Caderno de resumos com programa completo

Transcrição

Instituto Nacional de C&T de Biologia Estrutural e Bioimagem - INBEB
O Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB) é um instituto de
pesquisa multicêntrico, constituído por uma entidade sede e por uma rede de grupos de pesquisa em Biologia
Estrutural e Bioimagem distribuídos em diversos estados brasileiros. A entidade sede do INBEB é o Centro
Nacional de Bioimagem (CENABIO), localizado na Universidade Federal do Rio de Janeiro (UFRJ). Em
novembro de 2013, o Cenabio conquistou o status de Órgão Suplementar da UFRJ, efetivando o professor
Adalberto Ramon Vieyra (IBCCF/UFRJ) como seu 1º diretor.
O INBEB tem a missão de criar e consolidar uma infraestrutura técnico-científica que permita o
desenvolvimento de pesquisas nacionais de qualidade, acerca das estruturas de sistemas biológicos, desde o
nível macromolecular até o imageamento de organismos inteiros. Para isso, o CENABIO conta com
equipamentos modernos e corpo técnico qualificado, oferecendo amplo suporte tecnológico para a
comunidade científica. Sua inserção no INBEB permite mobilizar e agregar, de forma articulada, os melhores
grupos de pesquisa na área, além de promover mais interações e oportunidades de formação profissional.
É neste âmbito que apresentamos no Programa do V Encontro Anual do INBEB a programação do evento,
assim como os resumos dos 142 pôsteres inscritos no encontro. Esperamos que todos possam aproveitar a
oportunidade para interagir, aperfeiçoar seus conhecimentos e estabelecer novas parcerias.
Prof. Adalberto R. Vieyra
Diretor do Cenabio
Prof. Jerson Lima da Silva
Coordenador do INBEB
Prof. Wanderley de Souza
Vice-coordenador do INBEB
Apoio:
V Encontro Annual do INBEB – www.inbeb.org.br
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Índice
Programação ........................................................................................................................................................................... 3
Quarta-feira - 07/05/2014 .................................................................................................................................................. 3
Quinta-feira - 08/05/2014 .................................................................................................................................................. 5
Sexta-feira - 09/05/2014..................................................................................................................................................... 7
Sessões de pôsteres ................................................................................................................................................................ 9
Data das apresentações ...................................................................................................................................................... 9
Certificados ......................................................................................................................................................................... 9
Listas de apresentadores .................................................................................................................................................. 10
Graduandos................................................................................................................................................................... 10
Mestrandos ................................................................................................................................................................... 11
Doutorandos ................................................................................................................................................................. 12
Pós-doutorandos .......................................................................................................................................................... 14
Pesquisadores ............................................................................................................................................................... 14
Resumos................................................................................................................................................................................ 15
Graduandos....................................................................................................................................................................... 15
Mestrandos ....................................................................................................................................................................... 24
Doutorandos ..................................................................................................................................................................... 35
Pós-doutorandos .............................................................................................................................................................. 53
Pesquisadores ................................................................................................................................................................... 60
V Encontro Anual do INBEB – www.inbeb.org.br
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Programação
Quarta-feira - 07/05/2014
Auditório do bloco N, CCS/UFRJ (Acesso pelo interbloco I/J e pela Rua
César Pernetta)
9:30 – 10:00h
Abertura.
– Diretor do CENABIO, Prof. Adalberto Vieyra (IBCCF/CENABIO/UFRJ).
10:00 – 10:30h
“Um breve histórico do Centro Nacional de Ressonância Magnética Nuclear”.
– Coordenador do INBEB, Prof. Jerson Lima da Silva (IBqM/CNRMN/CENABIO/UFRJ).
10:30 – 10:50h - Coffee-break.
10:50 – 11:50 h
“A Personal View of the History of NMR in Structural Biology and Medical Diagnosis”.
– Nobel de Química de 2002 Prof. Kurt Wüthrich (The Scripps Research Institute/Eidgenössische Technische
Hochschule Zürich/Prof. Visitante do IBqM/UFRJ/INBEB).
11:50 – 12: 10h
Inauguração dos novos magnetos e visitação ao Centro Nacional de Ressonância Magnética Nuclear.
12:10 – 14h – Almoço.
14:00 -14:40h
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“As pesquisas desenvolvidas no Centro Nacional de Ressonância Magnética Nuclear”.
– Prof. Fabio Almeida (IBqM/CNRMN/CENABIO/UFRJ).
14:40 – 15:20h
“Novas capacidades do Centro Nacional de Ressonância Magnética Nuclear”.
– Profª. Monica Freitas (IBqM/CNRMN/CENABIO/UFRJ).
15:20 -16h
“Determinação de estrutura de proteínas de membrana por Ressonância Magnética Nuclear em estado
sólido”.
– Prof. Barth-Jan van Rossum (Leibniz Institute für Molekulare Pharmakologie, Berlin, Germany).
16:00 – 18:00h – Sessão de pôster com coffee-break.
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Quinta-feira - 08/05/2014
César Pernetta)
9:30 - 10:30h
“Tutorial na determinação da estrutura de proteínas de membrana por RMN em estado sólido: 1ª parte”.
10:30 - 10:50h - Coffee-break.
10:50 - 12:30h
“Tutorial na determinação da estrutura de proteínas de membrana por RMN em estado sólido: 2ª parte”.
12:30h – 14:00h – Almoço.
14:00h – 15:00h
“Traditional and new technologies combined for a dynamic portrait of Toxoplasma gondii”
- Profª. Marcia Attias (IBCCF/INBEB/UFRJ).
15:00h – 17:30h
Mesa-redonda: “PAPEL DO INBEB NOS GRUPOS DE PESQUISA DE FORA DA SEDE”.
1. “A consolidação do grupo de pesquisa em Biologia Estrutural (LA14) na Região Norte do país com apoio
do INBEB”.
– Dra. Ana Paula Rodrigues/Pós-doc/UFPA/INBEB;
2. “O Inbeb na Ilha de Santa Catarina”.
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– Dr. Hernán Terenzi/Prof. Associado/UFSC/INBEB.
3. “Estudo da Estrutura das Chaperonas Hsp40”
– Dra. Regina Adão/Pós-doc do IQ/UNICAMP/INBEB.
4. “Efeitos do Sildenafil no Sistema Nervoso Central: Potencial Aplicação Farmacológica.”
– MsC. Ana Karolina Santana Nunes/UFPE/INBEB
5. “O papel do INBEB na estruturação do laboratório de Bioinformática e Modelagem Molecular da UFBA”.
– Dr. Marcelo Castilho/Prof. Associado/UFBA/INBEB
17:30h – 19:30h - Sessão de pôster com coffee-break.
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Sexta-feira - 09/05/2014
César Pernetta)
10:00h – 12:00 h
Mesa-redonda: “JOVENS PESQUISADORES DO INBEB”.
1. “Estrutura e dinâmica do domínio RRM1 do regulador pós-transcricional HuR: implicações para o
reconhecimento específico de RNAm”.
- Prof. Anderson Pinheiro (IQ/INBEB/UFRJ).
2. "The three-dimensional structure of the cytostome-cytopharinx complex of Trypanosoma cruzi
epimastigotes".
– MsC. Carolina de Lima Alcantara (IBCCF/INBEB/UFRJ).
3.“Terapia celular com células mesenquimais de medula óssea em cães com cardiomiopatia chagásica
crônica”.
– Profª. Adriana Bastos de Carvalho (IBCCF/CENABIO/UFRJ).
4. “Minha experiência no laboratório 17 do INBEB no programa Ciências sem Fronteiras”.
– Profª. Karine Verdoorn (UFRJ- Campus Macaé/INBEB).
12:00h -13:30h – Almoço.
13:30h – 16:00h
Mesa-redonda: “PARCERIAS PÚBLICO-PRIVADAS”.
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- Representante do Instituto DÓr de Pesquisa e Ensino (IDOR), Dr. Jorge Moll;
- Representante do Banco Nacional do Desenvolvimento (BNDES), Dr. Pedro L. Palmeira Filho;
- Representante da Fundação do Cancer, Dr. Marcos Moraes;
- Representante da Bruker Corporation, Dr. Mark Chaykovsky;
- Representante do Hospital Sírio e Libanês, Dr. Fabio Gregory.
16:00h -16:20h - Coffee-break.
16:20h -17:20h
“Impacto do INBEB medicina translacional”.
- Prof. Antônio Carlos Campos de Carvalho (IBCCF/CENABIO/UFRJ/DECIT/MS).
17:20 – 18:00h
Encerramento com premiação dos pôsteres.
- Diretor do CENABIO, Prof. Adalberto Vieyra e Comitê Gestor do CENABIO/INBEB.
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Sessões de pôsteres
Data das apresentações
Trabalhos de número ímpar: dia 07/05
Trabalhos de número par: dia 08/05
Certificados
Após a arguição dos avaliadores, serão entregues certificados de apresentação apenas em nome dos
apresentadores inscritos que forem avaliados. Os demais autores terão este caderno de resumos como
certificado de inscrição dos trabalhos no evento.
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Listas de apresentadores
Os resumos estão separados por titulação e ordenados alfabeticamente pelo primeiro nome do apresentador.
