIH

TRC4_Metal fluoride complex of plasma membrane calcium pump: Effect of fluoride-
stabilized phosphoenzyme analogues
Siciliano, N., Ferreira-Gomes, M., Rossi, JPFC. and Mangialavori, IC.
IQUIFIB- Dr. Paladini -Departamento de Qumica Biolgica, Universidad de Buenos Aires
The plasma membrane calcium pump (PMCA) belongs to the P-type ATPase family of active cation
pumps. According to the conventional E
1
E
2
theory, E
1
and E
2
respectively refer to high-affinity and low-
affinity states to Ca
2+
. Gating of the ion pathway is coupled to the phosphorylation and dephosphorylation
of the ATPase. Phosphoryl transfer from ATP to an Asp in the cytoplasmic domain (i.e., E
1
to E
1
P) closes
the cytoplasmic gate, and the release of ADP triggers a change in affinity of the Ca
2+
binding sites (i.e.,
E
1
P to E
2
P) and opening of the luminal gate. Hydrolysis of the aspartylphosphate (E
2
P to E
2
) closes the
gate. Metal fluorides like magnesium, beryllium, and aluminum fluorides act as phosphate analogues and
inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are
analogous to specific phosphoenzyme intermediates. Thus, MgF
x
, AlF
x
, and BeF
x
stabilize analogues of
the E
2
P product state (E
2
MgF
4
2-
), the E
2
P transition state (E
2
AlF
4
-
), and the E
2
P ground state (E
2
BeF
3
-
),
respectively. The interaction of PMCA with these complexes has never been characterized; therefore in
the present work we study the interactions of various metal fluorides (MeF) with purified preparations of
PMCA. Our results show that (1) PMCA Ca
2+
-ATPase activity is totally inhibited in the presence of MeF;
(2) Inhibition of PMCA Ca
2+
-ATPase activity in the presence of NaF and as a function of AlCl
3

or BeCl
2

concentrations, was well described by a rectangular hyperbola while as a function of MgCl
2
a more
complex behavior was observed; (3) The apparent inhibitory affinity of PMCA for these MeF was AlF
4
-
<

BeF
3
-
<

MgF
4
2-
and;

(4) inhibition was highly dependent of the pH of the reaction medium.
With grants of ANPCYT, CONICET and UBACYT

TRC SAB 2013
96

TRC5_A mathematical model approach dissects properties of interacting PIP aquaporins
Yaneff A
a,c
, Sigaut L
b,c
, Marquez M
a,c
, Alleva K
a,c
, Pietrasanta LI
b,c
, Amodeo G
a,c

a
Instituto de Biodiversidad y Biologa Experimental, CONICET - UBA and Departamento de Biodiversidad
y Biologa Experimental, FCEN, UBA, Argentina.
b
Centro de Microscopas Avanzadas and Departamento
de Fsica, Facultad de Ciencias Exactas y Naturales, UBA, Argentina.
c
Consejo Nacional de
Investigaciones Cientficas y Tcnicas (CONICET), Argentina.

The plant aquaporin PIP subfamily represents one of the main gateways for water exchange at the level
of the plasma membrane (PM). Among them, a fraction of this subfamily, known as PIP1, does not reach
the PM unless they are co-expressed with a PIP2 aquaporin. Although ubiquitous and abundantly
expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here we unravel
how FaPIP1;1 -a fruit specific PIP1 aquaporin from Fragaria x ananassa- contributes to modulate
membrane water permeability (P
f
) using a mathematical model approach applied to experimentally
obtained P
f
from different co-expression ratios of FaPIP1;1 and FaPIP2;1 or a FaPIP2;1 mutant. For the
first time we dissect the individual contribution of each PIP, showing that i) PIP1 has also high water
transport capacity when co-expressed with PIP2; ii) PIP2 water permeability is enhanced when it
physically interacts with PIP1; iii) PIP1-PIP2 seem to form heterotetramers with a random stoichiometry
arrangement.
In this context, we propose FaPIP1;1 is a key participant in the regulation of the water movement across
the membrane of cells expressing both aquaporins.

Acknowledgements: This work was supported with grants to GA (Prstamo BID PICT 2239; UBACyT11-
14 and PIP CONICET) and KA (Prstamo BID PICT10-02042; PIP0190 and UBACyT10-12 659).

TRC6_Functional and structural characterization of three different PIP aquaporins
Scochera, F.
1
,Jozefkowicz, C.
1,2
, Soto, G.
2,3
, Ayub, N.
3
, Amodeo, G.
1,2
, Alleva, K
1,2
.
1
IBBEA and DBBE, FCEN, UBA,
2
CONICET,
3
CICVyA-INTA, Buenos Aires, Argentina.

Among plant aquaporins, three subfamilies are involved in the modulation of the permeability of the
plasma membrane (NIP, XIP and PIP). Within these, only PIP are known for their capacity to form
heteromeric complexes by the interaction of different members of the subfamily. PIP members are usually
known as PIP1 and PIP2, but we recently showed that they are better described as three PIP-like clusters
instead of two: i- PIPCLI, corresponding to the classical PIP1, ii- PIPCLII and iii- PIPCLIII, involving most
of PIP2 (1).).It was shown that pairs of PIPs from different clusters can interact. However, when
expressed alone, important differences in functionality of these channels was addressed (2, 3).With the
aim of exploring how these three clusters can provide us information regarding these interactions, we
analyzed the functional and structural profile of three Beta vulgaris PIP, one corresponding to each
cluster, BvPIP2;1 (PIPCLIII), BvPIP1;1 (PIPCLI) and BvPIP2;2 (PIPCLII). We compare their water and
solute transport activity, inhibition, and molecular structures. The dissection of each channel particularities
is a key tool to understand their participation in the functionality not only of homomeric complexes but
also when assembling as heterotetramers.
Acknowledgements: This work was financed PICT10-02042, CONICET PIP 0190 & UBACYT 2013 0159
grants to K.A and UBACyT11-14 and PICT11-2239 to GA.
1-Soto et al., 2012; 2- Bellati et al., 2010; 3- Jozefkowicz, C. et al., PlosOne. 2013

SAB 2013 TRC
97

TRC7_Lipid structure affects the activity of integral membrane proteins: PMCA as a
model
Pignataro, MF; Dodes Traian MM; Gonzlez Flecha,FL; Mangialavori, IC and Rossi, JPFC.
IQUIFIB- Dr. Paladini -Departamento de Qumica Biolgica, Universidad de Buenos Aires

Integral membrane proteins have central roles in a vast number of vital cellular processes. The enzymic
activity of these proteins is affected by the structures of the lipid molecules that surround them in the
membrane. The effects of lipid structure on a membrane protein are likely to be complex and unique for
each membrane protein. As a model, we chose the plasma membrane calcium pump (PMCA), an integral
membrane protein that regulates the intracellular calcium level. In the present work, purified PMCA was
reconstituted into different phospholipid-detergent mixed micelles to relate Ca
2+-
ATPase activity with the
lipid environment. We found that: ATPase activity depends on the carbon chain length and on the molar
fraction (X
PL
) of the phospholipid in the mixed micelle. From X
PL
= 0.4, there is a drop in ATPase activity
for the majority of the assayed phospholipids, except for 1,2-Dilauroyl-sn-glycero-3-phosphocholine
(DLPC). To understand this behavior, we studied the biophysical properties of those reconstitution
systems and found that there was a micelle-vesicle transition starting from X
PL
=0.3 (followed by
Fluorescence Correlation Spectroscopy and Static Light Scattering) for most of the phospholipids. DLPC
was also the exception and the transition was found at higher X
PL
. These results provide insights to
develop a model that links the biophysical properties of the system with the enzymatic activity.
With grants of CONICET, FONCyT and UBA.




TRC8_Aquaporin channel heterotetramerization studied by dimeric tandem subunits
Jozefkowicz, C.
1,3
, Scochera, F.
1,3
, Sigaut, L.
2,3
, Pietrasanta, L.
2,3
, Amodeo, G.
1,3
, Alleva, K
1,3
.
1
IBBEA y DBBE, FCEN, UBA,
2
CMA y DF, FCEN, UBA,
3
CONICET. Buenos Aires, Argentina.
Research done in the last years strongly supports the hypothesis that certain plant plasma membrane
aquaporin (PIP) can form heterooligomeric assemblies, specially combining PIP2 and PIP1 subunits. We
have already confirmed the existence of PIP heterotetramers by studying structural elements involved in
the ruling of homo versus heterotetramerization organization between Beta vulgaris PIP aquaporins (1).
To further investigate the biophysical properties of PIP1-PIP2 heterotetramerization process, and as the
assembly of some aquaporins seemed to proceed as a dimerization of dimers (2), we co-inject a series of
cRNA tandem constructs encoding for covalently linked BvPIP1;1 and BvPIP2;2. The constructions
expressed in the plasma membrane of Xenopus oocytes formed by tandem dimers show identical
biophysical characteristics, i.e. osmotic water coefficient (P
f
) and pH regulation, than the co-expressed
individual subunits. Our results show for the first time that PIP1-PIP2 dimers can form functional
heterotetrameric channels.
Acknowledgements: This work was financed by PICT 10-02042, CONICET PIP 0190 & UBACYT 2013
0159 grants to K.A and UBACyT11-14 y PICT11-2239 to GA.
1- Jozefkowicz, C.et al., Plos One. 2013.
2- Veerappan A. et al., Biochemistry. 2011.

TRC SAB 2013
98

TRC9_Functional characterization of the YiiP transporter of Shinorizobium melliloti
Raimunda D., Elso-Berberin G.
Instituto de Investigacin Mdica M. y M. Ferreyra, INIMEC-CONICET, Universidad Nacional de Crdoba.
Transition metals (copper, nickel, cobalt, manganese, etc.) are essential micronutrients. In bacteria they
participate as co-factors in various fundamental enzymes required during infection to overcome initial
oxidative burst imposed by the host. However, intracellular transition metal accumulation leads to toxicity
and cell death. Therefore bacterial cells strive to keep null the free transition metal concentration while
assuring their quota for metalloprotein synthesis in different compartments. Thus, a direct role of
membrane transporters in periplasmic metalloprotein synthesis can be postulated. The legume symbiont
bacterium S. meliloti, is responsible of the biological N
2
fixation occurred in the nodules of alfalfa. S.
meliloti Rm1021 possesses two homologous genes codifying for Cation Difussion Facilitator (CDF)
transporters of unknown transport specificity and unknown functional roles during plant infection. In this
line, we first generated the deletion mutant strain of the SmYiiP (smyiip) and compared its resistance to
transition metal to that of the wild type (WT) strain. The growth fitness in vitro of the mutant strain was
reduced only in presence of Mn
2+
. Supporting the role of SmYiiP as Mn
2+
exporter the incubation of
smyiip and WT cells with sub-lethal Mn
2+
concentrations resulted in 2-fold increase of the metal in the
first strain. Normal levels of resistance to Mn
2+
were attained by complementation with the gene under
regulation of its promoter, discarding polar effects. Nodulation assays in alfalfa plants showed that the
strain smyiip induces less number of nodules (nod
-
) compared to plants infected with the WT strain. This
correlates with the lower weight of plants inoculated with the mutant strain. The results indicate that
SmYiiP is a Mn
2+
exporter and that this metal homeostasis is relevant in S. meliloti for infection and
nodulation.
Acknowledgements: CONICET (G111220090100063)



TRC10_Study of the mechanosensitive properties of plant aquaporins PIP2 and TIP2
Goldman, R.P.
a
, Jozefkowicz, C.
b
, Politi M.T.
a
, Sutka, M.
b
, Alleva, K.
b
, Ozu, M.
a


a
Laboratorio de Biomembranas, Departamento de Ciencias Fisiolgicas, Facultad de Medicina,
Universidad de Buenos Aires, Argentina.
b
Instituto de Biodiversidad y Biologa Experimental y Aplicada,
Facultad de Ciencias Exactas y Naturales, Consejo Nacional de Investigaciones Cientficas y
Tecnolgicas (CONICET), Universidad de Buenos Aires, Argentina.

The functions of aquaporins (AQPs) are as wide as their presence in cells and tissues of organisms from
all living Kingdoms [1]. Several regulatory and gating mechanisms have been described in different
members of the family. It was recently demonstrated that human AQP1 (hAQP1) closes when membrane
tension () increases [2]. In this work we proposed to study the mechanosensitive properties of PIP2 and
TIP2, both AQPs from the plant B. vulgaris. Since these AQPs have different functional characteristics,
and since their sequences show differences in some putative mechanosensitive sites, the purpose of this
work is to elucidate whether these two AQPs have dissimilar functional responses to changes in , and if
such possible differences are related to their distinct sequences. Preliminary results show that PIP2
responds in a similar way as hAQP1 to increments. This is interesting because hAQP1 and PIP2 show
high homology in their sequence. However, the sequence of TIP2 shows differences in some glycines,
which could be putative sensors of mechanical gating.