Graduandos
G 1
- Ana Beatriz Padilha de Figueirêdo
G 16 - Lilian Hernández Alvarez
G 2
- Antonio Leonardo Freitas Casalinho
G 17 - Luciana Domett Siqueira
G 3
- Bruno Jennings de Almeida
G 18 - Matheus Campello P. L. P. Baptista
G 4
- Caio Bidueira Denani
G 19 - Mirian Kelley Alves
G 5
- Carlla Assis de Araujo e Silva
G 20 - Nathali Pereira da Costa Campos
G 6
- Clarice S C Machado
G 21 - Raiane França Delvalle dos Santos
G 7
- Claudia Monteiro da Rocha
G 22 - Ramon Pinheiro Aguiar
G 8
- Dahienne Ferreira de Oliveira
G 23 - Raysla Alves Pires
G 9
- Daniel Meira dos Anjos
G 24 - Roger Borges dos Santos
G 10 - Fenando Limoeiro Lara de Oliveira
G 25 - Stéphanie Casali Rocha
G 11 - Hector Franco Barbosa Rhault Loponte
G 26 - Stephanie Fernanda Fulaz
G 12 - Juliana Bosco Santos
G 27 - Stephanny Miranda Alves de Souza
G 13 - Juliana Santos Santana
G 28 - Wesley Júnio Alves da Conceição
G 14 - Lara Soares Junqueira
G 15 - Leandro Coelho Teixeira Pinheiro
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Mestrandos
M 1
- Alan Cesar Nunes de Moraes
M 20 - Luiz Carlos dos Santos Ribeiro
M 2
- Alessandro Miranda de Souza
M 21 - Luiza Fernandes
M 3
- Aline Araujo Alves
M 22 - Murilo Martins Pedrote
M 4
- Aline Miyoko Sakaguchi Yamashita
M 23 - Nathália Rocco Machado
M 5
- Ana Carolina Loyola Machado
M 24 - Paula Cristina Rodrigues Frade
M 6
- Barbara Barbosa Succar
M 25 - Paula Mattos da Silva
M 7
- Beatriz Bastos Fonseca
M 26 - Pedro Ernesto Lopes Leão
M 8
- Bruno José Martins da Silva
M 27 - Rafael Mesquita Stoque
M 9
- Caroline Martins Almeida
M 28 - Raquel Gama Gomes Leite
M 10 - Cristine Saibert
M 29 - Rodrigo Dias Requião
M 11 - Cristóvão Freitas Iglesias Junior
M 30 - Talita Stelling de Araujo
M 12 - Dany Naranjo Feliciano
M 31 - Thamires Quadros Froes
M 13 - Diana Pelizzari Raymundo
M 32 - Tiago Souza Salles
M 14 - Gabriel Nascimento dos Santos
M 33 - Verônica Leite Queiroz
M 15 - Gabriela Veras de Moraes
M 34 - Victor da Conceição David
M 16 - Glauber Ribeiro de Sousa Araujo
M 17 - Júlia Araújo de Freitas
M 18 - Lienne Silveira de Moraes
M 19 - Lucas Vellasco
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Doutorandos
D 1 - Alane Bernardo Ramos
D 20 - Franco Henrique Andrade Leite
D 2 - Almir Jordão da Silva Junior
D 21 - Gustavo Monnerat Cahli
D 3 - Ana Clara Vicente dos Santos
D 22 - Isalira Peroba Ramos de Góes Freitas
D 4 - Ana Karolina De Santana Nunes
D 23 - Ivone Rosa de Andrade
D 5 - André Luiz Oliveira Poleto
D 24 - João Guilherme de Moraes Pontes
D 6 - Anita Leocadio Freitas Mesquita
D 25 - Josineide Pantoja Da Costa
D 7 - Antonio Real Hohn Neto
D 26 - Juliana Cunha Vidal
D 8 - Aracelys López Castilla
D 27 - Juliana Dias Alves Pinto
D 9 - Banny Silva Barbosa
D 28 - Julliana Ferreira Santanna
D 10 - Beatriz Ferreira de Carvalho Patricio
D 29 - Larissa Nogueira de Almeida
D 11 - Bruno Macedo da Silva
D 30 - Laura Machado de Faria
D 12 - Carlos Henrique Dumard
D 31 - Leonardo Maciel de Oliveira Pinto
D 13 - Davi Marcos Souza de Oliveira
D 32 - Leonardo Vazquez
D 14 - Edlene Lima Ribeiro
D 33 - Luana P. Borba-Santos
D 15 - Elaine da Conceição Petronilho
D 34 - Luis Henrique Seabra de Farias
D 16 - Elaine Hilário de Souza
D 35 - Luiza Helena Daltro Cardoso
D 17 - Erivan Schnaider Ramos Junior
D 36 - Luiza Villarinho Pereira Mendes
D 18 - Estefania Azevedo
D 37 - Luzia da Silva Sampaio
D 19 - Flora Magno de Jesus Oliveira
D 38 - Mara Cristina Pimenta dos Santos
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D 39 - Marcelle de Souza Ferreira
D 50 - Reinaldo Souza de Oliveira Júnior
D 40 - Maria Micaela Lopez Alarcón
D 51 - Rosemberg de Oliveira Soares
D 41 - MARIANA G. A. T. CUNHA
D 52 - Sabrina Ribeiro Gonsalez
D 42 - Moema Monteiro Batista
D 53 - Samir Pereira da Costa Campos
D 43 - NATALIA DO CARMO FERREIRA
D 54 - Sura Wanessa Santos Rocha
D 44 - Nathalia dos Santos Alves
D 55 - Tácio Vinício Amorim Fernandes
D 45 - Paula Viegas Pereira Signoretti
D 56 - Tatiana Christina Paredes
D 46 - Paulo César Arantes
D 57 - Thais Russo Abrahão
D 47 - Pedro Henrique Monteiro Torres
D 58 - Tiago Bortolotto
D 48 - Rachel Santos de Menezes
D 49 - Raísa da Rocha Reis
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Pós-doutorandos
PD 1 - Aline A. Zuma
PD 11 - Luana Heimfarth
PD 2 - Carla Maria de Souza Menezes
PD 12 - Lucio Ayres Caldas
PD 3 - Carlos Alberto Marques de Carvalho
PD 13 - Mariangela Dametto
PD 4 - Carolina Galvão Sarzedas
PD 14 - Paulo André da Silva
PD 5 - Cherley Borba Vieira de Andrade
PD 15 - Pedro Alexandre de A. G. L. Loureiro
PD 6 - Clarissa Rodrigues Nascimento
PD 16 - Rafael Soares Lindoso
PD 7 - Clelton Aparecido dos Santos
PD 17 - Shana Priscila Coutinho Barroso
PD 8 - Douglas Ricardo Norberto
PD 18 - Tatiana Kelly Silva Fidalgo
PD 9 - Fernanda Magalhães Ferrão
PD 19 - Victor do Valle Pereira Midlej
PD 10 - Humberto Muzi Filho
Pesquisadores
P 1
- André Meyer Alves de Lima
P 2
- Diego Enry Barreto Gomes
P 3
- Manuela Leal da Silva
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Resumos
Graduandos
G1
ANÁLISE POR RESSONÂNCIA MAGNÉTICA DA PERMANÊNCIA
DE CÉLULAS DE MEDULA ÓSSEA NO VÍTREO APÓS LESÃO DO NERVO ÓPTICO
1,2-FIGUEIRÊDO, A.B.P; 1,2-MESENTIER-LOURO,L;1,2-ZAVERUCHA-DO-VALLE,C; 1,2NASCIMENTO-DOS-SANTOS,G; 2-TEIXEIRA,C;2-TOVAR-MOLL,F;1,2-MENDEZ-OTERO,R;
1,2-SANTIAGO,M.F
1-Instituto de Biofísica Carlos Chagas Filho;2-Intituto Nacional de Ciência e Tecnologia
de Biologia Estrutural e Bioimagem,INBEB
Em mamíferos adultos, lesões no nervo óptico provocam degeneração retrógrada das
células ganglionares da retina e morte progressiva, especialmente por apoptose. Várias
abordagens terapêuticas vem sendo estudadas para aumentar a sobrevida neuronal e
regeneração axonal daqueles neurônios em diferentes modelos de lesão, incluindo a
terapia celular. Nesse sentido, em trabalhos anteriores demonstramos que tanto as
células mononucleares quanto as células mesenquimais derivadas da medula óssea
geram neuroproteção e aumentam a extensão axonal após lesão no nervo óptico. No
presente trabalho, procuramos, então, analisar o destino das células transplantadas in
vivo por imagem através de ressonância magnética e ex vivo através de reações
histoquímicas.
Para isso, utilizamos ratos adultos da variedade Lister-hooded e as células derivadas da
medula óssea foram marcadas com nanopartículas superparamagnéticas (SPIO Feratrack) e injetadas no vítreo dos animais imediatamente após lesão no nervo óptico.
Os animais foram então analisados por ressonância magnética 1 dia e 2, 14 e 18
semanas após o transplante.
Demonstramos, então, que por ressonância magnética foi possível observar um sinal
hipointenso derivado das células marcadas em todos os períodos analisados e não
houve redução evidente de sinal ao longo do experimento. Além disso, foi possível
identificar células marcadas com SPIO ex vivo 2 e 18 semanas após a injeção através da
reação de azul da Prússia. Dezoito semanas após a injeção, observamos uma
concentração de células marcadas no vítreo próximas à região do disco óptico. Isso
sugere a permanência a longo prazo das células injetadas nos olhos dos animais
hospedeiros, demonstrando a eficiência do transplante e levantando novas questões
sobre o possível efeito terapêutico dessas células."
CNPQ,FAPERJ,INBEB
G2
STRUCTURAL AND PHYSICOCHEMICAL STUDIES OF THE
HEPATITIS C VIRUS CORE PROTEIN
Braga, V.L.A.1; Casalinho, A. L. F.1; Albernaz, F. P. 1; Mendes-Silva, A.1; Bianconi, M. L.1,
Rangel, L. P.1; Peabody, D.2; Silva, J.L .1; Gomes, A.M.O.1; Souza, T.L.F.3 & Oliveira,
A.C.1
1Programa de Biologia Estrutural/IBqM/UFRJ, Brasil. 2Department of
Molecular Genetics and Microbiology and Cancer Research and Treatment
Center/UNMSM/USA. 3Faculdade de Farmácia/UFRJ/Brazil.
"Introduction: Hepatitis C virus (HCV) is the major cause of chronic liver disease. Cell
culture systems for virus propagation are few and recent, so the core protein (HCVcp)
has been described as a great target of studies. HCVcp is involved in the formation of
the nucleocapsid, viral infection, and different cellular processes. Two different sizes of
core protein, 179 and 124 amino acids, are involved with HCV assembly and
pathogenesis. Small regions of the HCVcp, comprised of peptides 22-39, 50-67 and 85102, were identified to be important for the nucleocapsid assembly. The structural
characterization, and the elucidation of the mechanisms involved in lipid and nucleic
acid interactions of HCVcp are new approaches to understand the HCV assembly
process.
Material and Methods: Our experiments were performed with the protein HCVcp124,
obtained in an E. coli expression system, or with the peptides 22-39, 50-67, and 85-102,
synthesized by Genscript, in the absence or in the presence of different membrane
models (micelles) and non-specific nucleic acids. Structural and physicochemical aspects
of the interaction of HCVcp with the RNA or with the viral envelope during HCV
assembly were investigated by circular dichroism (CD), fluorescence spectroscopy and
isothermal titration calorimetry (ITC).
Results and Discussion: Although HCVcp is an intrinsically unstructured protein,
denaturation studies by CD and fluorescence spectroscopy demonstrated the presence
of few structural elements. Only the peptide 85-102 of HCVcp adopted an alpha-helix
structure in n-octylglucopyranoside and SDS micelles with partial internalization of its
tryptophan residues. HCVcp124 interacts with different DNAs and is able to form
nucleocapsid-like particles (NLPs). The presence of all three peptides does not prevent
the formation of these NLPs. ITC measurements showed that the peptide 50-67 was the
only one able to interact with DNAs.
Conclusions: Our data are relevant to better understand the assembly mechanisms and
can contribute to the identification of new targets for inhibitors of HCV viral infection."
FAPERJ, CAPES, CNPq, Pronex and INBEB.
G3
THE AXIS NALP3-ASC-CASPASE-1-IL-1BETA IS DECISIVE FOR
THE PERITONITIS INDUCED BY BACTEROIDES FRAGILIS AND STERILE CECAL CONTENTS
1-DE ALMEIDA, B.J.; 1-CASTELPOGGI, J.; 2-LOBO, L.; 2-FERREIRA, E.O.; 2-DOMINGUES,
R.M.P.; 3-ZAMBONI, D.; 2-BELLIO, M.; 1-SCHARFSTEIN, J.; 1-OLIVEIRA, A.C.
1- Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro; 2- Instituto de
Microbiologia Paulo de Góes, UFRJ, Rio de Janeiro; 3 - Faculdade de Medicina de
Ribeirão Preto, USP, São Paulo.
The cytokine IL-1β has pleiotropic effects in several cells and tissues and plays a crucial
role in amplifying the host inflammatory response during sepsis and abscess
development. Its secretion is dependent of inflammasome activation, a multiprotein
cytoplasmic complex that activates caspase-1 which converts pro-IL-1β into its mature
form. Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium frequently
Página 15
isolated from clinical infections, such as intra-abdominal abscesses and bacteremia.
When intestinal integrity is disrupted, B. fragilis and colonic contents escape into the
peritoneum, featuring a virulent bacteria-host interaction. The murine model of abscess
induction consists of ip. inoculum of B. fragilis + sterile cecal contents (SCC). Our aim
was to evaluate the inflammasome activation and IL-1 secretion induced by B. fragilis
and/or SCC. Initially, we observed that SCC alone induces in vitro IL-1β production by
BMDC and BMDM in a MyD88-dependent manner, whereas no effect was observed
with B. fragilis alone, which was confirmed in in vivo assays. Next we used macrophages
deficient in Nalp3, ASC and caspase-1, and a drastic reduction in the IL-1 production
was observed. Consistently, in vivo analyses showed that caspase-1, ASC and IL-1R
knockout mice were protected from decrease on body weight and exhibit reduced
inflammatory abscesses, as compared to WT mice. Besides, WT mice present an intense
cellular infiltration into the peritoneal cavity in response to B. fragilis + SCC, which was
dramatically reduced in caspase-1-, ASC- and IL-1R-deficient mice. Taken together, our
results strongly suggest that the development of abdominal peritonitis is critically
dependent on inflammasome/IL-1β pathway by colonic contents. Based on our mice
studies, we suggest that the development of such abscesses in susceptible patients
might be controlled by procedures that restore the epithelium barrier, preventing the
transposition of colonic contents to the peritoneal cavity, and/or by inhibitors of the
inflammasome/IL-1β pathway.
CNPq, FAPERJ, INBEB, PRONEX
G4
THE ROLE OF BOVINE LACTOFERRIN/HEPARAN SULPHATE
INTERACTION ON HUMAN RHINOVIRUS 14 INFECTION
1 - DENANI, C.B.; 1 - SANTOS, R.A.; 2 - CARVALHO, C.A.M.; 2 - SILVA, J.; 2 - OLIVEIRA,
A.C.; 2 - GOMES, A.M.O.; 1 - GONÇALVES, R.B.
1 - Departamento de Bioquímica, Universidade Federal do Estado do Rio de Janeiro; 2 Instituto de Bioquímica Médica Leopoldo DeMeis, Universidade Federal do Rio de
Janeiro.
Rhinovirus, the causative agent of the common cold, is responsible for enormous
damages to the world economy and health. Lactoferrin (Lf), a glycoprotein present in
mucosal secretions of mammals, is widely studied for its antiviral activity. The
mechanisms of Lf antiviral activity already described include intracellular biochemical
processes, interaction with cell surface and competition for virus receptors. Our aim is
to study the interaction of lactoferrin with HeLa H1 cells and how it interferes with the
HRV14 infection cycle, investigating the possible interaction of Lf with molecules
present at the cell surface such as glycosaminoglycans and ICAM-1 receptors. We used
confocal microscopy to investigate the interaction between HRV14 and HeLa-H1 cells.
Virus infectivity and Lf ability to bind nonspecifically to other cellular surface molecules,
like heparan sulfate, were also investigated by the use of fluorescent labeling of Lf and
sulfation inhibition. Time-lapse microscopy experiments showed that bLf causes
morphological changes to the cellular surface. Structures similar to transitional blebs are
observed immediately after bLf addition. After 30 minutes, bLf is observed bound to the
cell membrane, while after 60 minutes a perinuclear distribution of bLf could be
observed. Cells treated with sodium chlorate for sulfation inhibition present less bLf
binding as compared to control cells, suggesting a role for heparan sulfate during bLf
binding to the cell. Our data show that bLf binds to HeLa H1 cells surface and is slowly
internalized (60 min) moving to the perinuclear region of the cell. Sulfation inhibition
suggests that the initial interaction of bLf is dependent on negative molecules, such as
heparan sulfate. The bLf binding to glycosaminoglycans may be the key for an unspecific
antiviral mechanism of bLf. The slow internalization of the molecule suggests that it may
interfere with intermediate to late stages of the viral infection cycle.
FAPERJ, CNPq, FINEP, CAPES
G5
TRATAMENTO COM CÉLULAS-TRONCO MESENQUIMAIS DA
MEDULA ÓSSEA EM UM MODELO IN VITRO DA DOENÇA DE ALZHEIMER
1-GODOY, MA; 2-SARAIVA L; 1-VASCONCELOS-DOS-SANTOS, A; 1-BEIRAL, HJV; 1-SINIS,
LC;; 1-SILVA, LRP; 1-BRAGA, CV; 1-LEAL, RB; 1-SILVA, CAA; 1-VIEYRA, A; 2-DE FELICE, FG;
2-FERREIRA, ST; 1-MENDEZ-OTERO, R
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro 2Instituto de Bioquímica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brasil
"A doença de Alzheimer é uma doença neurodegenerativa cuja incidência tende a
aumentar com o envelhecimento da população mundial. Até o momento, não existem
alternativas terapêuticas eficazes. Os oligômeros abeta são considerados as principais
neurotoxinas para o desencadeamento dos efeitos deletérios nas sinapses e estresse
oxidativo presentes na doença de Alzheimer. A terapia celular é uma perspectiva para o
tratamento desta desordem neurodegenerativa. Na medula óssea existem célulastronco hematopoiéticas, que dão origem à linhagem hematopoiética e células-tronco
mesenquimais (MSCs), que originam as células do tecido conectivo da medula óssea. A
principal hipótese sobre o mecanismo de ação das células mesenquimais é sua ação
parácrina, liberando fatores tróficos que poderiam atuar na estimulação de
sobrevivência neuronal, neurogênese e modulação da inflamação.
Nosso objetivo é avaliar o potencial neuroprotetor das células-tronco mesenquimais da
medula óssea sobre os efeitos deletérios gerados pela exposição aos oligômeros abeta
em neurônios hipocampais, assim como a interação dos oligômeros com as células
mesenquimais.
Para isso, foram estabelecidas culturas de MSCs provenientes de tíbias e fêmures de
ratos Wistar adultos e após 3 passagens, foi realizada a cocultura por 24h dessas células
com neurônios dissociados hipocampais, obtidos do hipocampo de embriões Wistar
com 18 a 20 dias. Após esse período foi realizada a exposição aos oligômeros abeta por
6h a 24h (500 nM) nas coculturas ou somente nas MSCs. Os principais parâmetros
avaliados serão: estresse oxidativo, integridade das sinapses, expressão de pTAU nos
neurônios, assim como a viabilidade, proliferação e estresse oxidativo nas MSCs
expostas aos oligômeros.
Nós concluímos até o momento que os oligômeros são capazes de se ligar as
mesenquimais, sem alterar sua viabilidade durante todo o período analisado (máximo
de 72h de exposição). As células mesenquimais são resistentes ao estresse oxidativo
gerado pelos oligômeros após 24h de exposição, diferente do observado nos neurônios.
Além disso, uma análise inicial sugere que a cocultura com células tronco mesenquimais
protege as culturas hipocampais contra o estresse oxidativo após a exposição por 6h aos
oligômeros abeta. Assim, o tratamento com células tronco mesenquimais parece ser
uma boa opção como abordagem terapêutica para os sintomas iniciais da doença de
Alzheimer."
CAPES, CNPq, INBEB, FAPERJ
G6
IN SILICO EVALUATION OF PHARMACODYNAMIC AND
PHARMACOKINETIC PARAMETERS AND IN VITRO MUTAGENICITY ANALYSIS OF ANTIPRION COMPOUNDS
1- MACHADO, C. S. C., 1- NATALIA C. FERREIRA, 1- PATRÍCIA N. FERNANDES, 1- WESLEY
A. CONCEIÇÃO, 2- ALESSANDRA MASCARELLO, 2- LOUISE D. CHIARADIA-DELATORRE, 2ROSENDO A. YUNES, 2- RICARDO J. NUNES, 1- YRAIMA CORDEIRO
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2= Departamento de
Química, Universidade Federal de Santa Catarina.
Página 16
"Introduction: Transmissible Spongiform Encephalopathies (TSEs) are a group of
neurodegenerative disorders that affect humans and other animals. TSEs develop after
the conversion of PrPC into a pathological isoform, PrPSc that can form aggregates,
which are deposited in the central nervous system inducing neurodegeneration. Until
now there is no treatment and they are invariably fatal. In an attempt to identify new
anti-prion compounds, we realized a screening in neuroblastoma cells infected with
scrapie (ScN2a) and we identified ~40 compounds effective in reducing the levels of
PrPSc .
Material and Methods: We analyzed in silico the possible interaction of PrPC with
selected active compounds. We used molecular docking seeking the less energetic
molecular complexes, considering electrostatic and geometric aspects of the molecules
involved. We used the SWISSDOCK server to evaluate the interactions between proteins
and small ligands. CHARMM force field was used to predict parameters like interaction
energy and stoichiometry. Since these compounds are potential drug candidates, we
performed in silico tests in order to predict the pharmacokinetic properties of these
compounds. Moreover, the Salmonella typhimurium reverse mutation assay (Ames test)
was done to validate the results from the in silico mutagenicity prediction.
Results and Conclusions: The ChemSilico predictor showed three chalcones with
potential for future drugs, due to the good results in the calculated pharmacokinetic
parameters. It was predicted that these molecules possess good ability to passively
cross membranes, and the blood brain barrier, are lipophilic and non-mutagenic.
Preliminary results from the AMES test confirmed that they are non-mutagenic, in
agreement with in silico predictions. Molecular docking showed that the compounds
may bind directly to PrP globular domain (1AG2.pdb), preferentially interacting in the
loop between helix 2 and β-sheets of mice PrPC. In summary, these chalcones possess
good pharmacokinetic and pharmacodinamycs characteristics that warrant their use as
anti-prion compounds."
CNPq, FAPERJ, PIBIC
G7
YELLOW
FEVER
VIRUS-INDUCED
MITOCHONDRIAL
DYSFUNCTION: CHANGES IN MITOCHONDRIAL ENERGETIC METABOLISM AND
APOPTOSIS INDUCTION
Daniel Sanches 1, Samir Pereira da Costa Campos 1, Claudia Monteiro da Rocha 1,
Luciane Pinto Gaspar 2, Marcos da Silva Freire 2, Mariana Figueiredo Rodrigues 1, Jerson
Lima da Silva 1, Andre Marco de Oliveira Gomes 1, Andréa Cheble de Oliveira 1
1 Universidade Federal do Rio de Janeiro (UFRJ) (Instituto de Bioquímica Médica), 2
Fundação Oswaldo Cruz - Bio-Manguinhos (Instituto de Tecnologia em Imunobiológicos)
"Yellow fever is a hemorrhagic disease of great relevance in Africa and South America,
where it occurs as small outbreaks. It is caused by the yellow fever virus (YFV), a
flavivirus such as the Dengue Virus, both being transmitted by mosquitos. It has been
described that the cytopathological effect observed during YFV infection is related to
apoptosis. During this process, the mitochondrial pathway has been reported to play an
important role in viruses-induced apoptosis. Once the mitochondrial pathway is
triggered, loss of mitochondrial membrane potential (Δym) occurs and caspases can be
activated, ending in apoptotic process. In this study, we investigate the role of
mitochondrial pathway of apoptosis in YFV infection and its consequence to
mitochondrial energetic metabolism. We infected Vero cells with YFV using a MOI=1.
We analyzed the cell viability by Live/Dead and LDH assay. Apoptosis was investigated
through PhosphatidylSerine (PS) exposure and TUNEL, while the role of mitochondrial
pathway was studied by the Δym through fluorescence microscopy. The role of the
mitochondrial pathway was examined by using Bongkrekic acid, an adenine nucleotide
translocator (ANT) inhibitor. The mitochondrial energetic metabolism was studied by
oxygraphy.
Apoptosis was detected 72 hours post infection (h.p.i.) through TUNEL and PS exposure.
Its dependence of caspases activation was observed using z-Vad-fmk, a pancaspase
inhibitor. Also, we observed loss of Δym 72 h.p.i., indication of the mitochondrial
pathway activation. Apoptosis seems to depend on ANT activity. Oxygraphy data show a
significant increase of oligomycin-sensitive oxygen consumption at 72 h.p.i., which
suggests an increase in oxygen consumption rate related to ATP synthesis. Our data
suggest that the mitochondrial pathway is activated, contributing partially for the
caspase-dependent cell death induced by YFV. Our data also demonstrate changes on
mitochondrial energetic metabolism associated to virus infection.
CNPq, CAPES, FAPERJ, FINEP, PRONEX, INBEB
G8
CARDIOPROTEÇÃO INDUZIDA POR FATORES HUMORAIS,
LIBERADOS PELO PRECONDICIONAMENTO ISQUÊMICO EM CORAÇÕES ISOLADO DE
RATOS.
1 - OLIVEIRA, F. D.; 1, 2 - BATISTA, G.; 1 - MACIEL, L.; 1- NASCIMENTO, J. H. M.
1 - Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho / Instituto de
Biofísica Carlos Chagas Filho , Universidade Federal do Rio de Janeiro 2 - Faculdade de
Farmárcia, Universidade Federal do Rio de Janeiro.