1- Soto et al,. Gene 503(1). 2012. P 165.
2- Ozu et al,. Biophys. J. 104(1). 2013. P 85.
SAB 2013 TRC
99

TRC11_Early effects produced by the interaction of sublytical concentration of alpha
hemolysin of E.coli with erythrocytes.
Romina Vazquez
1
, Fernanda Carrizo
1
, Sabina Mat
1
, Laura Baks
1,2
and Vanesa Herlax
1

1-INIBIOLP, CCT-La Plata, Facultad Ciencias Mdicas, UNLP 60 y 120 La Plata, Argentina.
2-Departamento de Ciencias Biolgicas, Facultad Ciencias Exactas, UNLP 47 y 115 La Plata
-hemolysin (HlyA) is one of the key virulence factors released by invading E. coli strains. It causes
lysis of various mammalian cells, including erythrocytes of different animal species. HlyA is a calcium
dependant toxin that at high concentration forms pores in target cells causing lysis of them. Nevertheless,
what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the
course of an infection. In fact, sublytic concentrations of HlyA may even be more physiologically relevant.
The aim of the present work is to study the intracellular effects triggered by the interaction of sublytical
concentration of HlyA with rabbit and sheep erythrocytes. These animal species erythrocytes were
chosen due to their differences in the lipid composition of their membranes. The calcium concentration
inside the erythrocytes was monitored while incubating with sublytic concentrations of HlyA by lifetime of
the calcium indicator Green 1 using fluorescence lifetime imaging microscopy (FLIM). HlyA induces
calcium concentration raise in both erythrocytes, but the increment in rabbit is 4 fold higher and faster
than in sheep erythrocytes. To study this thoughtfully, the hemolytic assays in the presence and absence
of different ionic channel inhibitors (amiloride, PPADS and KN 62) were done. Results demonstrated that
the influx of calcium in both erythrocytes species seems to be through purinergic channels. The
morphological changes observed where quite different, while rabbit erythrocytes shrinkage before lysis,
sheep erythrocytes did not. To further study the differences observed in both animal species more
experiments are needed, as measurement of the kinetic released of ATP, hemolytic activity in the
presence of clotrimidazole (agonist of Ca
+2
activated K
+
efflux) and measure of erythrocytes volumes
changes by flow cytometry of both animal species erythrocytes treated with sublytical concentration of
HlyA

TRC12_Role of Mg
2+
and magnesium fluoride in the induction of the E2P-like state in the
Na,K-ATPase
Centeno, M.; Ferreira-Gomes, M.S.; Rossi, R. C.; Montes, M. R.

Instituto de Qumica y Fisicoqumica Biolgicas y Departamento de Qumica Biolgica Dr. Paladini.
Universidad de Buenos Aires. mmontes@qb.ffyb.uba.ar
The Na,K-ATPase is a membrane-bound ion pump that generates electrochemical gradients for Na
+
and
K
+
across the plasma membranes of animal cells. The enzyme oscillates between two major
conformations, E1, which has high affinity for Na
+
and E2, with high affinity for K
+
. The crystal structure of
the Na,K-ATPase in the E2P form stabilized by magnesium fluoride with occluded K
+
((E2.MgF
4
[K
2
]) was
the first complex described (1). In order to establish the relationship between structure and function, this
work studied by kinetic studies the changes in conformational transitions produced by the binding of
magnesium fluoride, magnesium and K
+
to the Na,K-ATPAse. Our results show that Mg
2+
binds to either
E1 and E2 states of the ATPase whereas magnesium fluoride binds to the E2-Mg state and then shifts
the conformation of the enzyme to the E2P-like structure.
With grants of ANPCYT, CONICET y UBACYT
(1) Morth et al. Nature. 2007. 1043

TRC SAB 2013
100

TRC13_Study of the effect of hypotonic stimuli on mouse epithelial sodium channel
(ENaC) activity in Xenopus laevis oocytes
Luciano Galizia, Alejandra Palma, Gabriela I. Marino, Basilio A. Kotsias
Laboratorio de Canales Inicos, Instituto de Investigaciones Mdicas Alfredo Lanari, Universidad de
Buenos Aires, IDIM-CONICET, Buenos Aires, Argentina.
The regulation of the epithelial Na
+
channel (ENaC) during cell swelling is relevant in cellular processes in
which cell volume changes occurs. Its sensitivity to hypotonic induced swelling was investigated in the
Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC (mENaC). We
used the voltage clamp technique to study the amiloride-sensitive Na
+
currents (INa
(amil)
) and video
microscopy methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 %
reduced hypotonicity) inward current amplitude decreased rapidly during 1.5 minutes. INa
(amil)
kinetics
analysis revealed a decrease in slower inactivation time constant during the hypotonic stimuli. We
evaluate the role of external Cl
-
in the inhibition of INa
(amil)
by hypotonicity. The reduction of extracellular
Cl
-
concentration did not affect the observed response. To study the role of actin cytoskeleton in this
response we evaluate short (20 minutes) and long term (2-5hs) effects of cytochalasin D pre-treatment on
hypotonic mediated decrease in ENaC activity. Neither short nor long term cytochalasin D treatment
affected the observed response. Oocytes expressing a DEG mutant -ENaC subunit (-S518K) with an
open probability of 1 showed a reduced INa
(amil)
hypotonic response in comparison to the oocytes injected
with the wild type ENaC subunits. During the hypotonic response the ENaC injected oocytes did not show
cell volume difference compared with water injected oocytes. On this basis we suggest that hypotonicity-
dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the
membrane and is independent of actin cytoskeleton.

TRC14_Unraveling mechanisms underlying partial agonism in 5-HT
3
A receptors.
Corradi J. and Bouzat C.
Instituto de Investigaciones Bioqumicas de Baha Blanca, Baha Blanca, Argentina.

Partial agonists are unable to elicit full maximal responses and they have emerged as attractive
therapeutic molecules since they can act both as agonists or antagonists depending on the concentration
of neurotransmitter. The characterization of a genuine partial agonist is complex because other
mechanisms, such as channel block, may also limit maximum open probability. Taking advantage of the
high conductance form of the 5-HT
3
A receptor, we here studied activation by two typical partial agonists:
2-Me-5HT and tryptamine at the single-channel level. For all ligands, activation appears as openings in
quick succession grouped in clusters showing high open probability (P
open
>0.9). Open time distributions
show three components; the slowest one is 6.5- and 3.5-fold briefer for 2-Me-5HT and tryptamine,
respectively, than for 5-HT. The duration of this component decreases as a function of agonist
concentration due to open-channel block. For 2-Me-5HT, the forward blocking rate is 10-fold faster than
for tryptamine and 5-HT. Our single-channel kinetic analysis shows that 2-Me-5HT is actually a full
agonist, its maximum response being limited by channel block. In contrast, tryptamine is a genuine partial
agonist and its low efficacy is mainly due to a slow transition from the fully-liganded closed state to a pre-
open state. After reaching the latter state, activation proceeds similarly as in the presence of 5-HT.
Molecular docking shows that interactions at the binding site are similar for 2-Me-5HT and 5-HT. In
contrast, the potential to form the cation-Pi interaction with W183 seems to be reduced for tryptamine.
Unraveling the mechanism underlying partial responses elicited by 5-HT
3
ligands has implications in the
design for novel therapeutic compounds. In addition, our results reveal the existence of multiple pre-open
states associated to partial agonism in a yet unexplored member of the Cys-loop receptor family.

SAB 2013 TRC
101

TRC15_The S6 transmembrane segment modulates channel opening in BK channels
Carrasquel-Ursulaez
1,2*
, W., Contreras
1,2*
, G. F., Seplveda
3,4
, R., Aguayo
3
, D., Gonzlez-Nilo
1,3
, F.,
Gonzlez
1
, C. and Latorre
1
, R.
1
Centro Interdisciplinario de Neurociencia de Valparaso, Facultad de Ciencias, Universidad de
Valparaso, Chile.
2
Doctorado en Ciencias mencin Neurociencia, Universidad de Valparaso, Chile.
3
Universidad Andres Bello, Centro de Bioinformtica y Biologa Integrativa, Facultad de Ciencias
Biolgicas, Santiago, Chile.
4
Doctorado en Biotecnologa, Universidad Andres Bello, Chile.
*To whom correspondence should be addressed: Ramn Latorre, Centro Interdisciplinario de
Neurociencia de Valparaso, Pasaje Harrington 287, Playa Ancha, Valparaso 2366103, Chile. E-mail:
ramon.latorre@uv.cl; Phone: 56-32-2508040; Fax: 56-32-250-8027

Large conductance Ca
2+
and voltage-activated K
+
channels (BK) open probability is enhanced by
increasing cytosolic Ca
2+
concentration and/or depolarization. These stimuli activate sensors that are
coupled by allosteric interactions to channel gating. Here, we report a point mutation of a phenylalanine
(F380A) located in the S6 transmembrane helix that produces a large rightward shift of the open
probability-voltage curve with minor changes in gating current. Interpretation of our results using an
allosteric model, suggests that the F380A mutation modifies mainly the channel closed-open equilibrium
and uncouples voltage sensors and calcium binding from channel opening. Based on these functional
studies, we propose a structural model that sheds light on the molecular nature of the coupling between
both stimuli and pore opening. Moreover, the mutation F380A could be used for detailed voltage sensor
studies in the presence of permeant cations. Acknowledgements: FONDECYT Grant 1110430 (R.L.) and
FONDECYT Grant 1131003 (F.G.N), doctoral thesis support fellowship AT- 24110157 (G.F.C.),
MECESUP-UV0804 doctoral fellowship (W.C.U.).


TRC16_Characterization of a PIB copper transport ATPase from a mesophilic bacteria
expressed in yeast.
Walter Bosich, Luis M. Bredeston; F. Luis Gonzalez Flecha
Laboratorio de Biofisica Molecular . IQUIFIB(CONICET-UBA), Departamento de Qumica Biolgica,
Universidad de Buenos Aires, Argentina.
Cu
+
transporting ATPases are integral membrane proteins that have an important role in cooper
homeostasis and detoxification. Here we report the production of CopA from Legionella pneumophila
(LpCopA) in Saccharomyces cerevisiae using a galactose induced system and GFP as an indicator. We
optimized the expression conditions of LpCopA measuring fluorescence in whole cells. Maximal
fluorescence was obtained growing the transformed cells in media containing 1% yeast extract, 1%
peptone, 0.1% glucose and induced with 1% galactose for 36 hs at 28
o
C. The GFP technology was
critical for monitoring the production and purification steps of this protein, and can be an useful tool for
other membrane proteins. The ATPase activity of the purified enzyme was determined as the initial rate of
Pi release using a colorimetric method based in the formation of a phosphate-malachite green complex,
optimized in our laboratory. The LpCopA ATPase activity was similar to that previously reported
(Gourdon, et. Al 2012), but was found to be only dependent on ATP, Mg
2+
Cu
+,
DTT and NaCl
concentration.
With grants from UBACyT and ANPCyT.
1. Gourdon et al. (2012) Nature 475, 59-64.
2. Drew el al. (2007) PNAS, 104, 13936-13941

TRC SAB 2013
102

TRC17_Mutation of two charged aminoacids in transmembrane helix 2 impairs UDP-
GlcNac transport activity of mouse SLC35A3
Toscanini MA, Levalle A, Bredeston LM
IQUIFIB (CONICET-UBA), Departamento de Qumica Biolgica, Facultad. de Farmacia y Bioqumica,
Universidad de Buenos Aires, Argentina. bredes@qb.ffyb.uba.ar

Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of
Golgi apparatus where glyconjugate biosynthesis take place. To these transport process an antiport
mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed. However the amino
acids residues involved in the binding and transport of NS, are unknown. In this work we studied the
mouse SLC35A3 transporter (MmSLC35A3), specific for UDP-GlcNac, as a model of NST. MmSLC35A3
is a 326 aminoacids protein with a predicted 10 transmembrane helices. The goal of the project is the
identification of aminoacids essentials for the transport process. For these porpoise we: a) engineered a
C-terminal GFP tagged SLC35A3 to detect expression in yeast, b) used a complementation assay in a
yeast mutant lacking the UDP-GlcNac transporter, to test activity and c) constructed an active mutant
devoid of cysteins (SLC35A3-CL) with the aim to obtained a collection of cysteine unique mutant. The
cystein scanning mutagenesis of 23 residues of SLC35A3, including the TM2 helix, from Leu38 (loop
betweeen TM1-2) to Lys60 (loop betweeen TM2-3) was performed. Expression of each mutant in yeast,
detected in whole cells, showed that all the mutants were expressed at similar levels of SLC35A3-CL.
Meanwhile the activity, tested by the binding of the lectin GSII to the cell surface GlcNac residues,
showed that two mutations E47C and K50C impairs the UDP-GlcNac transport activity of SLC35A3. More
conservative substitutions E47D or K50R not recover the wild type full activity. Double mutant E47K/K50E
showed a residual activity, indicating the residues are not interchangeable. All together these results
suggest that both charged aminoacids are important for transport mechanism.

TRC18_Is the ATPase activity of the P5-ATPase rSpf1 stimulated by Ca
2+
?
Czysezon, N. A., Corradi, G. R., Adamo, H. P.A.
Instituto de Qumica y Fisicoqumica Biolgicas (IQUIFIB) Departamento de Qumica Biolgica, Facultad
de Farmacia y Bioqumica, Universidad de Buenos Aires, Junn 956, 1113 Buenos Aires, Argentina.
email: hpadamo@qb.ffyb.uba.ar
The transporters known as P5-ATPases have been associated with the early development of Parkinsons
disease and other neurodegenerative disorders. They belong to the family of ATP fuelled ion pumps
which cycle between two major conformations E
1
and E
2
and form an acylphosphate (E~P) species as
intermediate during transport. The ion transported by the P5-ATPases is not known. We have recently
begun to characterize the catalytic cycle of the P5-ATPase Spf1 from Saccharomyces cerevisiae
obtained by overexpression in the same yeast (Corradi et al., 2012). Here we have investigated the effect
of Ca
2+
on the ATPase activity of the pump, as well as its role in the EP formation. At 37 C and in the
presence of 3 M Ca
2+
the purified recombiant Spf1 with the GFP fused to its N-terminus (rSpf1) was
capable of hydrolyzing ATP at a nearly linear rate of about 5.7 mol Pi/mg min for 7.5 minutes. In contras
in the absence of Ca
2+
(0.5 mM EGTA) the ATPase activity rapidly decreased. Maximum ATPase activty
was achieved in the presence of 3 M Ca
2+
and remained high at concentrations of Ca
2+
up to 1 mM. The
incubation of rSpf1 with ATP for 30 s at 4 C resulted in the formation of the phosphoenzyme
intermediate. Noteworthy, the formation of the EP intermediate was higher in the presence of EGTA and
decreased in the presence of 10 M Ca
2+
. If the ATPase activity and EP formation are both ascribed to
the rSpf1 enzyme, these results may indicate that Ca
2+
stimulates the ATPase activity of rSpf1 by
accelerating the turnover of EP. Acknowledgments: This work is being supported by ANPCyT Prestamo
BID 2134, UBACYT y CONICET. CONICET PIP 2022.
SAB 2013 TRC
103

TRC19_Screening of yeast and bacterial expression of SLC35A3 transporters based on
GFP technology
Favarolo B, Saldaa C, Peppe S, Zarycki M, Fuentealba J, Esersky I, Bredeston LM
IQUIFIB (CONICET-UBA), Departamento de Qumica Biolgica, Facultad de Farmacia y Bioqumica,
Universidad de Buenos Aires, Argentina. bredes@qb.ffyb.uba.ar

Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of
Golgi apparatus, where glyconjugate biosynthesis take place. To these transport process an antiport
mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed. The main limitation to
structure-function studies of NST is the production of high levels of protein. Recently was described a
methodology, based on GFP fusion to membrane proteins, for a rapid screening of expression and
purification conditions.
In this work we studied the expression of UDP-GlcNac-SLC35A3 transporters from diverse species
tagged to GFP in three different systems, P. pastoris, S. cerevisiae and E. coli. In S. cerevisiae, C03H5.2
the C. elegans A3-homolog, showed the higher expression levels (in compassion to mouse-A3 and
Xenopus-A3) measuring by fluorescence in whole cells and quantified by a purified yEGFP standard (2-3
mg C03H5.2-GFPhis8/L of culture). Preliminary attempts to purified the protein showed a yield of 1.1
mg/L culture. Expression of mouse SLC35A3-GFP in Pichia pastoris showed similar levels to S.
cerevisiae in an initial screening. The advantage of this system is the obtention of stable transformants
and multiple insertion events (that occurs spontaneously at about 1-10% of single insertion). Now we are
screening a large number of clones looking a small number of highly expressing clones. Finally
expression of mouse SLC35A3-GFP in E. coli showed slight expression than yeast under initial
conditions. Optimization of IPTG concentration, temperature and time could be important to improve the
final yields.

TRC20_ABCB1 expression in Brazilian breast cancer samples
Joo Marcos de Azevedo Delou
1
, Taiana Sousa Lopes da Silva
2
, Mrcia Alves Marques Capella
1
,
Rosane Vianna Jorge
2,3

1
Programa de Bioqumica e Biologia Celular, IBqM, UFRJ;
2
Programa de Farmacologia, Coordenao de
Pesquisa, INCA;
3
Programa de Inflamao e Farmacologia Celular, ICB, UFRJ
ABCB1 is the most studied transporter of the ABC superfamily and confers multidrug resistance
phenotype, a major complication in cancer chemotherapy. Three ABCB1 gene polymorphisms
(rs1128503, rs2032582 e rs1045642) have been associated to diminished protein expression, altered
protein conformations with different substrate recognition and activity. We aimed to characterize ABCB1
tumor expression in Brazilian breast cancer patients evaluating the impact of gene polymorphisms on
clinical and histopathological variables. Cohort of 713 women is under investigation. Paraffin-embedded
tumor samples were processed for immunohistochemistry and blood samples for genotyping. Protein
expression scores considered both tumor positivity and intensity of reaction. ABCB1 positivity in tumor
samples was 86.3%. Multivariate forward stepwise logistic regression model of the independently
associated variables concluded that ABCB1 expression is associated to: triple negative subtype (OR
0.239, CI95 0.127-0.451); hypertension status (OR 0.420, CI95 0.243-0.726); lymph node status pN2
(OR 3.679, CI95 1.407-9.624) and tumor size 5 cm (OR 0.548, CI95 0.321-0.935). In conclusion, we
observed that ABCB1 expression in Brazilian breast cancer samples is frequent, independent of the three
gene polymorphisms investigated, associated to molecular tumor variables such as hormonal receptors
and HER-2, as well as to clinical individual characteristics, such as systemic arterial hypertension.
Acknowledgements: CNPq, FAPERJ.
Sealizacin y Dinmica Intracelular (Signaling and Intracellular dynamics ) SAB 2013
104

SDI1_Characterization of organelles bidirectional transport: a key to understand
intracellular traffic
De Rossi MC
1
, Bruno L
2
, Wetzler D
1
, Sued M
3
, Rodriguez D
3
, Levi V
1

1
Departamento de Qumica Biolgica, FCEN-UBA-IQUIBICEN-CONICET
2
Departamento de Fsica, FCEN-UBA-IFIBA-CONICET
3
Instituto de Clculo, FCEN-UBA-CONICET

Cell survival and maintenance of several biological functions depend on an effective intracellular
transport. Molecular motors transport a wide variety of cellular cargoes positioning them in the cytoplasm
with high spatialtemporal precision. These proteins bind to specific cargoes and step along cytoskeletal
filaments using energy provided by ATP hydrolysis. In particular, microtubule-dependent-motors, dynein
and kinesin, drive organelles bidirectionally; while dynein transports cargoes toward the minus end of the
microtubule, kinesin does it towards the plus end. Therefore, these opposite-polarity motors compete with
each other to determine the final direction of motion. In this work, we study the bidirectional transport of
fluorescent peroxisomes in Drosophila S2 cells. These cells can be induced to form long processes filled
with uniformly oriented microtubules when actin filaments are depolymerized. Using single particle
tracking, we registered peroxisomes trajectories inside the processes to study the mechanical properties
of molecular motors (dynein and kinesin-1) and their contribution to the bidirectional transport dynamics.
The velocity distribution obtained for both opposite-polarity-motors indicates that there is a heterogeneous
population of motors driving the organelles which may be associated with the number of active motors
attached to them. Furthermore, the run length distribution obtained for dynein differs substantially from
that of kinesin-1. These facts can be interpreted considering that the collective behavior of plus and
minus-directed motors are different.

SDI2_Observing the competition between two Ca
2+
dyes to study the interaction between
geometry and dynamics during intracellular Ca
2+
signals.
Piegari, E.,Lpez, L. and Ponce Dawson, S.
Departamento de Fsica (FCEN-UBA) and IFIBA (CONICET-UBA)

Ca
2+
signaling is ubiquitous across cell types. Ca
2+
liberation through inositol 1,4,5-trisphosphate
receptors (IP
3
Rs) is a key component of the Ca
2+
signaling toolkit. The specificity and universality of
these signals rely on the variety of spatio-temporal patterns of the intracellular Ca
2+
concentration. Various
factors shape these patterns, among them, the receptors kinetics over which Ca
2+
plays an inhibitory and
an activating role, the heterogeneous distribution of IP
3
Rs and the Ca
2+
removal mechanisms. Among the
latter, Ca
2+
buffering by large molecules is the fastest. It has been studied introducing exogenous buffers
in the cells and observing the signals using a Ca
2+
dye. It has been observed that buffers with the same
dissociation constant but diverse kinetics modulate the signals differently because of the different ways in
which they interfere with the communication between channels that are more or less distant. In this work
we study how these different effects may arise combining the observation with two dyes of IP
3
R-mediated
Ca
2+
signals and extensive numerical simulations of a model of the intracellular Ca
2+
dynamics. For the
experiments we use a multi-spectral confocal microscope and two dyes (Fluo4 and Rhod2) that differ in
their binding kinetics and emission spectra. In this way we observe calcium signals with Fluo4 and Rhod2
simultaneously, focusing on localized signals called puffs. From a characterization of their properties we
conclude that the slow dye, Rhod2, has a similar effect to that of the slow buffer, EGTA, on the
distribution of the calcium-bound Fluo4 concentration. In particular, the latter increases in the presence of
Rhod2, for a certain range of Rhod2 concentrations. Inspection of the calcium-bound Rhod2
concentration allows us to study possible causes for this behavior.
SAB 2013 SDI
105

SDI3_FRAP Analysis: mechanical forces alter zyxin binding kinetics within focal
adhesions of living cells
Bianchi, M.
1,2,
*, Sigaut, L.
1,2,3
, von Bilderling, C.
1,2
, Gastaldi, L.
1
and Pietrasanta, L.I.
1,2,3

1
Centro de Microscopas Avanzadas, FCEN, UBA,
2
Departamento de Fsica, FCEN, UBA
3
Consejo
Nacional de Investigaciones Cientficas y Tcnicas (CONICET), Buenos Aires, Argentina.
*mbianchi@df.uba.ar
Focal adhesions (FAs) are large, multiprotein complexes that provide a mechanical link between the
cytoskeletal contractile machinery and the extracellular matrix, and are involved in cell proliferation,
migration and motility [1]. FAs exhibit mechanosensitive properties: in the presence of a mechanical
stimulus, FAs are dynamically assembled and disassembled. To investigate the influence of mechanical
forces on the molecular binding kinetics of focal adhesion proteins, we use an approach based on
Fluorescence Recovery After Photobleaching (FRAP) [2]. In this work, we study the kinetics of the FA
protein zyxin - a suggested mechanosensitive protein - tagged with green fluorescent protein (GFP) and
expressed in epithelial mammalian living cells (HC11), in response to a mechanical stretch. Controlled
and repeatable mechanical stimuli were performed using a stretching device [3] that allows both live
fluorescence imaging and mechanical equibiaxial stretching of the cells cultured on silicone elastic
membranes. From FRAP recovery curves, assuming that the binding kinetics is much slower than the
diffusion time, we estimate the dissociation constant and fraction mobile of the protein.
Acknowledgements: This work was financed by ANPCyT and UBA.
1- Shemesh, T. et al., PNAS, vol. 102, No. 35 (2005), p. 12383.
2- 2- Lele, T. et al. MCB, vol.1, no.3 (2004) p. 181. 3- Quaglino, A. et al. BMC Cell Biology, vol. 10
(2009) p.55.

SDI4_Cellular mechanotransduction: use of RNA interference technique to study focal
adhesion proteins
Gastaldi L. M.
1
*, Bianchi M.
1
, von Bilderling C.
1,2,4
, Sigaut L.
1,2,3
and Pietrasanta L. I.
1,2,3

1
Centro de Microscopas Avanzadas y
2
Departamento de Fsica, FCEyN, UBA, Argentina.
3
Consejo
Nacional de Investigaciones Cientficas y Tcnicas (CONICET), Argentina.
4
IFIBA, CONICET, Argentina.
*lgastaldi@df.uba.ar.
Cell adhesion to the extracellular matrix involves multiple components that are dynamically interacting
with each other (focal adhesions, FA). Cells sense the extracellular environment using adhesion
receptors (integrins) linked to the intracellular actin cytoskeleton through a large repertoire of
mechanosensitive and signaling components in their cytoplasmic scaffold, including cytoskeletal and
adaptor proteins
1
(such as actin, vinculin, paxillin, zyxin, Talin, a-actinin) and enzymes (such us focal-
adhesion kinase). The presence of a mechanical stimulus, activates signal transduction pathways
involving the adhesion components, which results in cascading events. These ultimately affect protein-
protein interactions allowing self-assembly and/or remodeling of the adhesion unit
2
. We used RNA
interference technique to knockdown individual FA proteins to study the effect on composition, dynamic
and molecular interactions of the whole set of adhesion proteins within FA of a living cell. This approach,
combined with mechanical stretching
3
of the cells cultured on silicone elastic membranes, help us to
dilucidate new interactions involved in the mechanotransduction process.
Acknowledgements. UBA and ANPCyT. L.G. has a fellowship from ANPCyT, C. v. B. and M. B. have
fellowships from CONICET.
1
Zaidel-Bar et al., Nat Cell Biol. 2007 9(8):857.
2
Chen C. S., J. Cell Sci. 2009, 121:3285.
3
Quaglino, A. et
al., BMC CellBiology 2009 10:55.
SDI SAB 2013
106

SDI5_Manganese (II) speciation in intact cells of Deinococcus radiodurans
determined by High Magnetic-Field EPR

Bruch MB, Un S and Tabares LC.

Service de Bionergtique, Biologie Structurale et Mcanismes, CEA-Saclay, France.
High magnetic-field high-frequency electron paramagnetic resonance techniques were used to measure
in situ Mn(II) speciation in Deinococcus radiodurans, a radiation resistant bacteria capable of
accumulating high concentrations of Mn(II) [1]. It was possible to identify and quantify the evolution of
Mn(II) species in intact cells at various stages of growth. Aside from water, 95 GHz high-field electron-
nuclear double resonance showed that the Mn(II) ions are bound to histidines, glutamates and phosphate
groups, mostly from fructose-1,6-bisphosphate but also inorganic phosphates and nucleotides. During
stationary growth phase, 285 GHz continuous-wave EPR measurements showed that the majority of the
cellular Mn(II) in D. radiodurans is bound to a yet unidentified protein and superoxide dismutase. This
manganese distribution is affected by the growth conditions but it appears to be not modified by gamma
irradiation.
We have extended this studies to other organisms and found that this approach is general and
establishes a method for studying Mn(II) speciation and homeostasis in intact cells.
1- Tabares LC and Un S. J. Biol. Chem. 2013, 288:5050-5.