As doenças do sistema cardiovascular são a causa predominante de morte nos países
industrializados. O pré-condicionamento isquêmico (PCI) é um mecanismo endógeno de
cardioproteção contra lesões de isquemia e reperfusão. Assim, é importante elucidar os
mecanismos do PCI para explorar sua possível utilização terapêutica. Objetivos: Avaliar
a atividade cardioprotetora dos fatores humorais liberados no precondicionamento
isquêmico. Método: Corações de ratos Wistar macho foram perfundidos com solução de
Krebs-Henseleit em sistema de coração isolado com fluxo constante de 10 ml/min. Um
balão de látex foi inserido no ventrículo esquerdo e conectado a um transdutor de
pressão para registro da pressão intraventricular. Os grupos experimentais: Controle
(n=5): submetidos a 30 min. de isquemia e 60 min. de reperfusão (I/R); PCI (n=5):
submetidos a 3 ciclos de 5 min. de isquemia e 5min. de reperfusão, antes do I/R; RECEP
(n=5): perfusão do efluente coletado durante o PCI, por 15 min. antes da I/R; Perfusão
do efluente coletado, durante o PCI por 15 min., antes da I/R; Antagonista (DPCPX
20nM, Naloxone 10nM, Gliburide 10µM, 5HD 100µM) (n=5). Ao final da reperfusão, os
corações foram seccionados e incubados com TTC (1%), para determinação planimétrica
da área de infarto. Resultados: Os resultados mostraram um aumento da PDVE nos
grupos PCI e RECEP, comparados ao grupo CTRL. este efeito foi atenuado nos grupos
com antagonistas. Em relação a área de infarto, houve cardioproteção nos grupos
RECEP e PCI. Nos grupos com DPCPX e Naloxone houve bloqueio da cardioproteção,
enquanto nos grupos Gliburide e 5HD este efeito foi apenas atenuado. Conclusão: O
efluente coletado do precondicionamento isquêmico é capaz de gerar cardioproteção e
os antagonistas dos receptores de opióides, adenosina A1, mKATP e KATP foram
capazes de atenuar ou reverter o efeito cardioprotetor do efluente, sugerindo um
possível envolvimento dessas vias no preconcionamento isquêmico.
FAPERJ, CNPq, CAPES
G9
POSSIBLE INVOLVEMENT OF INNATE IMMUNITY IN PRION
PROPAGATION MECHANISM: AGGREGATION TRIGGERED BY NEUTROPHIL
EXTRACELLULAR TRAPS (NETS)
1-DOSANJOS, D. M. ; 1-AZEVEDO, E. P. ;1- VIEIRA, T. C. R. G. ; 1-FOGUEL, D. ; 2-SARAIVA,
E. M. ; 3 - MORAES, M. C. ; 1-SANTOS, J. B ; 1-SILVA, J. L.
Página 17
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Instituto de
Microbiologia, Universidade Federal do Rio de Janeiro; 3 - Instituto de Química,
Universidade Federal Fluminense
"Prion diseases are fatal neurodegenerative protein-misfolding diseases related to a
protein known as prion protein (PrP). The constitutive cellular isoform of PrP (PrPC),
present in the cell surface, can be converted into its abnormal, β-sheet-rich isoform
(PrPSc) that undergoes aggregation. PrPSc can be transmissible and infectious.
Evidences suggest that adjuvant factors may play a role in the conversion process. Our
group previously reported that DNA can convert PrPC into a β-sheet conformation
leading to its aggregation.
Innate immune system cells have been proven to play critical roles in the incunabular
pathogenic events of prion diseases and may be involved in its progression. Following
peripheral exposure, the replication of prions within the lymphoid tissue has been
shown to be important for the efficient spread of the disease to the brain.
Neutrophil extracellular traps (NETs) are large DNA networks decorated with histones
and granule proteins that trap and kill pathogens, being extruded by activated
neutrophils in response to several stimuli. NETs were reported of being released in
lymphoid tissues. Furthermore, NETs were recently found in association with amyloid
fibrils in tissues of patients with other protein-misfolding diseases and these fibrils
triggered the release of NETs.1 Regarding such observations and considering the large
quantity of DNA in NETs we asked if it could induce PrPC aggregation in vitro. Murine
recombinant PrP23-231 was added into the supernatants from activated human
neutrophils containing NETs. Aggregation was measured by light scattering and electron
microscopy. The results show NETs dose-dependently triggering an instantaneous PrPC
aggregation. It gradually declined remaining detectable after 1 hour. The aggregates
showed amorphous morphology and absence of fibrils. NETs previous treatment with
DNase I inhibited PrPC aggregation, pointing out the importance of the DNA integrity for
its effect. Our results show that NETs are able to trigger stable PrPC aggregation and
that its DNA networks are crucial for this process. Differences in NETs protein
composition among different donors seems to be related to distinct aggregation
features. PrP-coated magnetic beads were used to “fish out” PrP-ligands from a pooled
sample containing NETs from 7 human donators. MALDI-TOF mass spectrum of proteins
as potential ligands isolated from NETs shows a mass ion at around 13.5 kDa.
Our data suggest that NETs are an intriguing factor that must be appraised in the studies
concerning prion propagation mechanisms."
INBEB, FAPERJ, CNPq, CAPES
G10
ANOTAÇÃO E MODELAGEM DE ENDOGLUCANASE DE
SERRATIA SP ORIUNDA DO METAGENOMA DO CARAMUJO AFRICANO ACHATINA
FULICA.
1,2-LARA, FLO ; 2- SOARES, RO ; 1-GOMES, DEB ; 1-DA SILVA, ML
1 - Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de
Metrologia, Qualidade e Tecnologia ; 2- Universidade Federal do Rio de Janeiro
"A biomassa lignocelulósica tornou-se um dos principais alvos da produção de
combustíveis de segunda geração devido ao seu potencial como matéria-prima para
fabricação de bioetanol. Por estar amplamente disponível no Brasil, principalmente
através do resíduo industrial da fermentação de etanol, estudar os organismos e / ou
enzimas com potencial para degradar biomassa lignocelulósica e extrair fermentáveis de
polissacarídeos de cadeia longa torna-se um alvo em potencial para tal fim.
Neste trabalho, analisamos com técnicas de bioinformática e modelagem molecular
uma das enzimas oriundas do metagenoma do suco gástrico do caramujo Achatina
fulica. Utilizamos diferentes programas e bancos de dados para anotação da mesma.
Através da utilização do BlastP (não redundante), KEGG, COG. supFam, CDD, UniProt,
MetaCyc e dbCAN identificamos a enzima em questão como uma endo-1,4-Dglucanase, membro da família 8 glicosil hidrolases, mais especificamente a Subunidade
Z da Celulose Sintase de Serratia sp. Além disso, utilizamos ferramentas para predição
de estrutura secundária (PSIPRED), presença de peptídeo sinal (SignalP), localização
celular (PSORT) e diferentes metodologias de alinhamento (PROMALS e ClustalW).
Na técnica de Modelagem Comparativa, utilizamos a Subunidade Z de celulose Sintase
de E. Coli K-12 (PDBID 3QXQ) como molde, tendo essa enzima 50% de identidade, 66%
de similaridade e 100% de cobertura com o alvo . Foram construídos 100 modelos
candidatos e para avaliar esses modelos analisamos os Gráficos de Ramachandran
(PROCHECK) , valores de RMSD entre molde e modelos (função super do PyMOL), bem
como os escores GA341 e DOPEscore (MODELLER).
O modelo escolhido para a Celulose Sintase obteve 89.8% aminoácidos em zonas
permitidas do gráfico de ramachandran, 0.5% em zonas proibidas, DOPEscore de 22368.76953 e RMSD relativo ao molde de 0.354 A. À partir deste modelo, poderemos
realizar estudos de docking e dinâmica molecular, de modo à entender as interações
que ocorrem no sítio ativo dessa enzima."
CNPq
G11
IN VIVO STUDIES OF THE HYPERGLYCEMIA INFLUENCE ON THE
GLYCOPHENOTYPE AND THE PROGRESSION OF COLON CARCINOME CELLS
1 - Loponte, H. F.; 1 - Vasconcelos-dos-Santos, A.; 1 - Mantuano, N. R.; 1 - Oliveira, I.A.;
2 - Moll, F.; 1 - Dias, W.B.; 1 - Todeschini, A.R.
1 - IBCCF, 2 - CENABIO. Rio de Janeiro, Brazil.
Cancer cells depend on altered metabolism and nutrient uptake to generate and keep
the malignant phenotype. Recent work suggests that the metabolite availability to the
hexosamine and Golgi O-glycosylation pathways exerts control over cell signaling, gene
expression and cell migration (Alisson-Silva etal., Plos One, 8: e60471, 2013). Indeed,
high glucose concentration increases O-glycosylation of FN (onFN), which generates the
onFN, and modulates tumorigenesis. Herein we implicate hyperglycemia in facilitating
cancer progression and increasing onFN expression in vivo. Cells from colon carcinoma
of C57BL/6 mice (MC-38 cell line) were subcutaneously inoculated in streptozotocin
(STZ)-treated or untreated C57BL/6 mice. Subcutaneous tumors from STZ-treated group
were larger and more vascularized when compared with vehicle group. Tumors from
STZ-treated mice presented high expression of α2-6-Neu5Ac and α1-3-/ α1-6-Fucose
(Fuc) containing glycoconjugates as well as increased expression of onFN. These data
suggest that metabolite availability modulates cell surface glycosylation contributing to
tumor formation and progression. CNPq, FAPERJ, CAPES
G
12
Prion Protein Coated Magnetic Beads: A New
Approach For Ligands Screening
1,2 Santos, J.B.;1,3 de Moraes, M.C.;1- dos Anjos,
D.M.; 1- da Silva, J.L.
1-Instituto de Bioquímica Médica, IBqM-UFRJ, RJ; 2- Faculdade
de Medicina, Unirio, RJ; 3-Departamento de Química Orgânica, UFF, Niterói/RJ
Prion diseases are characterized by prion protein (PrP) aggregation and
eventual neurodegeneration. The conversion of the constitutive cellular isoform of PrP
(PrPC) into an abnormal one - scrapie PrP (PrPSc) - is the most important step leading to
the development of these diseases [1]. The mechanism of conversion is still unclear.
Therefore, the development of analytical tools for the identification of PrP-ligands,
which can act as cofactors or inhibitors in the conversion mechanism of PrP, has
become an interesting task. The main purpose of this work is the development of PrPligands screening assay based on bioaffinity chromatography, to rapidly identify ligands
which may contribute to understand and completely elucidate this conversion
mechanism, which probably requires a cellular cofactor. This work describes the PrPC
Página 18
covalent immobilization onto the surface of silica-based magnetic beads (Bioclone) to
isolate ligands from complex mixtures. PrP was labeled with fluorescein isothiocyanate
prior to immobilization so as to evaluate the success of the immobilization procedure by
fluorescence microscopy of the PrPC coated-beads. To validate the purposed bioaffinity
assay, the PrPC-coated magnetic beads were used to “fish out” ligands from an
equimolar mixture of thiamine (Syrian hamster PrP-ligand), quinacrine (human PrPligand) and caffeine (negative control). An UHPLC-MS/MS method was developed and
validated to quantify the amount of isolated ligands by the bioaffinity assay. The
selected ratio PrP:beads amount was 1:3 (mg), which yielded around 60% of PrP
immobilization. The binding of labeled PrPC was observed by fluorescence microscopy
and showed a substantial coating. In the bioaffinity assays, the PrP-coated magnetic
beads isolated 40% of the quinacrine from the equimolar (300nM) compounds mixture.
The next step is applying this method for screening new compounds, cellular products
and extracts. The developed method represents an useful tool to investigate the affinity
and interaction of PrP with cellular components and complex matrices.
FAPERJ, CNPq
G13
DESENVOLVIMENTO DE ESTRATÉGIAS PARA O ESTUDO DE
PROTEÍNAS MAL ENOVELADAS POR RESSONÂNCIA MAGNÉTICA NUCLEAR
1-SANTANA, J.S.; 1-SOUSA, A.R.N.; 1- SANT'ANNA, R.O.; 1- FOGUEL, D.; 1- FREITAS, M.S.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Instituto
Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro,
Brasil.
"Doenças amiloidogênicas, em sua maioria, estão relacionadas com o mal
enovelamento de determinadas proteínas. A doença de Alzheimer, doença de
Parkinson, doença de Creutzfeldt-Jakob, doença do Neurônio Motor, Polineuropatia
Amiloidótica Familiar e a doença de Huntingnton são exemplos de amilóidoses e são de
grande impacto na saúde pública. Os estudos com as proteínas envolvidas nas doenças
supracitadas tem possibilitado um ganho na compreensão quanto aos mecanismos
envolvidos no mal enovelamento proteico. A importância de se estudar a má
conformação através da caracterização da estrutura tridimensional, possibilita observar
o que acarreta o mal enovelamento, favorecendo o desenvolvimento de farmacos,
terapias ou até mesmo estimar possíveis grupos propensos a desenvolver tais doenças.
O objetivo do nosso trabalho tem sido monitorar padrões estruturais relacionados a
formação de pequenos e grandes oligômeros que são causados pelo enovelamento
incorreto, visando estabelecer um protocolo de experimentos In cell onde possamos
melhorar o compromisso entre o tempo de aquisição e resolução dos espectros de
Ressonância Magnética Nuclear.
Com o intuito de estudar algumas das doenças supracitadas, foram usadas como
modelos as proteínas: Transtirretina , Transtirretina monomérica, Prion e α-Sinucleína.
Todas expressas In cell com o objetivo de propiciar um ambiente celular na tentativa de
analisar possíveis mudanças em relação aos experimentos realizados In Vitro com a
proteína purificada. Foi utilizada deuteração da amostra com uso de sequências de
pulso que minimizam os efeitos inseridos pelo acoplamento dipolar devido a
viscosidade do ambiente intracelular que leva a uma redução do T2 que
consequentemente, leva ao alargamento de linha.
Levando em consideração os aspectos apresentados, esse trabalho, visa estabelecer um
protocolo experimental, In cell, que reduzirá o tempo de preparo da amostra, otimizará
o tempo de análise dos dados, podendo trazer novas informações sobre a interação da
proteína com o micro ambiente intracelular."
CNPq, FAPERJ, AvH, CAPES, DAAD
G14
EFEITOS DE CICLOSPORINA A E CICLOHEXIMIDA SOBRE O
CONDIONAMENTO E EXTINÇÃO DE MEMÓRIAS AVERSIVAS
1- JUNQUEIRA, S. L.; 2- MOULIN, T. C.; 3- ALMEIDA-CORRÊA, S. 4- AMARAL, O. B.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Instituto
de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 3-Instituto de
Bioquímica Médica, Universidade Federal do Rio de Janeiro; 4-Instituto de Bioquímica
Médica, Universidade Federal do Rio de Janeiro
"Memórias aversivas não são estáticas, podendo ser labilizadas ou modificada após sua
consolidação a partir de processos como reconsolidação e extinção. Dentre diversas
moléculas envolvidas, trabalhos anteriores descrevem a proteína fosfatase calcineurina
(CaN) como uma enzima crucial no processo da extinção do condicionamento aversivo
contextual, contudo seu exato papel sobre este tipo de memória ainda é contraditório.
Neste estudo, investigamos os efeitos do bloqueio de CaN pelo fármaco ciclosporina A
(CsA) e os comparamos com a influência da cicloheximida (CHX), um inibidor de síntese
proteica, mecanismo esse necessário para a aquisição da memória. Para isso,
condicionamos aversivamente camundongos Swiss machos a um contexto e em
seguida, os reexpomos ao mesmo local para a extinção desta memória. Em diferentes
grupos aplicamos os fármacos CsA e CHX 1h pré-extinção ou pré-treino por injeção
intracerebroventricular (ICV) freehand. A medida utilizada para calcular o medo foi a
contagem do tempo de congelamento diante do contexto aversivo (freezing) durante as
sessões de extinção e teste."
FAPERJ, CNPq.
G15
ANÁLISE TRIDIMENSIONAL DO SISTEMA NERVOSO CENTRAL
POR MICROSCOPIA ÓTICA APÓS CLARIFICAÇÃO TECIDUAL.
1- TEIXEIRA-PINHEIRO, L.C. 1- NASCIMENTO-DOS-SANTOS, G. ; 1- MENDEZ-OTERO, R.; 1SANTIAGO, M.F.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
"O tecido nervoso possui camadas densas de lipídios que o fazem ser pouco permeável
à luz e a anticorpos usados em imunohistoquimica, o que limita a visualização por
microscopia ótica. Sendo assim, faz-se necessária a secção mecânica do tecido para a
visualização de áreas que de outra forma não seriam acessíveis. Novas técnicas de
clarificação tecidual, como CLARITY e 3DISCO, vem sendo desenvolvidas com o objetivo
de permitir a visualização de camadas mais internas de tecidos intactos. Neste trabalho
propomos a comparação de dois protocolos distintos de clarificação tecidual em
diferentes tecidos do SNC com o objetivo de aplica-las e adapta-las aos principais
modelos de estudo utilizados por nosso grupo (retina e nervo ótico).
Camundongos foram perfundidos com PBS seguido de uma solução de 4% de
paraformoldeido, 4% de acrilamida, 0.05% de bis-acrilamida e 0.25% de iniciador VA044 em PBS (solucao de hidrogel). Ratos Lister foram divididos em 3 grupos, o grupo 1
foi perfundido em solução de 4% de paraformoldeido e pós fixados em solução de
hidrogel, o grupo 2 foi perfundido em solução de hidrogel e o grupo 3 perfundido em
solução de hidrogel sem bis-acrilamida. Os cérebros foram dissecados e armazenados
na solução de hidrogel correspondente a cada grupo e colocados em 37°C por 3 horas
para a polimerização da solução. O tecido em solução polimerizada foi cortado em fatias
de 1, 2 e 3mm ou mantido intacto. O cérebros e as fatias foram então colocados em
uma solução de acido bórico a 200mM com 4% de dodecil sulfato de sódio (solução de
clareamento) a 37°C ou temperatura ambiente ate estarem visivelmente transparentes.
As amostras foram então lavadas em PBS, marcadas por imunohistoquímica e colocadas
em 80% de glicerol para serem visualizadas por microscopia confocal."
CAPES, FAPERJ, INBEB, CNPq.
Página 19
G16
RATIONAL DRUG DESIGN FOR FASCIOLA HEPATICA
CATHEPSIN L3
1,2-HERNANDEZ, L.A. ; 3- VALIENTE, P.A. ; 4- GOMES, D.E.B. ; 2-PASCUTTI, P.G.
1- Centro Nacional de Sanidad Agropecuaria; 2- Universidade Federal do Rio de Janeiro
; 3- Universidad de La Habana ; 4- Instituto Nacional de Metrologia, Qualidade e
Tecnologia
"Cathepsin L3 of Fasciola hepatica (FhCL3) is a cystein protease considered as an
attractive target for the design of fasciolicidal drugs, but its crystallographic structure is
still unknown. In order to search for potential inhibitors of FhCL3, we calculated a
homology model of its 3D structure based on the primary sequence of this protease
using the 3D structure of F. hepatica cathepsin L1 (PDB:2O6X) as template. Our model
suggests that the binding site of FhCL3 has distinctive structural features compared to
those of both human and bovine cathepsins L which, in turn, indicates the feasibility of
designing selective inhibitors of FhCL3. A docking study was then conducted based on
the FhCL3 model using. MYBRIDGE compound database against FhCL3 and both bovin
and human cathepsin L, and five compounds selective to FhCL3 and with high energy
scores were found. Subsequently, molecular dynamics (MD) simulations were
performed to study the stability and structural properties of the free enzyme and the
five ligand-enzyme complexes. Besides the five ligand-enzyme complexes, the model of
FhCL3 in complex with nitrile, a well-known inhibitor of the cystein protease family, was
included as a positive control. MD simulations were used as well to refine the binding
modes of the ligands in the enzyme pocket. Hydrogen bonds present in the ligandenzyme complexes were analyzed, as well as contact residues. Finally, MM-GBSA
binding free energy calculations were performed based on the MD trajectories
generated for each complex. These calculations suggest that two out of the five
compounds
identified through virtual screening have favorable binding free energy values
comparable to that of nitrile, the positive control."
CAPES
G17
ESTUDO DOS MECANISMOS DE DEFICIT COGNITIVO
ASS I
EN E E I EN : P PE
IN
SIS
I
1- D'AVILA, J.C.; 1-SIQUEIRA, L.D.; 3- AZEVEDO, E.P.; 1- ARAÚJO, J.P.; 3- FOGUEL, D.; 2BOZZA, F.A.
1- Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz - FIOCRUZ; 2- Instituto de
Pesquisa Clínica Evandro Chagas - FIOCRUZ; 3- Instituto de Bioquímica Médica,
Universidade Federal do Rio de Janeiro
en elhecimento o ulacional é um fen meno global. en elhecimento é o maior
fator de risco ara doen as neurodegenerati as, ue t m como caracter stica comum a
resen a de neuroinflama o caracteri ada ela ati a o da microglia, as células
imunol gicas residentes do sistema ner oso central. urante o rocesso de
en elhecimento, a efic cia das fun es da microglia se deteriora e esta disfun o
microglial ode estar relacionada aos danos cogniti os associados ao en elhecimento.
s mecanismos de les o res ons eis ela disfun o microglial associada ao
en elhecimento ainda n o s o bem com reendidos. ossa hi tese é de ue a
disfun o microglial associada ao en elhecimento en ol e déficits em ias de
degrada o fagocitose e autofagia e de gera o de es écies reati as de o ig nio
P o idases e mitoc ndrias defeituosas ue odem determinar o ac mulo de
prote nas neurot icas e estresse o idati o. ste trabalho tem como ob eti o in estigar
os mecanismos da neuroinflama o e dano tecidual em um modelo de en elhecimento
com re u o cogniti o, com nfase no erfil de ati a o microglial e nos mecanismos
de les o tecidual. disfun o microglial foi indu ida com a inflama o sist mica cr nica
de bai a intensidade, rodu ida atra és de in e es intra eritonais semanais de bai as
doses de li o olissacarideo. ados reliminares demostram ue h ati a o microglial
mais ronunciada em idosos, tanto nai e uanto desafiados com P cronicamente, em
com ara o com os gru os o ens. s testes de com ortamento mostraram ue o
desafio inflamat rio cr nico de bai a intensidade afeta o desem enho cogniti o dos
animais idosos e n o dos animais o ens frente ao desafio com P cr nico, re elando
assim, uma maior susce tibilidade dos idosos inflama o sist mica cr nica. s dados
obtidos neste estudo oder o contribuir ara o desen ol imento de tera ias mais
efica es ara doen as neurodegenerati as associadas ao en elhecimento.
FAPERJ, CNPq, CAPES
G18
REGULATION OF GLUTAMATE AND D-SERINE LEVELS IN MICE
CEREBRAL CORTEX
1- BAPTISTA, M.; 1- POLETO, A.; 1- MADEIRA, C.; 1- PANIZZUTTI, R.
1- Instituto de Ciências Biomédicas, UFRJ
D-serine is the main co-agonist for the glutamate activation of the N-methyl D-aspartate
receptor (NMDAr), an ionotropic receptor important for memory and neuroplasticity. In
vitro studies using neuron and astroglial cultures have shown that activation of
glutamate receptors can stimulate the release of D-serine. Even though the mechanisms
that regulates the release of D-serine in vivo has not been established. Also, it is
unknown whether D-serine can regulate the extracellular levels of glutamate in vivo.