SDI6_FCS experiments to infer calcium dye properties.
Villarruel C, Sigaut L, Ponce ML and Ponce Dawson S
Departamento de Fsica and IFIBA (CONICET), FCEyN-UBA, Ciudad Universitaria, Pabelln I, (1428)
Buenos Aires, Argentina.
Optical techniques provide a relatively non-invasive means to study intracellular calcium signals. Although
the fluorescence amplitude increases with the free calcium concentration, it is only linearly related to the
calcium-bound dye concentration. Therefore, it is key to know the reaction rates and diffusion coefficients
of the dyes to extract quantitative information on the free calcium concentration that underlies the signals
observed. In this work we show how Fluorescence Correlation Spectroscopy (FCS) experiments can be
used to estimate these parameters from fits to the autocorrelation function that is obtained from the data.
We first present an analytic approximation to this function that is valid for a fast calcium-dye reaction.
We then present the results of numerical simulations with which we analyze the limits of applicability of
this approximation and the errors introduced in the estimation of some parameters when the reaction is
not fast enough. We subsequently discuss briefly the results of its application to FCS experiments
performed in solution using calcium and a dye. Finally, we show the results of experiments performed in
oocytes with which we determine the factor by which the coefficients estimated in solution should be
multiplied to obtain those that hold in the cytoplasm.

SAB 2013 SDI
107

SDI7_Transcription factor dynamics in living mouse embryos and stem cells revealed by
Monte Carlo simulations and fluorescence correlation spectroscopy

Angiolini, Juan F.; Kaur, Gurpreet; Mocskos, Esteban; Bruno, Luciana ; Plachta, Nicols*; Levi,
Valeria*.
Departamento de Qumica Biolgica, FCEN, UBA. IQUIBICEN-CONICET. European Molecular Biology
Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute, Monash University, Australia.
Departamento de Computacin, FCEN-UBA.CSC-CONICET Departamento de Fsica. FCEN-UBA.*
Equal contribution

Transcription factors (TFs) control cell differentiation during embryonic development, yet their diffusion
dynamics in vivo remain poorly understood.
TF diffusion has recently been measured in living mouse embryos using fluorescence correlation
spectroscopy (FCS) (Kaur et al., Nat Comm 4, 1637, 2013). Here, we developed a model considering that
TF diffuses in the nucleus and binds to DNA with different affinities. Moreover, we run Monte Carlo and
FCS simulations to confirm the model predictions. Our findings reveal two populations of DNA binding
sites with different TF affinities.
By using the model proposed and a global fitting routine, we analyzed the FCS data of the TF Oct4 in
embryonic stem cells and recovered the fractions of this TF bound to different sites. Additionally, we
determined the diffusion coefficient, k
on
*
and k
off
values for each binding population. Finally, we analyzed
FCS data obtained in the presence of drugs that modify chromatin condensation and using an Oct4
mutant unable to bind DNA. Our model provides a biophysical explanation of TF dynamics a complex
mammalian in vivo system.

SDI8_Microtubule organization in living cells, using a new single filament tracking
routine
Pallavicini, C
1
; Levi, V
2,4
; Wetzler, D. E.
2,4
; Angiolini, J. F.
2
; Benseor, L.
3,4
; Despsito, M. A.
1,4
and
Bruno, L.
1,4

1
Departamento de Fsica, FCEyN, UBA, Argentina;
2
Departamento de Qumica Biolgica, FCEyN, UBA,
Argentina
3
Fundacin Instituto Leloir, IIBBA-CONICET, Buenos Aires, Argentina
4
Consejo Nacional de
Investigaciones Cientficas y Tcnicas, Argentina.
The cytoskeleton is involved in numerous cellular processes and provides the tracks for transport driven
by molecular motors. Hence, the quantification of the mechanical behavior of cytoskeletal filaments is
essential for understanding cell mechanics and organization. Tracking individual fluorescent filaments in
living cells is extremely complex. We introduce a new tracking algorithm that allows recovering the
coordinates of microtubule (MT) segments in living cells with 5-10nm precision. Using this algorithm we
studied the curvature distributions of fluorescent MT in living cells. Fourier analysis of microtubule shapes,
revealed that the curvatures followed a thermal-like distribution as previously reported with an effective
persistence length (lp*) of ~15m, significantly smaller than that measured in vitro. We found
depolymerization of actin or intermediate filaments decreased lp* while expression of the microtubule-
associated protein XTP increased it. We also recovered trajectories of microtubule segments in actin or
intermediate filament depleted cells, and observed a significant increase of their motion with respect to
untreated cells showing that these filaments contribute to overall organization of the microtubule network.
Moreover, the analysis of trajectories of MT segments in untreated cells showed that these filaments
presented slower but more directional motion in the cortex than in the perinuclear region. We also could
study the correlation between organelle motion and MT dynamics.

Nuevas tcnicas en biofsica (New techniques in biophysics) SAB 2013
108

NTB1_Studying interfaces and protein conformation with a photochemical probe
Gmez, G.E.; Monti, J.L.E.; Bernar, E.M. and Delfino, J.M.
IQUIFIB (UBA-CONICET), Junn 956, (C1113AAD), Buenos Aires, Argentina.
Diazirine (DZN) is a photoreactive gas similar to water in size and shape. Upon photolysis it generates
methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds.
Thus, chemical modification (methylation) depends primarily on the solvent accessible surface area
(SASA) of the polypeptide chain.
3
H-DZN was used in our laboratory for investigating protein folding and
for mapping interfaces in protein complexes (1-3). More recently, we explored the feasibility of DZN
labeling coupled to detection by mass spectrometry (MS, 4). Here, we applied this technique for studying
interfaces using the complex calmodulin (CaM)melittin (Mel) as a model. We observed that the extent of
methylation (EM) of each complexed partner decreases, in correlation with the expected decrement in
SASA due to complex formation. Additionally, we probed the increment in SASA experienced by CaM in
the presence of Ca
2+
. Finally, we are providing evidence of site-specific labelling of CaM, showing
differences due to (i) the CaM-Mel complex formation or (ii) the conformational change triggered by
calcium binding. This method -which relies on a novel application of MS-, allows a straightforward
measurement of SASA.
1- Ureta DB et al. Biochemistry (2007) 46, 14567-14577
2- Craig PO et al. J. Mol. Biol. (2009) 394, 982993.
3- Gmez GE et al. Protein Sci. (2006) 15, 744-752.
4- Gmez GE et al. J Am Soc Mass Spectrom. (2012) 23, 30-42.


NTB2_Use of a solvent mimetic probe in the study of protein conformation
Ainciart N, Gmez GE, Delfino JM.
IQUIFIB (UBA-CONICET), Junn 956, (C1113AAD), Buenos Aires, Argentina.
In proteins, biological function is intimately related to the three dimensional structure. The accessible
surface area (SASA) of the polypeptide chain is a key parameter to understand the architecture of a
protein, but is not directly measurable in an experimental fashion. Diazirine (DZN) is a photoreactive gas
similar in size to water. On photolysis it generates methylene carbene (:CH
2
) which reacts unselectively
with its molecular cage, inserting even into C-H bonds. The absence of chemical specificity of :CH
2
and
the minimal size of the precursor (DZN) makes it possible for the first time to obtain an experimental
estimate of SASA. DZN was used in our laboratory to probe the topography of the surface and inner
space in proteins (1-3). Recently, the sensitivity and high resolution of mass spectrometry techniques
allowed us to derive a quantitative signal proportional to the extent of modification (EM) of the sample (4).
E. coli thioredoxin (TRX) is a protein extensively used as a model to study conformational changes. In this
work, we analyzed the differences between TRX wt and the mutant L103A, which have an increased
cavity size. In addition, enzymatic fragmentation of these proteins allows us to locate the EM along the
amino acid sequence, enabling the construction of a map of solvent accessibility.
1- Craig PO et al. Protein Sci. (2002) 11, 1353-1366
2- Craig PO et al. J Mol Biol (2009) 394, 982-993
3- Gmez GE et al. Protein Sci. (2006) 15, 744-752
4- Gmez GE et al. J Am Soc Mass Spectrom. (2012) 23, 30-42.

SAB 2013 NTB
109

NTB3_Study of physicochemical and flow behavior properties of pectins extracted from
the tomato industry.
Matas M. Alancay
1
, Manuel O. Lobo
2
& Laura B. Iturriaga
1

(1)CITSE-CONICET-Universidad Nacional de Santiago del Estero. Laboratorios Centrales
matiasalancay@yahoo.com
(2)Facultad de Ingeniera-Universidad Nacional de Jujuy.

Pectin is responsible of the texture of fruit and plants due to its ability cementing. The Food industry uses
pectin as a gelling forming and/or thickening agent. The by-product from the tomatoes industry (BTI)
represents an alternative source for commercial pectins. The objective was to determine in pectins
extracted from BTI (PBTI) by acid hydrolysis, high methoxyl (HM) and low methoxyl amidated (LMA)
commercial pectins: a) intrinsic viscosity [] of dispersed samples in NaCl 50 mM; b) pectin solubility at
pH 4,5 and 6,5, c) degree of esterification (DE%) and flow properties of aqueous solutions of pectins at
pH 4,5;6,5 and natural pH. [n] showed the following increasing order: HM, PBTI and LMA. Solubility at pH
4,5 and 6,5 were higher than 70 (g/100g) in both cases and for all pectins. The DE% at natural pH and pH
6,5 were 76,8, 58,3 and 55,9% and 21,22, 27,89 and 22,52% for HM, PBTI and LMA respectively,
reflecting the effect of pH. Intermediate values were obtained at pH 4,5. The flow behavior of the samples
to different pH was fitted to Ostwald-de Weale model. The consistency coefficient (K) was higher for
PBTI, it didnt show changes for LMA, decreased with the increase of pH in the HM and PBTI and would
be associated with the change of DE% of pectins at different pH. Values of flow behavior index (n)
showed that PBTI at pH 4,5 and 6,5, were more pseudoplastic than HM and LMA. The results present
physicochemical and flow promising properties for PBTI under the conditions of this study.
Acknowledgements: CITSE-CONICET

NTB4_A combined AFM-Fluorescence experiment: adhesion proteins recruitment in
response to a local and functional mechanical stimulus
von Bilderling C.
1,3,5
*, Caldarola M.
2,3
, Bragas A. V.
2,4,5
and Pietrasanta L. I.
1,3,4

1
Centro de Microscopas Avanzadas,
2
Laboratorio de Electrnica Cuntica y
3
Departamento de Fsica,
FCEyN, UBA, Argentina.
4
Consejo Nacional de Investigaciones Cientficas y Tcnicas (CONICET),
Argentina.
5
IFIBA, CONICET, Argentina. *catalina@df.uba.ar.
Mechanical forces play an important role in the organization, growth, maturation and function of living
tissues [1]. At the cellular level, many of the biological responses to external forces originate at two types
of specialized structures: focal adhesions and adherent junctions. In order to explore the dynamic
regulation of adhesive contacts, we are focused on the relationship between local force applied to the cell
and the assembly, mechanics and dynamics of focal adhesions (FA). The goal of our work is to study the
dynamic response of FA proteins when a precise and local force is applied. In this context, we integrate
Fluorescence Microscopy and Atomic Force Microscopy (AFM) in a new combined microscope [2] to
investigate the recruitment of adhesion proteins in response to an applied local and functional stimulus.
With this novel combined microscope we studied the temporal evolution of the fluorescence from fusion
GFP-FAs proteins (FAK, vinculin and zyxin), in response to the mechanical stimulus applied with a
fibronectin functionalized AFM force-probe. We were able to observe the remodeling of mature FAs and
the recruitment of FAK and vinculin to form nascent FAs in response to the locally applied nN forces.
Acknowledgements: UBA and ANPCyT. C. v. B. and M. C. have fellowships from CONICET.
[1] Chen C. S., J. Cell Sci. 2009, 121:3285. [2] Caldarola M. et al., Frontiers in Optics, Rochester, NY,
October 14, 2012, Microscopy and OCT III (FTu1C).
NTB SAB 2013
110

NTB5_Design of sensors to measure macromolecular crowding in vivo
Labanda, M. S.
1
, Craig, P. O.
2
, Caramelo, J. J.
1
1
Instituto de Investigaciones Bioqumicas de Buenos Aires (IIBA-CONICET) y Fundacin Instituto Leloir,
Av. Patricias Argentinas 435, Capital Federal C1405BWE
2
instituto de Qumica y Fisicoqumica
Biolgicas (IQUIFIB-UBA/CONICET), Junin 956, Capital Federal 1113
The interior of cells is characterized by a high content of macromolecules which occupy between 20 and
40% of the total volume
1
. Due to the mutual impenetrability of particles, this volume fraction is unavailable
to other molecules, producing a steric repulsion that generates important kinetic and thermodynamic
consequences on processes occurring in vivo
2
. This makes macromolecular crowding a physiological
parameter of great relevance that should be considered during in vitro experiments. The aim of this work
is to develop a probe to measure macromolecular crowding in vivo. We started from a chimeric protein
consisting of two fluorescent proteins (CFP and YFP) linked by a natively unfolded linker, in which
intramolecular fluorescence resonance energy transfer (FRET) can be induced
3
. To analyze the effects
of crowding on FRET efficiency of the protein, we measured fluorescence spectra of the protein in media
with increasing concentrations of PEG 8000. Interestingly, FRET efficiency increases as PEG
concentration increases, showing a sharp and cooperative change. In order to understand these
observations, we performed coarse grained molecular dynamics simulations of the protein at various
fractions of volume occupied by an inert crowding agent. The analysis of the trajectories shows that the
average distance between chromophores decreases as crowding level increases. These results suggest
that in crowded conditions the protein adopts compact conformations, decreasing the distance between
donor and acceptor, and hence, increasing FRET.