The goal of this project is to study the effect of glutamate receptors activation on the
extracellular levels of D-serine and glutamate in the mice cerebral cortex. To do so, we
performed microdialysis in freely moving mice and used high performance liquid
chromatography to analyze the extracellular levels of amino acids. Preliminary data
show that increasing concentrations of D-serine in the perfusate led to higher
concentrations of glutamate in the dialysate. On the other hand, increasing
concentrations of glutamate in the perfusate did not affect the concentrations of Dserine in the dialisate. In vivo brain microdialysis provides a proper model to analyze the
interaction of these neuroactive amino acids and how they modulate each other’s
release. Further studies using specific agonists or antagonists of glutamate receptors are
needed to clarify the mechanism involved in such interactions.
FAPERJ
G19
USO DE UMA RESINA FUNCIONALIZADA PARA PURIFICAÇÃO
DE FIBRAS AMILÓIDES
MIRIAN KELLEY1, DANIELA GAMA1, DEBORA FOGUEL1, FERNANDO L. PALHANO1
[1] Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brasil
Fibras amilóides são responsáveis por uma série de doenças degenerativas, como
Parkinson e Alzheimer, que atingem cada vez mais pessoas devido ao envelhecimento
da população mundial. O tratamento dessas doenças, quando existe, é pouco eficaz.
Acredita-se que isso se deve, em parte, ao diagnóstico tardio, sendo de grande valia o
desenvolvimento de métodos de diagnóstico precoce. Uma técnica com potencial
aplicação no diagnóstico precoce de doenças amilóides é a cromatografia de afinidade,
sendo interessante a associação da molécula de penta tiofeno à resina de agarose, pois
esta molécula tem grande afinidade por fibras amilóides. Objetivamos então produzir
uma resina funcionalizada e um protocolo eficaz que sejam capazes de detectar fibras
amil ides com alta sensibilidade e es ecificidade. Para tal, usamos fibras de β1-40 e αsinucleína produzidas in vitro, associadas respectivamente às doenças de Alzheimer e de
Parkinson. Como controle usamos albumina solúvel. Para detecção das amostras
usamos a técnica de Dot-Blot. bser amos ue as fibras de β1-40 se ligam tão
Página 20
fortemente à resina com penta tiofeno que apenas ácido fórmico 96% foi capaz de
deslocar um pequeno percentual dessa fibra da coluna, ficando a maior parte desta
resa na mesma. J a α-sinucleína, demonstrou ter se deslocado da resina com eluição
em 8M de uréia pH 2,2+2% SDS. Por outro lado, a albumina eluiu facilmente da resina
com solução de lavagem (PBS+1% Triton X-100+0,5M NaCl). Nossas perspectivas são
encontrar um sol ente eficiente ra elui o da fibra de β1-40, avaliar se a
es ecificidade ara α-sinucleína ocorre apenas com fibras ou também com monômeros
e fazer testes usando fibras produzidas in vivo usando como modelo leveduras com ou
sem agregados amiloides.
FAPERJ, CNPq, CAPES.
G20
DOES RESVERATROL PREVENTS P53 AGGREGATION?
1,2- COSTA, D.C.F.; 1,2- CAMPOS, N.P.C.; 2,3- ZANPHORLIN, L.; 2,3- RAMOS, C.H.I.; 1,2SILVA, J.L.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e
Bioimagem, Rio de Janeiro, Brazil; 3- Instituto de Química, Universidade Estadual de
Campinas, São Paulo, Brazil.
p53 has an essential role in preventing cancer development by inducing cell cycle arrest
and/or apoptosis in response to cellular stress. Mutations in the p53 gene are described
in over 50% of all human cancers. Besides the mutations, cellular aggregation of p53 can
also inactivate the protein, leading to malignancy. Resveratrol, a natural polyphenol
found in grapes and red wine, is able to induce p53-dependent cell death in a variety of
cell lines. Although several indirect mechanisms of p53 activation by resveratrol have
been proposed, there is no evidence that this bioactive compound can interact with
p53. Thus, we investigated a possible interaction between resveratrol and the p53 core
domain (p53C). In addition, we tested the potential of resveratrol in preventing wildtype and mutated p53 aggregation in vitro and in tumor cells. Experiments were
performed by using fluorescence spectroscopy and fluorescence microscopy
techniques. Our data suggest that an interaction between resveratrol and the wild-type
p53C does occur. We also found that resveratrol has the ability to inhibit the wild-type
p53 core domain as well as the R248Q p53 mutant to undergo in vitro aggregation.
Additionally, resveratrol (50 and 100 µM) induces the formation of nuclear p53
aggregates in MDA-MB-231 human breast cancer cells. Our study provides evidence
that resveratrol can directly modulate p53 and may pave the way for a better
understanding of the mechanisms involved in p53 protein aggregation as a therapeutic
strategy for cancer treatment.
FAPERJ, CNPq, INCT/INBEB
G21
-
DISSECTION OF PRION PROTEIN AND LIPID INTERACTION
1,2,3-Santos, R.F.D; 1,2,3-Vieira, 1,2,3-TCRG; Silva, JL.
1-Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, 2-Instituto de
Bioquímica Médica Leopoldo De Meis, 3- Instituto Nacional de Ciência e Tecnologia de
Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
21941-902, Brazil.
Introduction: Transmissible spongiform encephalopathies are a group of fatal diseases,
which affect mammals, caused by an abnormal isoform of the prion protein (PrP).
Conversion of cellular PrP (PrPC) into the pathological conformer, PrPSc, involves
contact between both isoforms and probably requires a cellular factor. Recombinant
PrP can be converted to an abnormal form via seeded polymerization in vitro
techniques in the presence of lipids. Objectives: The importance of lipid molecules for
conversion has been revealed, but little is known about the structural features implicit
in this interaction. A detailed understanding of this interaction may provide new insights
into toxic mechanisms associated with this disease. Material and Methods: In the
present work, we used Rayleigh and Dynamic Light Scattering, and fluorescence
measurements in order to provide information on the chemical and physical properties
of
the
murine
recombinant
PrP
(rPrP
23-231)
interaction
with
Phosphatidylethanolamine (PE) and Phosfatidic Acid (PA) vesicles. Results and
iscussion: We found that increasing concentrations of hos holi ids’ esicles raised
rPrP light scattering, forming large aggregates (>1 μm). The tryptophan fluorescence
emission intensity was increased and blue-shifted, showing change of the
environment/properties of the protein tryptophan groups. The observed effect of
phospholipids on the tryptophan fluorescence could arise from the lipid-induced
conformational change of PrP, or membrane protein insertion. Conclusions: Taken
together, our results indicate that PE and PA induce PrP oligomerization followed by
formation of large aggregates. The conformational changes observed could play a
critical role in nucleating the formation of toxic species.
CNPq, FAPERJ
G22
UNRAVELING THE FUNCTION OF THIOREDOXIN-INTERACTING
PROTEIN (TXNIP) THROUGH STRUCTURAL STUDIES
Aguiar, R. P.; Amorim, G. C.; Valente, A. P.; Almeida, F. C. L.
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
"Thioredoxin-interacting protein (TXNIP), also known as vitamin D up-regulated protein,
is strongly up-regulated by vitamin D. TXNIP belongs to the α-arrestin protein family and
functions regulating oxidative stress response and glucose uptake. TXNIP binds directly
to thioredoxin (hTrx) controlling all Trx-dependent regulatory processes, such as
apoptosis and response to oxidative stress. It modulates inflammatory response, cardiac
function, cell signaling and apoptosis. TXNIP plays a crucial role in several pathological
conditions such as cancer, diabetes and cardiovascular diseases.Little structural
information is available for this class of proteins. Our goals are to solve the structure of
TXNIP domains and understand details of the interaction with hTrx.
We cloned and expressed the N-terminal (1-149, D1) and C-terminal domain (163-301,
D2) using the native sequence and mutating all cysteines by serine, with the exception
of Cys63 for D1 and Cys247 for D2. We used standard methods to isotope label the
cysteine-mutated domain (15N and 15N/13C). D1 and D2 have been expressed in E. coli
and purified. Nuclear magnetic resonance triple resonance experiments were collected.
The software NMRPipe and CCPN-NMR were used for processing and assignment.
We successfully expressed native and cys-mutated D1 and cys-mutated D2. D1 showed
low-affinity interaction with hTrx. We almost fully assigned the resonances of D1 and
map its interaction with hTrx. We constructed a NMR-derived model of the complex D1Trx1. Contrary to D1, D2 showed strong interaction with hTrx.These data is crucial for
understanding the mechanism of interaction with hTrx. We showed that D1 binds to
hTrx through Cys37, possibly regulating the interaction, which occurs primarily thought
D2, the hTrx-binding domain."
CNPq, FAPERJ
G23
ANOTAÇÃO E MODELAGEM ESTRUTURAL DA ENDO-1,4-DGLUCANASE DE STENOTROPHOMONAS SP. PROVENIENTE DO METAGENOMA DO
CARAMUJO ACHATINA FULICA.
1,2-R A PIRES, 2-M L da SILVA
1- Universidade Federal do Rio de Janeiro, 2- Instituto Nacional de Metrologia,
Qualidade e Tecnologia – INMETRO
Página 21
O Brasil é referência mundial na produção de etanol, porém o etanol produzido a partir
de biomassa (celulósico) ainda não é viável economicamente. Devido a suas vantagens,
como a redução do uso de petróleo, minimização da poluição ambiental, e a
convivência com a cultura de alimentos sem concorrência, pesquisas nesse setor tem
crescido consideravelmente, representando desta forma uma solução próspera para os
problemas atuais do uso de combustíveis fósseis. Desta forma, é necessário desenvolver
o conhecimento acerca da hidrólise enzimática com uso de enzimas para que esta
tecnologia sustentável se torne viável e com valores interessantes para a indústria. Este
estudo analisou sequências obtidas pelos métodos de pirosequênciamento da
microbiota do suco gástrico do caramujo africano Achatina fulica para a triagem de
enzimas potencialmente envolvidas na degradação de celulose e sua modelagem
estrutural através da técnica de modelagem comparativa. Inicialmente, das enzimas
obtidas no metagenoma, optou-se por analisar estruturalmente a endo-1,4-D-glucanase
de Stenotrophomonas sp.. Essa proteína teve seu modelo construído com base na
estrutura da endoglucanase de Escherichia coli k-12 (PDBCod 3QXQ). A escolha pelo
melhor modelo dentre os 100 candidatos construídos pelo MODELLER foi feita
utilizando os critérios de RMSD entre molde e modelo e Gráfico de Ramachandran.
Obteve-se um modelo tridimensional de ótima qualidade para a enzima endo-1,4-Dglucanase de Stenotrophomonas sp., com RMSD 0,182 Å entre molde e modelo, Gráfico
de Ramachandran com 95,8% dos aminoácidos nas regiões favoráveis (Vermelha) 3,6%
nas regiões permitidas (Amarela) 0,6% nas regiões generosamente permitidas (Bege) e
0.0% nas regiões proibidas (Branca). Com o modelo gerado em mãos, a próxima etapa
do trabalho consistirá em desenvolver estudos de Dinâmica Molecular para melhor
elucidar o potencial catalítico na degradação de celulose.
CNPq
G24
CALORIMETRIC AND STRUCTURAL ANALYSIS OF THE
INTERACTION OF INHIBITOR OF APOPTOSIS PROTEIN XIAP WITH SMAC MIMETIC
COMPOUNDS
1- SANTOS, R.B.; 2- OLIVEIRA, A.C.; 1- SOUZA, T.L.F.
1- Faculdade de Farmácia, UFRJ, Rio de Janeiro, Brazil. 2- Programa de Biologia
Estrutural, IBqM, UFRJ, Rio de Janeiro, Brazil.
Apoptosis resistance, in cancer, can be generated by high expression of inhibitor of
apoptosis proteins (IAPs) family, which is responsible by the inhibition of initiator and
effector caspases. Smac/DIABLO is an endogenous inhibitor of XIAP due to its direct
interaction on the XIAP-BIR3 domain. Smac mimetics have been considered potential
drug candidates, owing to its capacity to bind on XIAP-BIR3 and to sensitize apoptosis in
cancer cell. The goal of this work is better understand the effects of the binding of Smac
mimetics on the structure and stability of the XIAP-BIR3 and to add useful information
to drug design. Smac mimetic compounds with structural similarities, but with different
activities, were selected to us investigate the individual thermodynamic contributions of
functional groups. We used circular dichroism (CD), fluorescence spectroscopy and
isothermal titration calorimetric (ITC) to perform the structural and thermodynamic
analyses. Denaturation with guanidine hydrochloride (GdnHCl) was used to estimate the
domain stability. Analysis of tryptophan fluorescence spectra showed that this domain
has a high stability and that the chemical denaturation occurs with two transitions. The
domain underwent a different stabilization in the presence of the different compounds
and the denaturation, differently, occurred with a single transition. Additionally, the
Smac mimetics promote a significant change in the CD spectra of free XIAP-BIR3
domain, indicating changes in the secondary structure content. Results obtained by ITC
revealed that, besides have different affinities, the enthalpy-entropy contributions of
each compounds are significantly different. The significant changes in the structure and
stability of XIAP-BIR3 promoted by the interaction with Smac mimetics, and that
contribute to the difference in the thermodynamic binding parameters, may be a
relevant factor to be considered in affinity optimization of anti-IAP drug candidates.
INBEB, FAPERJ, CNPq, CAPES.
G25
ANÁLISE DOS EFEITOS ESTRUTURAIS DE ÍONS COBRE E
ÁCIDOS NUCLEICOS NA PROTEÍNA PRÍON
1- CASALI, S.; 1- MACEDO B.; 1- GOMES, M.P.; 1- CORDEIRO, Y.
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro
Com o aumento da expectativa de vida média em alguns países, crescem também as
preocupações quanto às doenças neurodegenerativas. Destacam-se neste grupo as
encefalopatias espongiformes transmissíveis (EETs), cujo agente etiológico é a proteína
príon (PrP). Sua forma celular (PrPC) sofre uma conversão estrutural, levando à
formação da proteína prion scrapie (PrPSc), responsável pela agregação e a propagação
de espécies tóxicas no sistema nervoso central, onde é majoritariamente expressa. A
PrP é considerada promíscua, graças à descrição de diversos ligantes para ela, dentre os
quais encontram-se o íon cobre e os ácidos nucléicos, sendo o Cu2+ o ligante mais
descrito na literatura. Esses ligantes poderiam estar envolvidos tanto na patogênese,
facilitando o processo de conversão, quanto na função da PrP nos mamíferos. É
importante destacar que a interação da PrP com esses dois ligantes poderia acontecer
ao mesmo tempo no ambiente celular. Nosso grupo procura entender os efeitos dos
íons cobre na agregação e na estrutura da PrP selvagem de camundongo (PrPWT),
obtida por expressão heteróloga em E. coli, levando em consideração também o efeito
de moléculas de DNA no sistema. Este projeto constitui uma continuação de um
conjunto de dados desenvolvido por nosso grupo, o qual inclui também peptídeos da
PrP. Observamos anteriormente que cobre e pequenas moléculas de DNA (D67 e D44)
apresentam efeito sinérgico quanto à indução da agregação e alteração das estruturas
da PrP, sendo este mais pronunciado para os DNAs. Atualmente, estamos refinando
esses dados e adicionando outros utilizando não mais domínios da PrP isolados, mas a
PrP recombinante inteira, cujos resultados ainda são preliminares, mas incluem
dicroísmo circular, espalhamento de luz e fluorescência, além de calorimetria e
microscopia eletrônica. Esperamos, assim, trazer mais informações a respeito da
interação dual da PrP com Cu2+ e ácidos nucleicos, que podem auxiliar a compreender a
patogênese das EETs.
INBEB, CNPq, FAPERJ
G26
NANOSILVER
INNATE SUPRAMOLECULAR INTERACTIONS IN BIOGENIC
BALLOTTIN, D.1; 1- FULAZ, S.1; TASIC, L. 1
Universidade Estadual de Campinas - Instituto de Química 1 - Laboratório de Química
Biológica.
The aim of this work was to characterize the capping proteins around silver
nanoparticles produced by biosynthetic method using Fusarium oxysporum fungus. The
AgNP were characterized by TEM (spherical morphologies) and DLS (106 nm) and zeta
potential (-37 mV). Adhered proteins were quantified using Bradford Test and BSA as
standard (1.91 mg mL-1 in the fungal filtrate and 0.14 mg mL-1 in AgNP suspension) and
analyzed by electrophoresis (MW from 14.0 to 103.3 kDa). FTIR experiment results
exhibited characteristic proteins bands as, for example, in the region 3400-3500 cm-1
(N-H stretching), 2920-2950 cm-1 (C-H stretching vibrations), from 1620 to 1650 cm-1
due to aromatic rings, and at 1380 cm-1 and 1030 cm-1 associated to C-N vibrations.
Raman spectroscopy gave information about the involviment of cysteine residues, free
amine and amide groups in the AgNP formation and stalilization (changes in the
Página 22
intensity of Ag-N (240 cm-1) and Ag-S (233 cm-1) bands). Fluorescence spectrum
showed maximums at 350 nm, 422 nm and 489 nm that correspond, respectively, to
amino acid residues from fungal proteins (350 nm, tryptophan, tyrosine, cystine) and
AgNP. Shift in the emission maximum and decreasing in intensity (quenching) indicates
the interaction of the protein with AgNP surface. Circular Dichroism analysis allowed to
assess not only the structural composition of proteins present in FF and AgNP, as well as
conformational changes when these proteins interact with nanoparticles. There was a
slight loss of secondary structure, since changes in the percentage contents of helix α
and random coil were observed.
INBEB, FAPESP
G27
PEPTÍDEO BIOATIVO EM CÂNCER LUNASINA: UMA
CARACTERIZAÇÃO FÍSICO-QUÍMICA E ESTRUTURAL
SOUZA, S.M.A.; LIMA, L.M.T.R.; SOUZA, T.L.F.
Faculdade de Farmácia da UFRJ
Lunasina é um peptídeo de 43 aminoácidos, o qual foi descoberto em soja por sua
atividade antimitótica, mas também é presente em outras sementes e grãos, tais como
cevada, trigo, amaranto e centeio. Sua principal ação é alterar a dinâmica de
acetilação/desacetilação de histonas de células que sofrem transformação levando-as à
morte. Lunasina também é capaz de modular a expressão de genes e tem sido mostrada
sua atividade hipocolesterolêmica, anti-inflamatória e antioxidante. Muitos estudos têm
mostrado sua atuação em diferentes linhagens de células tumorais em modelos in vitro
e in vivo. Visto a sua promissora utilização como um biofármaco, e a escassez de
informações estruturais deste peptídeo, o objetivo do nosso trabalho foi analisar sua
estrutura em diferentes pHs simulando condições fisiológicas. Para essas análises
utilizamos dicroísmo circular (CD), espectroscopia de absorção no infravermelho
utilizando transformada de Fourier (FITR), espectroscopia por emissão de fluorescência
e cromatografia líquida de alta eficiência (CLAE). Medidas de dicroísmo circular
-hélice. Medidas por CD e FTIR indicam que
Lunasina é majoritariamente desestr
-hélice, a
qual sofre ligeira estabilização em pHs ácidos. Os resultados obtidos nos experimentos
de espectroscopia por emissão de fluorescência sugerem que o triptofano se encontra
exposto ao solvente e que não sofre alterações nos diferentes pHs testados. Análises
por filtração em gel (CLAE) demonstraram uma diminuição do raio hidrodinâmico do
peptídeo com a diminuição do pH, sugerindo que em pHs ácidos ocorra uma
compactação estrutural. Diante desses resultados, concluímos que a Lunasina possui
estrutura majoritariamente desordenada em solução com a presença de um pequeno
conteúdo de alfa-hélice que se torna mais estruturada em pHs ácidos. Esta flexibilidade
estrutural pode estar relacionada com as diferentes funções exercidas pela Lunasina.
INBEB, FAPERJ, CNPq.
G28
CONFORMATIONAL ENSEMBLES OF PRION PROTEIN: A
STRATEGY FOR MAPPING INTERACTIONS WITH PHYSIOLOGICAL AND THERAPEUTIC
LIGANDS
1,2-WESLEY ALVES; 1- REINALDO OLIVEIRA; 1- PEDRO TORRES; 2- BRUNO MACEDO; 1RAFAEL LINDEN; 2- YRAIMA CORDEIRO; 1- PEDRO PASCUTTI
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Faculdade de Farmácia, Universidade Federal do Rio de Janeiro
The prion protein (PrPC) is ubiquitously expressed in mammals, and anchored by GPI on
the outer leaflet of the plasmatic membrane. Three alpha helices, one short anti-parallel
beta-sheet, a large highly flexible domain, and two glycans compose the structure of
PrPC. The functional properties of PrPC are controversial, although a number of its
natural ligands have been described. Laminin receptor precursor (LRP), stress inducible
protein-1 (STI1) and neural cell adhesion molecule (NCAM) are three such proteins
ligands for which cognate interaction domains have been mapped onto each pair of
ligands. Our goal is to test whether the main function of PrPC may be to scaffold
signaling modules at the cell surface, through allosteric interactions with its protein
ligands, thus leading to effects on cell proliferation, differentiation and death. We use
techniques of molecular modelling, docking and dynamics to generate conformational
ensembles including PrPC with its flexible N-terminus, and to stabilize structural models
of interaction of PrPC together with its ligands. In addition, we use circular dichroism
(CD), X-ray small angle scattering, fluorescence anisotropy and calorimetry for in vitro
assays of binding and conformational effects among recombinant mouse PrPC and
synthetic cognate peptides from each ligands. CD and calorimetric experiments showed
apparent conformational changes undergone by PrPC in the presence of cognate
peptides added in a particular order, consistent with results from docking, and
suggesting that the affinity of the globular domain of PrPC for a given peptide is distinct,
depending on the previous binding of another ligand peptide. A novel protocol of
molecular dynamics was produced, specific for the generation of conformational
ensembles based on PrPC, including parameters for glycans. Promissory data are
consistent with the hypothesis that PrPC scaffolds cell surface protein ensembles, and
additional spectroscopy and simulation will provide further testing of allosteric
regulation of PrPC structure.
Página 23
Mestrandos
M1
ANÁLISE DAS ALTERAÇÕES OVARIANAS INDUZIDAS PELA
QUIMIOTERAPIA PARA O TRATAMENTO DO CÂNCER DE MAMA
1-MORAES, ACN; 2-ANDRADE, CBV; 2-RAMOS,IP; 3-SALATA,C; 1,4- NASCIMENTO ALR; 1CARVALHO, JJ; 1-STUMBO,AC
1-Departamento de Histologia e Embriologia, Universidade do Estado do Rio de Janeiro;
2 Laboratório de Cardiologia Celular e Molecular do Instituto de Biofísica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro; 3 - Laboratório de Ciências Radiológicas,
Universidade do Estado do Rio de Janeiro
"Introdução: Uma das mais utilizadas estratégias de tratamento para o câncer de mama
(CM) é a quimioterapia. O regime TC (docetaxel e ciclofosfamida) é o mais recente,
portanto existem poucos estudos relacionados a ele em relação a efeitos tardios, como
a osteoporose precoce. Mulheres na pré-menopausa submetidas à quimioterapia para o
tratamento do CM apresentam significante perda óssea a partir do primeiro ano após o
início do tratamento. A mais provável causa desta perda deve-se ao fato destes
quimioterápicos levarem a menopausa precoce. Como regra geral, a quimioterapia para
câncer de mama parece acrescentar cerca de 10 anos de idade ao ovário em termos de
função reprodutiva. Sabendo-se que alguns quimioterápicos para o tratamento do CM,
aumentam o risco de menopausa precoce, espera-se neste estudo, determinar se o
recente regime quimioterápico TC pode induzir danos ovarianos.