1- Zimmerman S.B. J. Mol. Biol. (1991) 599-620.
2- Zhou H. Annu. Rev. Biophys. (2008) 375-397.
3- Miyawaki A. Nature (1997) 882-887.

NTB6_Traction Force Microscopy: A tool for quantifying cell-matrix interaction
lvarez, YD
1,4
*, Tarkowski, N
2
, Tvez, N
2
, Sigaut, L
1,2,3
, Bianchi, M
1
, Stefani, F
2,3,4
and Pietrasanta, L I.
1,2,3

1
Centro de Microscopas Avanzadas, FCEyN, UBA.
2
Departamento de Fsica, FCEyN, UBA.
3
CONICET.
4
CIBION.*ayanina79@gmail.com

Cell exert traction forces on the extracellular matrix (ECM) to which they are adhered through the
formation of focal adhesion (FAs)
1
. Spatial-temporal regulation of traction forces is crucial in cell
adhesion, migration, cellular division and remodeling of the ECM. For quantitative measurements of the
direction and magnitude of cellular traction force are required biophysical techniques. Traction Force
Microscopy (TFM)
2
is a unique tool to assess the changes in the average traction force exerted by
individual cells. TFM utilizes elastic substrates and optical microscopy to track substrate surface
displacements through the spatial correlation of fluorescent particles embedded in the substrate. In this
work, we present the results obtained for epithelial mammalian living cells (HC11) cultured on fibronectin-
coated polyacrylamide gel sustrates. We were able to record cell-induced substrate displacements by
using a digital image correlation algorithm
3
, which allowed us to quantify traction forces.

Acknowledgments: UBA and ANPCyT. Y.A. and M.B. have fellowships from CONICET.


1
Baker, E. et al., Journal of Biomechanics, 43: 3844, 2010.
2
Dembo, M et al. Biophysical Journal
70:2008-2022 (1996).
3
Danowski, B.A. et al. Cell Biol.118:14111420 (1992).

SAB 2013 Biofsica: Aplicaciones biotecnolgicas (Biophysics: Biotechnological applications )
111

BBA1_Cytotoxic effects of light-activated Merocyanine 540 products (pMC540) for PDT
tumor therapy
Calzetta, N.L.; Farinola, J.; Maranzana, E.S.B.; Alonso, S. del V.
Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
Photodynamic therapy (PDT) usually involves the administration of a photoactive compound to the
subject to be treated followed by exposing the specific target location or target organ of the subject to
light. The potential of various photoactive compounds as antiseptic, antibacterial, antiviral, and antitumor
agents is well established. The ability of these compounds to become excited on exposure to light has
been exploited in vitro and in vivo [1]. However, simultaneous exposure of the target to the photoactive
agent and to light is a prerequisite for photodynamic action, thereby limiting the application of PDT to
accessible targets, e. g., localized solid tumors to which the light could easily be delivered. To overcome
this limitation, the pre-activation of photoactive compounds with light prior to their use in biological targets
is an alternative strategy [2]. This process involves controlled exposure of photoactive compounds to light
prior to their use, resulting in the formation of heretofore unknown photoproducts. The biological activity of
pre-activated merocyanine 540 (pMC540) has been documented. The results of these studies showed
that pMC540 was cytotoxic to certain tumor cell lines and enveloped viruses [2-4].
The present study was carried out to obtain MC540 products and evaluate the in vitro toxicity of pMC540
and compare structure-function to MC540. We report the results of investigation conducted to study the
toxicity of pMC540 in breast tumor cell line as well its half-time to evaluate if pMC540 retains its
therapeutic activity subsequent to photoactivation.
1- Gulliya K.S., Pervaiz S. (1989) Blood 73:1059; 2- Gulliya K.S.; Chanh T.; Newman J.; Pervalz S.;
Matthews J.L. (1990) Eur J Cancer 26:5; 3- Chanh T.C.; Allan J.S.; Pervaiz S.; et al (1992) J Acquired
Immune Defic Syndr 5:188; 4- Anderson G.S.; Miyagi K.; Sampson R.W.; Sieber F. (2002) J Photochem
Photobiol 68:101


BBA2_BSA/GFP hybrid nanoparticle functionalization
Farinola J., Grasselli M. and Alonso S. del V.
Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
Drug delivery has focused on providing new pharmacokinetic characteristics to biologically active
compounds since 1950
1
. A key aspect of this process is to generate complexes capable of targeting a
specific tissue in the body, is for this reason that nanoparticle (NP) functionalization is crucial for the
generation of its targeting properties
2
.
Our research group has developed a BSA-based NP using ionizing radiation so that the use of
interchanging reagents such as formaldehyde and bromide cyanogens is avoided
3
. In this new project we
aimed to incorporate to the NP synthesis process surface ligands of protein nature, so firstly we proposed
to attach GFP as a component of the NP in order to assess the molar relationship between BSA and
other protein ligands and easily measure the new characteristics through fluorescence spectroscopy. We
have optimized the purification of the NPs from the rest of the synthesis mixture using ultra-filtration or
ultra-centrifugation, so that the NPs have a more similar weight in between. We have also achieved NPs
with GFP attached to its surface via a couple of methods and demonstrated that it retains some of its
fluorescence characteristics. The NPs size was determined using Dynamic Light Scattering and we also
show that the proteins, as building blocks of the NPs, preserve its native conformation analyzing its
circular dicroism spectra.
Taking into account this results we expect to develop a BSA-based NP with selected ligands that will
provide a targeting function to the NP.
1- Rosen, H. & Abribat, T. Nature reviews. Drug discovery 4, 3815 (2005); 2- Hrkach, J. et al. Science
translational medicine 4, 128ra39 (2012); 3- Soto Espinoza, S. L., et al. Radiation Physics and Chemistry
81, 14171421 (2012).
BBA SAB 2013
112

BBA3_Study of pH-response of fluorescent nanoporous polymeric membranes
S.L. Soto Espinoza,
1
G. Esposito
1
, Claudia Albeirtman
2
y M. Grasselli
1

1)
Laboratorio de Materiales Biotecnolgicos, UNQ -IMBICE/ CONICET, Argentina.
2)
Gerencia de Investigacin y Aplicaciones, TANDAR-CNEA, Argentina
ssoto@unq.edu.ar
Our goal is to develop pH-responsible nanoporous membranes by tuning the chemical and physical
properties and explore their potential applications such as sensor devices.
Fluorescent track-etched polyethylene terephthalate (PET) membranes were synthesized from PET foils
which were swift-heavy-ion irradiated. After alkaline etched, membranes were chemically modified by
remaining-radical grafting technique and immobilized fluorescent molecule (Fluorescein and Green
Fluorescent Protein (GFP)).
In the present work we analysed the behaviour of fluorescent membranes to different environmental pH
conditions. The response was measured by direct fluorescence onto the membrane by Nanodrop
spectrofluorometer. Different fluorescent behaviours were found for GFP- and fluorescein-modified
membranes in comparison to free molecules.
S.L.S.E. thanks the ANPCyT for the fellowship. M.G. is researcher from CONICET. This work was
supported by grants from Universidad Nacional de Quilmes, IAEA and MINCyT.


BBA4_Lyophilized albumin-based nanoparticle carrier. Biophysical characterization for
pulmonary delivery.
Siri M.
a
, Fernndez Ruocco J.
a
, Risso V.
c
, Chiaramoni, N.
a
, Grasselli M.
b
, and Alonso S del V.
a
LBM
a
, LaMaBio
b
, LEPP
c
, UNQ-GBEyB IMBICE-CONICET, Quilmes, Buenos Aires

The main objective of this study was to investigate the properties of the lyophilized preparation of water-
soluble albumin nanoparticle (BSAn) as protein carrier of the photosensitive marker Merocyanine (MC
540). Nanoparticles were prepared by -irradiation of bovine serum albumin (BSA) in ethanol 40% (v/v)
1
.
The lyophilized albumin nanoparticle (BSAn-lyo) was characterized as potential delivery system for lung
therapy. Size and Z potential of BSAn-lyo were investigated by Light Scattering and Z Potential, along
with 4th derivative UV-Vis spectroscopy, pH changes and Sudlow site affinity study for MC540.
Conservation of structure-function characteristics were studied onto BSAn-lyo by MC540 interaction.
Before lyophilization the BSAn were spherical in shape and had a smooth surface c.a. 20-40 nm and Z
potential of -10 mV. However, after lyophilization BSAn-lyo solubilize as higher particles in the range of
100 and 1000 nm and Z potential between 15 mV (pH=2) and -15 mV (pH=9). BSAn-lyo has a 50 fold
increase in size respect to non-lyophilized one. If MC540 concentration is held constant at 7M and
BSAn-lyo has a variable concentration range from 1.25 to 30 M an isosbestic point at 530 nm is
obtained while BSA point is shifted towards 536 nm.
The present study suggests that BSAn-lyo could be used as inhaled suspension for respiratory illness as
a potential pulmonary protein delivery system (PPDS), according to findings that confirm enhanced
stability of serum albumins upon binding MC540.
Acknowledgements: to CONICET, MINCyT-CAPES, UNQ
1. Soto Espinoza, S. L., et al. Radiation Physics and Chemistry 81, 14171421 (2012).
SAB 2013 BBA
113

BBA5_Light absorption measurements for explaining tumor cell killing efficiency in
photodynamic therapy
Etcheverry, ME
1
, Pasquale, MA
2
y Garavaglia, M
1,3

1
Departamento de Fsica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP)
2
Instituto de Investigaciones Fisicoqumicas Tericas y Aplicadas (INIFTA) (CCT CONICET La Plata,
UNLP y CIC)
3
Centro de Investigaciones pticas (CIOp) (CCT CONICET La Plata, y CIC)

In this contribution we present results from a LED lamp at 637 nm and with an average width of 8 nm,
used for PDT in our previous works. The efficiency of light absorption for solutions with different
photosensitizer concentrations (Themoporfin) and light intensity was evaluated with and
spectrophotometer (Ocean Optics HR2000 + ES) coupled to an optical fiber and from photometric data
employing an electronic eye mounted in an optical bench.
Results were consistent with the improve in PDT efficiency obtained from HeLa cells (human cervical
carcinoma) cultures treated with increasing photosensitizer concentrations (0.05 g/ml, 0.25 g/ml and
1g/ml) and illuminating times (3min, 6min and 12 min) times. The absorption spectrum for temoporfirin
exhibited a characteristic peak centered at 650nm. Spectrophotometric measurements at the employed
conditions in theses work, showed that near the region of the absorption peak, the signal exhibited
significant differences when using two concentrations and four light intensities, the latter obtained varying
the distance of the lamp to the detector. Indeed, the higher the drug concentration the lower the
spectrophotometric record, which would correspond to a greater number of photons absorbed by HeLa
cells.


BBA6_Binding kinetics between soluble -Gal and anti--Gal immobilized on chemically
nanopatterned surfaces with fractal topography.
Clop, PD
a
, Marchesini GR
b
, and Perillo, MA
a
.
a
Instituto de Investigaciones Biolgicas y Tecnolgicas (IIByT),CONICET-UNC, Crdoba, Argentina.
b.
JRC IHCP Biosensing on nanostructures, Ispra (VA), Italy. e-mail: dclop@efn.uncor.edu
The present work was aimed at providing experimental support to the correlation between topographic
and kinetic dimensions of protein function we described previously. Through electronic and colloidal
nanolitographic techniques we produced surfaces exhibiting a fractal-like pattern with a pre-determined
topographic dimension of domains capable to bind proteins in a covalent manner (Ab
Anti -Gal
). These
surfaces were used as sensors to reversibly bind -Gal and enabled the kinetic study of Ab
Anti -Gal
--Gal
complex formation by surface plasmon resonance (SPR) spectroscopy. Compared with the behavior of
control sensors (homogeneous topography), the Ag-Ab binding kinetics in sensors produced by
nanolithography showed higher capacity and broader dispersion of binding sites characterized by a more
diffuse attractor in the k
d
vs. K
d
phase space. Our results contributed to the development of a nano-
biosensor for -Gal, to a strategy to enhance the sensitivity of a SPR sensor and to endorse the cross-
talk between supramolecular structure and dynamics in heterogenous systems. Additionally, a catalytic
activity could be measured for the first time by a SPR based method where the insoluble product of a -
Gal catalyzed reaction interfered with the evanescent field on the sensors surface.
Acknowledgements: Work financed with grants from CONICET, Foncyt, SeCyT-UNC.


BBA SAB 2013
114

BBA7_Magnetic Fluid Hyperthermia for Bladder Cancer: A Preclinical Dosimetry Study
Oliveira,T.R.
1,2
, Stauffer,P.R.
2
, Lee,C.T.
2
, Landon,C.D.
2
, Etienne, W.
3
,McNerny, K. L.
4
,Mashal, A
4
,
Maccarini,P.F.
1
, Inman, B.
3
, Dewhirst, M.W.
2

1
Instituto de Fsica, Universidade de So Paulo, So Paulo, Brazil;
2
Department of Radiation Oncology,
Duke University Medical Center, Durham, NC, USA;
3
Division of Urology, Duke University Medical
Center, Durham, USA;
4
Actium Biosystems, Boulder, CO, USA.
Background: The high recurrence rate associated with the treatment of Bladder cancer has motivated the
search for more efficacious therapeutic approaches such as thermochemotherapy. Iron oxide
nanoparticles have been investigated as a method to deliver local hyperthermia. The objective of this
preclinical study was to investigate the feasibility of treating bladder cancer with magnetic fluid
hyperthermia (MFH) by analyzing the thermal dosimetry and biodistribution of nanoparticles in a rat
bladder model. Materials and Methods: The bladders of twenty-five female rats were instilled with
magnetite-based nanoparticles, and hyperthermia was induced using a novel small animal magnetic field
applicator (Actium Biosystems, Boulder, CO). We aimed to increase the bladder lumen temperature to
42
o
C in <10 min and maintain that temperature for 60 min. Temperatures were measured throughout the
rat with seven fiberoptic temperature probes (OpSens Technologies, Quebec). Results: Thermal
dosimetry data demonstrated our ability to raise and control the temperature of rat bladder lumen
>1C/min to a steady-state of 42
o
C with minimal heating of surrounding normal tissues.MRI scans
confirmed the homogenous nanoparticle distribution throughout the bladder. Conclusion: These data
demonstrate that our MFH system with magnetite-based nanoparticles provide well-localized heating of
rat bladder lumen with effective control of temperature in the bladder and minimal heating of surrounding
tissues.

BBA8_Mucilage gum (OpuntiaficusindicaL.Mill) stabilizes oil/water emulsions by
enhancing repulsion surface forces.
C. Quinzio
1
; M. A. Fras
2
, L. Iturriaga
1
.
1. Laboratorio de Ciencia y Tecnologa de Alimentos.(CITSE, UNSE-CONICET).
2. Laboratorio de Biointerfases y Sistemas Biomimticos. Centro de Investigacin y Transferencia, de
Santiago del Estero (CITSE, UNSE-CONICET).
Mucilage is a mucopolyscharide present in the tissue of Opuntiaficusindica L.Mill. which can be used as a
natural hydrocolloid to stabilize oil/water emulsions.
In this work, we determined the stabilizing properties of this mucilage by measuring the storage stability.
The volume of oil phase separated from the emulsion was recorded at various time intervals within 3
months. The stability was also determined by a centrifugation assay. The emulsion stability was
calculated as percentage: (remaining emulsion height /initial emulsion height) x100.
In comparison with guar gum, mucilage is more effective in isoviscous conditions. This denotes that due
to its amphiphilic character the mucilage may confer other properties to the system. For this reason, we
further analyzed the effect of this polysaccharideon the surface tension and the dipole potential of water
dispersions. The results show that the surface tension is reduced which explains the formation of oil
drops in the continuum water media covered by the polysaccharide. In addition, the dipole potential is
increased, which is interpreted as a consequence of the organization of the mucilage on the oil/water
interphase of the dispersed oil drops. The enhanced stability is explained as a result of the repulsion
forces due to the organization of dipoles and charges on the surface.

Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
SAB 2013 BBA
115

BBA9_Nanomechanical characterization of biointerfaces by Peak Force QNM


*
Diaz, C.
1,2,3
, Min, A.
1
, Pissinis, D.
1
, Schilardi, P.
1,3
, Pietrasanta, L.I.
2,3,4

1
Instituto de Investigaciones Fisicoqumicas Tericas y Aplicadas, CONICET -UNLP.
2
Centro de
Microscopas Avanzadas, FCEyN-UBA.
3
Consejo de Investigaciones Cientficas y Tcnicas, Argentina.
4
Departamento de Fsica, FCEyN-UBA. *cdiaz@df.uba.ar
The design, development and characterization of substrates with different physical, chemical and
mechanical properties are critical for the study of biologically active surfaces and biointerfaces
1
. These
kinds of devices involve the interaction of synthetic materials with biological systems and require a
complete understanding of the physical properties of molecules, composites and nanostructures. Since its
development, AFM is a powerful diagnostic and investigation tool. Nowadays, Peak Force QNM


2
allows
quantitative nanomechanical mapping of material properties, including modulus, adhesion and
dissipation, while simultaneously imaging sample topography at high resolution. In order to study cellular
mechanostransduction, we characterize interfacial arquitectures and micro-nano structured platforms
based on molecular recognition.
Acknoledgments: UBA, ANPCyT, UNLP. Min and Pissinis have fellowships from CONICET.

1- Agheli, H., J. Malmstrm, et al. "Nanostructured biointerfaces." Materials Science and Engineering: C
2006. 26(5-7): 911-917.
2- Bede Pittenger, Natalia Erina, Chanmin Su. Quantitative Mechanical Property Mapping at the
Nanoscale with PeakForce QNM. 2010. Veeco. Solutions for a nanoscale world.



BBA10_Atomic Force Spectroscopy of single adhesin on living Bordetella pertussiss
surface
L.Arnal
1
, Giovanni Longo
3
, Federico Castez
2
, Osvaldo M. Yantorno
1
, Sandor Kassas
3
and Maria E. Vela
2
.

1
INIFTA-CONICET-UNLP.Suc. 4 CC 16, (1900) La Plata Argentina
2
CINDEFI-CONICET-50 y 115 CP (1900). La Plata-Argentina.
3
IPSB, colePolytechniqueFdrale de Lausanne, Lausanne, Switzerland.

Pertussis is a humans respiratory tract disease caused by Bordetella pertussis (Bp), a Gram
negative bacterium. Recent publications indicate that Bp might grow as biofilm attached to the upper
respiratory tract as a mean to persist in the host. Filamentous haemaglutinin (FHA) is the mayor adhesin
of Bp and is involved in different steps of biofilm formation.
Here, by using Atomic Force Spectroscopy, we studied single interactions between purified FHA and
a specific antibody attached to Si
3
Ni
4
cantilevers and we also detected single FHA molecules in the
surface of living BpTohama I(reference strain) cells. A scan rate of 500nm/s was used for single cell
spectroscopy measurements. We used BpTohama living cells, several individual cells were mapped and
the recorded Force Volume (fv) images were analysed. Force interaction maps were built, showing
specific interactions on bacterial surface for wild type cells but not for FHA- strain (FHA defective mutant
strain used as control). The analysis of the results allowed us to observe that the localization on the
bacteriums surface of FHA proteins is not homogeneous and dynamic; proteins seem to be localized into
nanodomains or clusters of adhesins. This distribution into nanodomains could mean that the bacterium
clusters FHA in certain regions of the membrane reaching biggest and strongest domains of interactions
which could be advantageous for cell-substrate and cell- cell interactions during biofilm development.
Serra, D. O.et all. PLoS ONE 2011,28811.
Yersin et all, PNAS, 2003, 8736.
BBA SAB 2013
116

BBA11_Study of the adsorptive performance of novel foam-based chromatographic
columns.
Mirna L. Snchez
[1,2]
, Leandro Martnez
[1]
, Marcelo Fernndez-Lahore
[2]
and Mariano Grasselli
[1]

mi.sanchez@jacobs-university.de
[1]
Universidad Nacional de Quilmes - IMBICE (CONICET).
[2]
Jacobs University Bremen.
Modern biotechnology heavily depends on the availability of efficient processes, which have to generate
competitive products in terms of quality and cost. Most commonly, the purification of bioproducts is based
on chromatographic platforms. However, the low productivity imposed by diffusional limitations, the need
for multiple separation steps, and the requirement of extensive solids separation, drives to a technological
situation characterised by low global bioproduct yields.
1

In a previous work, we fabricated reticulated open-pore polyurethane foams supporting a functional
hydrogel layer. Preliminary studies showed that the adsorptive properties of these materials allow the
reversibly binding of proteins by ion exchange mechanisms.
Dynamic binding capacity tests (DBC) were performed employing Lysozyme (HEWL) on a cation
exchanger foam. DBC experiments were carried out as a function column length, compression degree
and flow velocity. Furthermore, bed hydrodynamics was studied via residence time experiments (Pe Nr)
and correlated to the observed amount of protein captured.
Experimental data indicated that optimum performance, as judged by Pe and DBC, was obtained for the
following conditions: 30 cm column length, non-compaction, and 210 cm/h linear flow rate. The novel
cation exchange foams show as promise matrix material for the direct capture and purification of
biotechnology-derived products.
Acknowledgements: M.L.S. thanks to CONICET and DAAD for the fellowship. M.G. is member of
CONICET.
1- DePalma A. Genetic Engineering News 29 (2009) 8

BBA12_Physical evidence justifying experimental biological results in Photodynamic
Therapy of Cancer
Etcheverry, ME
1
, Pasquale, MA
2
y Garavaglia, M
1,3

1
Departamento de Fsica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP)
2
Instituto de Investigaciones Fisicoqumicas Tericas y Aplicadas (INIFTA) (CCT CONICET La Plata,
UNLP y CIC)
3
Centro de Investigaciones pticas (CIOp) (CCT CONICET La Plata, y CIC)

In order to verify the efficiency of absorption of radiation emitted by an LED lamp at 637 nm with an
average width of 8 nm in certain concentrations of drug, in this paper we present results recorded with a
spectrophotometer (Ocean Optics HR2000 + ES) coupled to an optical fiber.
From preliminary results performed on cultured HeLa cells (human cervical carcinoma), using a
temoporfirin as photosensitizer and the lamp was obtained the proportion of cell death for different drug
concentrations (0.05 mg / ml, 0.25 mg / ml and 1g/ml) and illumination times (3min, 6min and 12min).
The absorption spectrum obtained for temoporfirin showed its characteristic peak centered at 650nm.
In order to justify the biological results, spectrophotometric measurements were performed which showed
that in the area of drug absorption differences are significant when using two concentrations and four light
intensities obtained varying the distance of the lamp to the detector. Indeed, the higher the drug
concentration is lower spectrophotometric record, which corresponds to a greater number of photons
absorbed by HeLa cells.

SAB 2013 BBA
117

BBA13_Adhesion Kinetics of Escherichia coli onto surface-modified HDPE: Looking for
Answers in DLVO theories.
Quiroga, F.Y, & Grasselli, M.
Laboratorio de Materiales Biotecnolgicos (LaMaBio), UNQ-IMBICE-CONICET, Quilmes, Argentina.

This work studies the interactions between recombinant Escherichia coli cells and high density
polyethylene (HDPE) surface-modified with different concentrations of cationic branches. The adhesion
kinetics of E. coli on cationic HDPE films was measured in situ by fluorescence detection of green
fluorescence protein (GFP) expressed by the bacteria attached to the film. The overall interactions
between cells and surface were determined by contact angle and zeta potential measurements and
quantified using the classical and extended Derjaguin-Landau-Verwey-Overbeek theories (DLVO and
xDLVO respectively) [1]. According to those theories, interfacial interaction between a colloid (bacteria)
and a surface in aqueous media correspond to Lifsithz.van der Waals interactions, Lewis acid-base
interactions, and electrostatic interactions [2]. The adhesion capacity of E. coli was found higher for those
polymers with the highest cationic and hydrophobic values.

Acknowledgements
FYQ thanks to CONICET for their fellowship. MG is a CONICET researcher.

1- M. Hermansson. Colloid and Surfaces B. 1999. 105
2- C. J. van Oss. Colloids and Surfaces B. 2007. 2


BBA14_Latex colored particles: synthesis and BSA absorption for potential applications
in immunochromatography
Femia, L*; Gonzalez, V.*; Minari, R.*; Alonso S. del V**; Gugliotta L.*
*INTEC (UNL-CONICET), Santa Fe, Argentina.
**Laboratorio de Biomembranas, Depto de Ciencia y Tecnologa, UNQ, Quilmes, Bs As, Argentina.
Colored latexes of polystyrene have acquired much interest in biological fields, since they are applicable
in many topics [1]. Several authors had reported the synthesis of these materials, and its interaction with
proteins [2]. There are several potential applications like immunochromatography, immunoagglutination
assays, particle designs for flux cytometry, protein-latex detection kits, etc. In view of the increasing
interest in the use of these particles, we have synthesized a new polymerizable colorant from basic
fuchsine (BF). The functionalized colorant was characterized by NMR, FTIR and ESI-mass spectrometry.
Latex colored particles were prepared with this color-monomer and with the non-functionalized colorant,
BF. The obtained particles were characterized in terms of size, color incorporation, conversion efficiency
in the synthesis process and protein adsorption (BSA). We have obtained purple colored latex particles
with a sized range from 80-110 nm, which were stable in the presence of emulsifier. The monomer
conversion was higher in the styrene/colored-monomer reaction than styrene/BF reaction. Color
incorporation was not significantly different but, color leakage was greater for polystyrene/BF system.
Acknowledgements: To CONICET, MinCyT, and UNL for the financial supports.
1. Piskin E, Tuncel A, Denizli A, Ayhan H (1994) J Biomat Sci Pol. 451.
2. Luck M, Pistel KF, Li YX, Blunk T, Muller RH, Kissel T (1998) J Control Release. 107.




BBA SAB 2013
118


Sab2013

119

ndice por autores / Author index


Abriata, L.A. BPA3 53
Adamo, H.P.A. TRC18 102
Aguayo, R. TRC15 101
Agudelo, W.A. BPA44, TMSB11 73, 86
Ainciart, N. NTB2 108
Alancay, M.M. NTB3 109
Alarcon, L. BLM15 36
Albeirtman, C. BBA3 112
Ale, N.M. BLM33 45
Alleva, K. TRC5, TRC6, TRC8, TRC10 96, 97, 98
Alonso de Armio, D.J. TMSB4, TMSB7 82, 84
Alonso, G.L. TMSB3 82
Alonso, S. del V.
BLM10, BLM17, BLM37, BLM40,
BBA1, BBA2, BBA4, BBA14
33, 37, 47, 48, 111,
112, 117
Altabef, A.B. BLM33, BLM34, TMSB5 45, 83
lvarez, R.M.S. BLM18, BLM19, TMSB4, TMSB7 37, 38, 82, 84
lvarez, Y.D. NTB6 110
Ambroggio, E. BLM8, BLM35 32, 46
Ambroggio, X. S4.1 23
Amodeo, G. TRC5, TRC6, TRC8 96, 97
Amundarain, M.J. TMSB13, TMSB22 87, 91
Andrade, X. BTE4 80
Angelani, C.R. BPA42 72
Angiolini, J.F. SDI7, SDI8 106
Antollini, S.S. BLM22, BLM23 39, 40
Appignanesi, G.A. BLM15, ENZ6 36, 76
Aptekmann, A. BPA1 52
Aramenda, P. F. P7 13
Arn, M. S2.3, BPA11, BPA30, BPA44 20, 57, 66, 73
Arcn, J.P. TMSB18 89
Argello, J.M. S1.4 18
Arias, J.M. BLM34, TMSB5 45, 83
Arnal, L. BBA10 115
Auzmendi, J.A. S3.2 21
Aveldao, M.I. BLM13, BLM22, BLM23 35, 39, 40
Avila, C.L. BPA25, BPA26, BPA27 64, 65
Ayub, N. TRC6 96
Azzoni, A. S5.2 25

SAB 2013
120

Bagatolli, L.A. P4 10
Baks, L. TRC11 99
Balbino, T. S5.2 25
Barbosa, L.R.S. BPA25, BPA26 64
Barbosa, S.C. BPA37 70
Barrantes, F.J. BLM22 39
Basaez, G. S2.2 19
Bazn, S. BPA16 59
Bea, E.A. BTE4 80
Bellomio, A. BLM19, BPA27, BPA36, BTE3 38, 65, 69, 80
Belluzo, B.S. BPA5, BPA7 53, 55
Beltramo, D. BLM46 51
Beltrandi, M. S2.1 19
Benedini, L. BLM12, BLM30 34, 43
Benseor, L. SDI8 107
Berberin, M.V. BLM42 49
Berlin, J.S. S3.4 22
Bernar, E.M. NTB1 108
Bertoncini, C. BPA35 69
Bianchi, M. SDI3, SDI4, NTB6 105, 110
Bisch,P.M. S2.4 20
Blackledge, M. S2.1 19
Blangy, S S2.1 19
Blocquel, D. S2.1 19
Bocco Gianello, M.D. BLM43 50
Boeris, V. BPA9 56
Boffi, A. BPA8 55
Bolean, M. BLM28 42
Bonafe, C.F.S. ENZ10 78
Bonamore, A. BPA8 55
Borioli, G. BLM44 50
Boron, I. BPA18 60
Bosich, W. TRC16 101
Bouchet, A.M. BLM39, ENZ2 48, 74
Bougis, K. BLM20 38
Bouzat, C. TRC14 100
Bragas, A.V. NTB4 109
Brandelli, A. BPA9 56
Brandenburg, K. BLM3 30
Bredeston, L.M. TRC16, TRC17, TRC19 101, 102, 103
Brito, T. TMSB9 85
Bruch, M.B. SDI5 106
Bruno, L. SDI1, SDI7, SDI8 104, 107
Sab2013

121

Bucci, H.A. BPA34 68
Burgos, I. S2.3, BPA11, BPA15 20, 57, 59
Bustillo, I. S2.2 19
Bustos, M. TMSB1, TMSB2 80
Cababie, L.A. ENZ5 76
Cabrera, I. BPA25 64
Calabr, V. BLM17, BLM40 37, 40
Caldarola, M. NTB4 109
Calzetta, N.L. BBA1 111
Capece, L. BPA18 60
Caramelo, J.J. BPA42, NTB5 72, 110
Carrasquel-Ursulaez, W. TRC15 101
Carrire, F. P2 8
Carnovale, C.E. MLB7 32
Carrer, D. BPA15 59
Carrizo, F. TRC11 99
Carrizo, M.E. BPA22, ENZ1 62, 74
Caruso, B. BLM4, BLM6, BLM35 30, 31, 46
Carvalho, R.V. S4.3 24
Castez, F. BBA10 115
Cattoni, D.I. S1.4 18
Cavalcanti, L. S5.2 25
Celej, M.S. BPA14, BPA23 58, 63
Centeno, M. TRC12 99
Ceoln, M. S5.4 26
Chaln, M.C. BTE3 80
Chaves, S. BPA27 65
Chehn, R. BPA26, BPA27 64, 65
Chemes, D.M. BLM18 37
Chaillou, L.L. ENZ3 75
Chiaramoni, N.S. BLM17, BLM37, BLM40, BBA4 37, 47, 48, 112
Chisari, L. BPA18 60
Ciancaglini, P. BLM28, BPA37, ENZ7 42, 70, 77
Cilli, E. BPA36, BPA37 69, 70
Ciocco Aloia, F. BLM42 49
Clop, E.M. ENZ10 78
Clop, P.D. BBA6 113
Colque, A. BLM1 29
Communi, G. S2.1 19
Contreras, G.F. TRC15 101
Cooper, A. S1.1 17
Crsico, B. S1.1, TMSB15 17, 88
Corradi, J. TRC14, TRC18 100, 102
SAB 2013
122

Correa, W. BLM3 30
Cossy Isasi, S. TMSB10 85
Costabel, M.D. TMSB13, TMSB15, TMSB22 87, 88, 91
Craig, P.O. BPA39, NTB5 71, 110
Crosetti, D. BLM7 32
Cunha, K.C. TMSB24 92
Curtino, J.A. BPA22, ENZ1 62, 74
Curto, L.M. BPA42 72
Cutro, A.C. BLM31, BLM32 44
Cybulski, L S1.2, BPA31 17, 67
Czysezon, N.A. TRC18 102
da Silva Sanches, P.R. BPA36 69
de Athayde Moncorvo
Collado, A. BLM25 41
de Azevedo Delou, J.M. TRC20 103
de La Torre, L. S5.2 25
de Mendoza, D. BPA31 67
De Rossi, M.C. SDI1 104
De Sautu, M. TRC3 95
Decca, M.B. S1.3, BPA12 18, 57
Defelipe, L.A. TMSB16, TMSB18 88, 89
Defonsi, E. BPA27 65
Del Boca, M. BLM44 50
Del Popolo, M.G. BLM42 49
Delfino, J.M. BPA32, BPA42, NTB1, NTB2 67, 72, 108
Despsito, M.A. SDI8 107
Dewhirst, M.W. BBA7 114
Di Lella, S. BPA17 60
Diaz, C. BBA9 115
Daz, S.B. BLM33, BLM34, TMSB5 45, 83
Diaz-Franulic, I S3.1 21
Disalvo, E. A.
BLM15, BLM31, BLM32, BLM36,
BLM39, ENZ2 36, 44, 46, 48, 74
Dodero, V.I.
BLM12, BLM30, BPA13, BPA14,
BPA24, BPA33, TMSB13
34, 43, 58, 63, 68,
87
Dodes Traian, M.M. BLM27, TRC7 42, 97
Dominighini, A. BLM7 32
Donnamaria, M.C. TMSB21 91
Dosnon, M. S2.1 19
Dumas, V.G. TMSB18 89
Dupuy, F. BLM45, BLM46 51
Durn, R. BPA38 70
Durand, E.S. BPA12 57
Elso-Berberin, G. TRC9 98
Sab2013

123

Emperador, A. TMSB26 93
Enoki, T.A. BLM41 49
Erales, J. S2.1 19
Ermcora, M. S2.3, S5.3, BPA2, BPA10, BPA11 20, 26, 52, 56, 57
Esersky, I. TRC19 103
Espada, R. BPA43 73
Espejo, F. BLM3 30
Espinosa, Y.R. BLM26 41
Esposito, G. BBA3 112
Estrin, D.A.
BPA8, BPA17, BPA18, TMSB4,
TMSB7, TMSB14 55, 60, 82, 84, 87
Etcheverry, M.E. BBA5, BBA12 112, 116
Etienne, W. BBA7 114
Falcn, V. BPA25 64
Fanani, M.L. BLM9, BLM12, BLM13, BLM23, BLM24 33, 34, 35, 40
Faraj, S.E. BPA28, BPA30 65, 66
Farinola, J. BBA1, BBA2 111
Favarolo, B. TRC19 103
Femia, L. BBA14 117
Fernandez Ruocco, M.J. BLM37, BBA4 47, 112
Fernndez, A.S. ENZ6 76
Fernndez, C.O. BPA35 69
Fernndez-Lahore, M. BBA11 116
Ferreira-Gomes, M. TRC1, TRC2, TRC3, TRC4, TRC12 94, 95, 99
Ferreiro, D.U. BPA43 73
Ferreyra, R.G. BPA2 52
Fidelio, G.
S2.3, BLM8, BLM35, BPA11, BPA38,
ENZ9
20, 32, 46, 57, 70,
78
Flores, S.S. ENZ4 75
Flores-Romero, H. S2.2 19
Folmer, A.P. BPA9 56
Franchini, G.R. S1.1 17
Fras, M.A. BLM31, BLM32, BLM36, BLM39, BBA8 44, 46, 48, 114
Frigini, E. BLM21 39
Fuentealba, J. TRC19 103
Futerman A. P6 12
Gaggiotti, M.C. BLM44 50
Galassi, V.V. BTE1 79
Galizia, L. TRC13 100
Gallea, J.I. BPA23 63
Galliano, S. MLB7 32
Gallo, M. S2.3, BPA11, BPA30, BPA44 20, 57, 66, 73
Galvn, A.E. BTE3 80
Gamarnik, A.V. BPA21, ENZ5 62, 76
SAB 2013
124

Garavaglia, M. BBA5, BBA12 113, 116
Garay, A.S. BLM29 43
Garbarino, E. TMSB6 83
Garca, G. BLM7 32
Garcia-Valero, J. S2.2 19
Gasperini, A. S5.2 25
Gastaldi, L. SDI3, SDI4 105
Gauto, D.F. TMSB18 89
Gebhard, L.G. BPA21, ENZ5 62, 76
Gennaro, A.M. BLM43 50
Gennis, R. BTE3 80
Gerbelli, B.B. BLM14, BLM20 35, 38
Germano, R. TMSB9 85
Giordani, C. BLM2 29
Giraldo, M. BLM2 29
Giudice, F. BLM9 33
Glisoni, R. TMSB17 89
Goldbaum, F.A. S5.1 25
Goldman, R.P. TRC10 98
Gmez Zavaglia, A. BLM33 45
Gmez, G.E. NTB1, NTB2 108
Gonalves da Silva, A.M.P.S. P6 12
Gonzlez Flecha, F.L.
S1.4, BLM27, BPA20, BPA29, BPA44,
TMSB11, TMSB12, TRC7, TRC16
18, 42, 61, 66, 73,
86, 97, 101
Gonzalez Montoro, M.A. BLM1 29
Gonzlez, C. TRC15 101
Gonzlez, D.A. TMSB3 82
Gonzlez, M.C. BPA22 62
Gonzalez, V. BBA14 117
Gonzlez-Lebrero, R.M.
BPA20, BPA21, BPA41, BPA44, ENZ5,
TMSB11, TMSB12
61, 62, 72, 73,76,
86
Gonzlez-Nilo, F. S3.1, TRC15 21, 101
Gonzalvez, J. BLM7 32
Granados, A. BLM5 31
Grasselli, M. BBA2, BBA3, BBA4, BBA11, BBA13 111, 112, 116, 117
Grasso, E.J. BPA4 53
Griesinger, C. BPA35 69
Grigera, J.R. BLM26, TMSB21 41, 91
Gualtieri, A.F. TMSB19 90
Gurin Aguilar, D.M. TMSB22 91
Gugliotta, L. BBA14 117
Guido, M.E. TMSB6 83
Habchi, J. S2.1 19
Hecht, J.P. TMSB19 90
Sab2013

125

Henriques, V.B. TMSB9 85
Heredia, V. BLM46 51
Herlax, V. TRC11 99
Herrera, F.E. BLM29, TMSB20 43, 90
Herrera, M.G. BPA14, BPA24, TMSB13 58, 63, 87
Hbarnter, C. BPA6 54
Hoylaerts, M.F. BLM28 42
Hoyos de Rossi, R. BML16 36
Hugo, M. TMSB14 87
Ibez-Shimabukuro, M. TMSB15 88
Incicco, J.J. BPA21, ENZ5 62, 76
Inda, M.E. BPA31 67
Ingaramo, M.C. BPA22 62
Inman, B. BBA7 114
Issoglio, F.M. ENZ1 74
Itri, R. BPA25, BPA26, BPA37 64, 70
Iturriaga, L.B. NTB3, BBA8 109, 114
Jozefkowicz, C. TRC6, TRC8, TRC10 96, 97, 98
Juarez, A.C. BLM18, BLM19 37, 38
Kassas, S. BBA10 115
Kaufman, S.B. BPA20, BPA21, ENZ5 61, 62, 76
Kaur, G. SDI7 107
Kennedy, M.W. S1.1 17
Kiffer-Moreira, T. BLM28 42
Klinke, S. S5.1 25
Kotsias, B.S. TRC13 100
Krause, R. BLM5 31
Labanda, M.S. NTB5 110
Lamy, M.T. BLM41 49
Landajuela, A. S2.2 19
Landeta, O. S2.2 19
Landon, C.D. BBA7 114
Lanterna, A. BLM5 31
Latorre, R. TRC15 101
Learte, S. BPA33 68
Lee, C.T. BBA7 114
Leiva, E.P.M. TMSB26 93
Lenilson, TMSB9 85
Leupen, S. TMSB2 81
Levalle, A. TRC17 102
Levi, V. BLM27, SDI1, SDI7, SDI8 42, 104, 107
Levstein, F. TMSB1 81
Lezama, G. TMSB4 82
SAB 2013
126

Lichtig, P.L. TMSB14 87
Lima, A. BPA38 70
Lins, R.D. S4.3, TMSB24, TMSB25 24, 92, 93
Llarrull, L.I. C1, BPA5, BPA7, ENZ8 14, 54, 55, 77
Llovera, R. BPA10 56
Lobo, M.O. NTB3 109
Lombardi, J. BPA9 56
London, N. S4.1 23
Lonez, C. BPA14 58
Longhi, S. S2.1 19
Longo, G. BBA10 115
Lopes da Silva,T.S. TRC20 103
Lopez, E.D. BPA34, TMSB16 68, 88
Lpez, L. SDI2 104
Luquita, A. MLB7 32
Maccarini, P.F. BBA7 114
Madoery, R. ENZ9 78
Maggio, B.
BLM5, BLM9, BLM12, BLM13, BLM16,
BLM23, BLM45, BLM46, BPA4
31, 33, 34, 35, 36,
40, 51, 53
Mangialavori, I.C. TRC1, TRC2, TRC3, TRC4, TRC7 94, 95, 97
Mangiarotti, A. BLM4, BLM6 30, 31
Manrique-Moreno, M. BLM3 30
Maranzana, E.S.B. BBA1 111
Marchesini, G.R. BBA6 113
Mariani, M.E. ENZ9 78
Mari, I. BLM30 43
Marino, G.I. TRC13 100
Marques Capella, M.A. TRC20 103
Marquez, M. TRC5 96
Marsanasco, M. BLM17, BLM40 37, 48
Marti, M.A. BPA8, BPA18, TMSB18 55, 60, 89
Martiarena, J. TRC2 94
Martin, O.A. BLM21 39
Martnez, L. BBA11 116
Martnez, S. S1.4 18
Martini, M.F. TMSB17 89
Mascareas, J.L. BPA13 58
Mashal, A. BBA7 114
Masias, E. BPA36 69
Mat, S. TRC11 99
Mayorga, L.S. BLM42 49
McNerny, K.L. BBA7 114
Medina, A.V. ENZ2, ENZ3 74, 75
Sab2013

127

Menegon Arantes, G. BTE1 79
Meneses, K. BLM2 29
Menzaque, A. BLM5 31
Miguel, V. TMSB8 84
Milln, J.L. BLM28 42
Minahk, C.J. BLM25, BPA27, BPA36, BTE3 41, 65, 69, 80
Min, A. BBA9 115
Minari, R. BBA14 117
Mocskos, E. SDI7 107
Moffatt, L. S3.2 21
Moglioni, A. TMSB17 89
Monachesi, S.N. TMSB21 91
Montanari, J. BLM10 33
Montes de Oca, J.M. ENZ6 76
Montes, M.R. REC12 99
Monti, J. MLB7, NB1 32, 108
Montich, G.G. BPA12, BPA16 57, 59
Mora, T. BPA43 73
Morand Sales, E. BPA26 64
Morero, R.D. BLM25 41
Moreschi, A.C. BPA12 57
Morgada, M.N. BPA3 53
Morini, M. BLM15 36
Mottola, M. BLM24 40
Nadra, A. BPA1, BPA8, BPA18 52, 55, 60
Nallet, F. BLM14, BLM20 35, 38
Naranjo, D. S3.1 21
Navailles, L. BLM14, BLM20 35, 38
Navarro, N. S3.1 21
Nazareno, M.A. BLM32, BLM39, ENZ2, ENZ3 44, 48, 74, 75
Nieto, P.S. TMSB6 83
Noguera, M.E. BPA2, BPA28, BPA40 52, 65, 71
Nolan, M.V. BPA19 61
Nomura, D.A. BLM41 49
Oliveira, C. S5.2 25
Oliveira, C.L.P. BLM14, BLM20 35, 38
Oliveira, E.A. BLM14, BLM20 35, 38
Oliveira, R.G. BLM11, BLM45, BPA4 34, 51, 53
Oliveira, T.R. BBA7 114
Oate, J. BLM3 30
Ontiveros, M.Q. TRC2 94
Orozco, M. TMSB26 93
Ouidja, O. BPA26 64
SAB 2013
128

Outeiro, T.F. BPA35 69
Ozu, M. TRC10 98
Pez, R. BPA25 64
Palacios, A.R. ENZ8 77
Pli, T. P5 11
Pallavicini, C. SDI8 107
Palma, A. TRC13 100
Pantano, S. S4.2 23
Paolorossi, M. BPA16 59
Papageorgiou, N. S2.1 19
Papy-Garcia, D. BPA26 64
Paris, G. S5.1 25
Pasquale, M.A. BBA5, BBA12 113, 116
Patio, E. BLM3 30
Pedroni, V. BLM25 36
Peluffo, R.D. S3.3 22
Pealva, D.A. BLM13, BLM22, BLM23 35, 39, 40
Peppe, S. TRC19 103
Perillo, M.A.
BPA15, BPA19, ENZ4, ENA10,
TMSB8, BBA6
59, 61, 75, 78, 84,
113
Perillo, V.L. BLM22 39
Peris, D. BPA5 53
Perrota, R. BLM37 47
Pessoa, Jr. A. BPA25 64
Piccinni, F.E. BPA8 55
Pickholz, M. TMSB17, TMSB23 89, 92
Piegari, E. SDI2 104
Pierini, A.B. TMSB5 83
Pietrasanta, L.I.
TRC5, TRC8, SDI3, SDI4, NTB4,
NTB6, NTB9, BBA9
96, 97, 105, 109,
110, 115
Pignataro, M.F. BLM27, TRC7 42, 97
Pinto, O.A. BLM36 46
Pinto, S.N. P6 12
Pinzn Barrantes, J. BLM16 36
Piotrkowski, B. BLM17, BLM40 37, 48
Pissinis, D. BBA9 115
Placenti, M.A. BPA20 61
Plachta, N. SDI7 107
Politi, M.T. TRC10 98
Ponce Dawson, S. SDI2, SDI6 104, 106
Ponce, M.L. SDI6 106
Porasso, R.D. BLM21 39
Portela, M. BPA38 70
Prieto M. P6 12
Sab2013

129

Primo, E. BPA10 56
Puiatti, M. TMSB5 83
Pusterla, J.M. BLM11 34
Quinzio, C. BBA8 114
Quiroga, F.Y. BBA13 117
Quirolo, Z.B. BPA13 58
Rabinovich, G.A. BPA17 60
Raimunda, D. TRC9 98
Raisman-Vozari, R. BPA26 64
Rakes, C. TMSB2 81
Rasia, R.M. BPA6 54
Recoulat Angelini, A.A. BPA29 66
Revelli, J.A. TMSB6 83
Rey, F S1.1 17
Rico, M. BTE2 79
Rigal, J.B. BPA40 71
Rinaldi, J.J. S5.1 25
Ringkjobing-Jensen, M. S2.1 19
Ros, A. BPA10 56
Risso, P. BPA9 56
Risso, V. BBA4 112
Ritacco, H. BPA14 58
Rodi, P. BLM43 50
Rodrigues, D. E. BLM29 43
Rodriguez, D. SDI1 104
Rodriguez-Fris, A. ENZ6 76
Roitberg, A.E. TMSB4, TMSB7 82, 84
Romn, E.A.
S4.4, BPA28, BPA39, BPA40, BPA41,
TMSB12 24, 65, 71, 72, 86,
Romn, M.D. TMSB6 83
Romero, J.M. BPA17 60
Romero, J.M. BPA22 62
Ronco, M.T. MLB7 32
Rossi, J.P.F.C. TRC1, TRC2, TRC3, TRC4, TRC7 94, 95, 97
Rossi, R.C. TRC12 99
Roveri, O.A. BTE2 79
Rubim, R.L. BLM14, BLM20 35, 38
Rubio, A. BTE4 80
Ruigrok, R.W.H. S2.1 19
Rusu, V.H. TMSB24, TMSB25 92, 93
Ruysschaert, J.M. BPA14, BPA31 58, 67
Saavedra, L. BPA36 69
Sabeckis, M.L. TMSB12 86
SAB 2013
130

Saffioti, N.A. TRC1 94
Salcedo, C.L. BLM31, BLM32, BLM39 44, 48
Saldaa, C. TRC19 103
Samus, I. BPA2 52
Sanchez, J.M. ENZ4 75
Snchez, M.L. BBA11 116
Santana, H. BPA25 64
Santos, J.
BPA28, BPA30, BPA32, BPA39,
BPA40, BPA41, BPA44, TMSB11
65, 66, 76, 71, 72,
73, 86
Schilardi, P. BBA9 115
Schurig-Briccio, L. BTE3 80
Scochera, F. TRC6, TRC8 96, 97
Seddon, J. M. P1 7
Seplveda, R. S3.1, TRC15 21, 101
Sequeira, M.A. BLM12, BLM30, BPA33 34, 43, 68
Sferco, S.J. TMSB20 90
Sfriso, P. TMSB26 93
Shimabukuro, M.I. S1.1, TMSB15 17, 88
Sica, M. S2.3, BPA11 20, 57
Siciliano, N. TRC4 95
Sierra, M.B. BLM15 36
Sigaut, L. TRC5, TRC8, SDI3, SDI4, SDI6, NBT6
96, 97, 105, 106,
110
Silva L.C. P6 12
Silva, E.R. BLM14, BLM20 35, 38
Silveira, N.P. BLM41 49
Simo, A.M.S. BLM28 42
Siri, M. BLM37, BBA4 47, 112
Smith, B.O. S1.1 17
Soba, A. BTE4 80
Socas, B. BPA26 64
Sorre, B P3 9
Sosa Morales, M.C. BLM18, BLM19 37, 38
Sosnik, A. TMSB17 89
Soto Espinoza, S.L. BBA3 112
Soto, G. TRC6 96
Stabeli, R.G. BPA37 70
Stauffer, P.R. BBA7 114
Stefani, F. NTB6 110
Suarez, I.P. BPA6 54
Sued, M. SDI1 104
Sutka, M. TRC10 98
Sycz, G. S5.1 25
Tabares LC C2, SDI5 15, 106
Sab2013

131

Takara, D. TRC3 95
Tamarit, F.A. TMSB6 83
Tarkowski, N. NTB6 110
Terrones, O. S2.2 19
Tvez, N. NTB6 110
Toledo, P. BPA10 56
Torchio, G. S2.3, BPA11 20, 57
Torres-Bugeau, C.M. BPA26, BPA27 64, 65
Toscanini, M.A. TRC17 102
Trujillo, M. TMSB14 87
Turjanski, A.G. BPA34, TMSB16, TMSB18 68, 88, 89
Tuttolomondo, M.E. BLM34, TMSB5 45, 83
Un, S. SDI5 106
Urli, L. MLB7 32
Valdez-Taubas, J. BLM1 29
Valiente Gabioud, A.A. BPA35 69
Valsecchi, W.M. BPA32 67
Vandenbranden, M. BPA31 67
Varela A.R. P6 12
Vasques Camargo da Silva,
J.P. BTE1 79
Vazquez, D.S. BPA44, TMSB11 73, 86
Vazquez, R. TRC11 99
Veciana, J. BPA25 64
Vela, M.E. BBA10 115
Ventosa, N. BPA25 64
Vera, C. BPA27 65
Veuthey, T.V. BPA24 63
Viana, I.F.T. S4.3 24
Vianna Jorge, R. TRC20 103
Vico, R. BLM5, BLM16, BLM24 31, 36, 40
Vila, A.J. BPA3, ENZ8 53, 77
Villanueva, G. BLM3 30
Villanueva, M. BLM24 40
Villarreal, M.A. TMSB8, TMSB26 84, 93
Villarruel, C. SDI6 106
Viso, J.F. TMSB13, TMSB22 87, 91
Vitale, A.J. BLM22 39
von Bilderling, C. SDI3, SDI4, NTB4 105, 109
Wetzler, D.E. BPA18, BPA34, SDI1, SDI8 60, 68, 104, 107
Whitworth, K. TMSB2 81
Wilke, N. BLM1, BLM4, BLM6, BLM13, BLM35 29, 30, 31, 35, 46
Wood, I. TMSB23 92
SAB 2013
132

Yaneff, A. TRC5 96
Yantorno, O.M. BBA10 115
Yoneda, J.S. ENZ7 77
Yunes Quartino, P.J. BPA38 70
Zamarreo, F. TMSB13, TMSB15, TMSB22 87, 88, 91
Zarycki, M. TRC19 103
Zeida, A. TMSB14 87




































Sab2013

133

NOTAS/NOTES

























SAB 2013
134

NOTAS/NOTES