Materiais e Métodos: Ratas Wistar, com 3 meses, foram divididas em 2 grupos, tratados
com quimioterapia (TC) e controle tratados com placebo (NaCl 0,9%). Os animais foram
eutanasiados 5 meses após o término do tratamento. Foi realizada a coleta de sangue,
ovários e dos úteros para posterior análise. As técnicas utilizadas foram: aferição da
massa uterina e ovariana e concentração de estradiol sorológico por radioimunoensaio.
Resultados/Discussão: Os animais do grupo TC demonstraram redução significativa
(p<0.01) da massa do útero em relação ao controle, sugerindo atrofia uterina gerada
pela baixa de estrogênio (p<0.05), devido à ação dos quimioterápicos. Esse dado é
corroborado pela redução dos níveis de estradiol (p<0,001). Não houve alteração da
massa ovariana entre os grupos. Futuramente serão realizadas análises por
imunohistoquímicapara caspase3, TGF-beta, Colágeno tipo I e III, além de análise
ultraestrutural por microscopia eletrônica."
CAPES, FAPERJ
M2
A INFLUÊNCIA DA MANIPULAÇÃO EXTRACELULAR DE NA+
SOBRE OS TRANSPORTADORES RENAIS DE NA+ E CA2+: ESTUDOS IN VITRO E IN VIVO.
1-SOUZA, A.M.; 2,3-FERRÃO, F.M.; 2,3-LOWE, J.; 1-CUNHA, V.M.N.; 1,3-LARA, L.S.
1- Instituto de Ciências Biomédicas, UFRJ, 2- Instituto de Biofísica Carlos Chagas Filho,
UFRJ, 3-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem.
O acúmulo de Na+ no organismo não é exatamente proporcional a retenção de água. O
aumento localizado da concentração de Na+ pode contribuir para a hipertensão e lesões
em órgãos alvos, inclusive nos rins. Pouco se sabe como o aumento da ingestão de sal
ativa vias de sinalização que contribuem para o desenvolvimento e progressão da lesão
renal. Determinar os mecanismos moleculares ativados pela presença de alta
concentração de Na+ que levam a progressão da necrose tubular. Para os ensaios in
vitro, células LLC-PK1 foram incubadas por 1 h com NaCl 140 mEq (controle) e 170 mEq
(alta [Na+]). Foram medidas as atividades das ATPases transportadoras de Ca2+, Na+.
Nos entudos in vivo, ratos Wistar machos foram utilizados no modelo DOCA/Sal (dieta
com 4% de NaCl e acetato de desoxocorticosterona 8mg/Kg 2x/semana). Após 4
semanas os animais foram separados em gaiolas metabólicas e sacrificados e os rins
removidos. A região cortical foi dissecada para medidas de atividade enzimática.
Aprovado pela CEUA da UFRJ. Nas células LLC-PK1, o aumento da [Na+] aumenta a
atividade Ca2+-1×min-1, n=3,
p<0,05), sendo este evento mediado pelo aumento da atividade da SERCA. Nesta
condição, a atividade da (Na++K+)ATPase aumenta 3 vezes, sendo a Na+-ATPase
insensível. No modelo animal, tanto o grupo DOCA quanto o DOCA/Sal apresentaram
um aumento na pressão sistólica arterial e diminuição da função renal, associada ao
aumento da proteinúria. A atividade da (Na++K+)ATPase do grupo DOCA/Sal é reduzida
as ATPases renais respondem ao aumento da concentração extracelular de Na+ de
formas diferentes, dependendo do tempo de exposição. Os ensaios in vitro sugerem
que modulação do Ca2+intracelular pode ser o segundo mensageiro chave da função
tubular renal mediada pela alta ingestão de sal.
FAPERJ, CNPq
M3
ACTIN ROLE ON TRANSFERRIN ENDOCYTOSIS BY
TRYPANOSOMA CRUZI EPIMASTIGOTES
1-ALVES, A. A.; 1-ALCANTARA, C. L.; 1-VIDAL, J. C.; 1-CUNHA-E-SILVA; N. L.
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.
Trypanosoma cruzi epimastigote forms present high endocytic activity, differing from
trypomastigote and amastigote forms. Epimastigotes have two endocytic portals
(Soares, Parasitol. Res. 99:321, 2006): the flagellar pocket and the cytostomecytopharynx complex, described as a funnel-shaped opening, the cytostome that
invaginates deeply, forming the cytopharynx (Milder & Deane, J. Protozool. 16:730,
1969). Our group showed recently that specialized microtubules support the cytostomecytopharynx, playing a crucial role on its ultrastructure. Corrêa and coworkers (Exp.
Parasitol. 119:58, 2008) have shown a drastic reduction in transferrin uptake and
alterations in cytopharynx structure after treatment with Cytochalasin. Nevertheless,
the role of the cytoskeleton on endocytosis by T. cruzi is not clear. In this work we
focused on actin filaments, which are poorly characterized in T. cruzi, and its role on
endocytosis. We have used transferrin-FITC as tracer in endocytosis assays by
epimastigotes pretreated with Cytochalasin D, which destabilizes actin filaments, and
Latrunculin B, which binds to G-actin and hinders its polymerization. The results were
quantified by fluorimetry and also observed by fluorescence microscopy. We further
performed immunoelectron microscopy on ultrathin sections of parasites embedded in
LR White using a polyclonal antibody anti-Tc actin produced by Cevallos et al (Exp.
Parasitol. 127:249, 2011). Our results showed that Cytochalasin D and Latrunculin B
were both able to decrease transferrin endocytosis by about 80 %, without affecting
parasite viability. By fluorescence microscopy, we found the endocytic tracer retained at
the cytostome. Using transmission electron microscopy, we observed that anti-TcActin
antibody recognized thin filaments not yet described at the cytostome opening, among
other structures. These data suggest that actin acts on the initial stages of the
endocytosis in T. cruzi.
PIBIC/CNPq, CAPES, FAPERJ, CNPq
M4
S-NITROSOGLUTATIONA
REDUTASE
MODULA
PROLIFERAÇÃO E FUSÃO DE MIOBLASTOS
1-YAMASHITA, A.M.S.; 1-FIGUEIREDO-FREITAS, C.; 2-MERMELSTEIN, C.S.; 2POSSIDONIO, A.C.; 2-SOARES, C.P.; 1-SORENSON, M.M.
A
Página 24
1-Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de
Janeiro; 2-Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro.
A miogênese é o processo de formação da fibra muscular. Durante a miogênese do
tecido muscular esquelético diversos fatores levam o mioblasto a sair do ciclo celular, se
alongar, se alinhar, se aderir e fundir, formando miotubos. O ambiente redox da célula
pode atuar regulando a diferenciação do mioblasto. Foi mostrado que o radical livre
óxido nítrico (NO) tem um papel importante durante a formação do tecido muscular
esquelético. Uma das vias de sinalização por NO envolve a oxidação dos grupos tiol em
proteínas, formando SNO-proteína. Para controlar o estado redox intracelular, todas as
células sintetizam antioxidantes como a glutationa (GSH). O excesso de NO pode levar a
um aumento na produção de S-nitrosoglutationa (GSNO). Uma enzima importante para
controlar os níveis de GSNO é a S-nitrosoglutationa redutase (GSNO-R), que reduz GSNO
para GSH promovendo a denitrosilação de proteínas. Desta forma a GSNO-R controla os
níveis de S-nitrosotióis (SNOs). Diversos tecidos expressam GSNO-R, porém muito pouco
se sabe sobre essa enzima no músculo esquelético. O objetivo deste trabalho é analisar
o papel da GSNO-R durante a diferenciação muscular esquelética. Foi utilizada cultura
primária de músculo esquelético peitoral de embrião de galinha. Foi encontrada
atividade e a expressão da GSNO-R no tecido muscular esquelético. A cultura foi
crescida por 24 h e tratada por 48 h com inibidor da GSNO-R, o composto C3 (4-{[2-[(2ciano benzil) tio]-4-oxotieno [3,2-d] pirimidina-3 (4H)-il]metil} ácido benzóico), Snitrosocisteína (CysNO), ou o inibidor da óxido nítrico sintase 1 (L-NAME). Os
tratamentos com o C3 ou com a CysNO levaram a um aumento dos SNOs.
Imunofluoresc ncia contra rote nas musculares desmina e α-actinina) e para núcleos
(DAPI) permitiram quantificar a proliferação e a fusão dos mioblastos. Os tratamentos
com o C3 ou com a CysNO levaram a um aumento do número de núcleos das células
mononucleadas (mioblastos) e uma diminuição do índice de fusão. O L-NAME reverteu
os efeitos do tratamento com o C3 e levou a um aumento da espessura dos miotubos.
Estes dados demonstram a importância do NO e pela primeira vez revelam o papel da
GSNO-R modulando a diferenciação dos mioblastos.
M5
ENDOSYMBIOSIS IN TRYPANOSOMATIDS: THE ASSOCIATION
BETWEEN THE SYMBIOTIC BACTERIUM AND THE GLYCOSOMES OF STRIGOMONAS
CULICIS, AS REVEALED BY MICROSCOPY TECHNIQUES
Machado, A.C.L1, Catta-Preta, C.M.C.1, De Souza, W.1, Motta,C.M.1
1Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF-UFRJ
Strigomonas culicis is a trypanosomatid protozoan that co-evolves in a mutualist
relationship with a symbiotic bacterium and constitutes an excellent model to study the
origin of organelles and cell evolution. An interesting aspect of this relationship is the
intense metabolic exchange between the eukaryotic host and the prokaruyotic
endosymbiont. Based in this fact, the motion of our study was to investigate the
association between the symbiont and energetic organelles, as the mitochondrion and
glycosomes. Different microscopy approaches were used to observe the ultraestructure
of S. culicis, as confocal microscopy, transmission electron microscopy (TEM) and three
dimensional (3D) reconstruction using electron tomography. The immunofluorescence
assays revealed the great proximity between symbiont and glycosomes, that formed
clusters around the bacterium.. This association was confirmed by TEM since the
bacterium envelope was seen juxtaposed to the glycosome membrane, however a
fusion was not observed between both structures, as revealed by electron tomography,.
Such association was not observed between the bacterium and the mitochondrion.
Moreover, some mitochondrial branches were sometimes observed parallel to the
bacterium outermambrane, but this was a rare event. Preliminary data with the
aposymbiotic strain of S. culicis showed that glycosomes were randomically distributed
in the cytoplasm, either in the anterior or in the posterior part of the cell body. Taken
together, our results suggest that glycosome distribution is oriented according to the
symbiont localization. In accordance to this idea, our previous studies revealead that the
bacterium enhances O2 consumption and can benefits from host glycosomes energy
production Our next step is to analyse the symbiont association with other host cell
structures by the focused ion beam technique
INBEB, CAPES, CNPq, FAPERJ
M6
EVALUATION OF ANTIPLATELET PROFILE OF SYNTHETICS
PEPTIDES
SUCCAR,B.B.1; SILVA,M.C.C1; GERALDO,R.B.1; JULIANO,M.A.2; WERMELINGER,L.S.3 E
ZINGALI,R.B.1
1-Instituto de Bioquímica Médica Leopoldo de Meis, IBqM-UFRJ, RJ; 2-Departamento de
Biofísica, Escola Paulista de Medicina-UNIFESP, SP; 3-Faculdade de Farmácia, DACTUFRJ, RJ, Brazil.
"Introduction: Thrombosis can occur in the arterial or venous circulation, being the most
common cause of cardiovascular disease associated death. Nowadays, antiplatelet
drugs are effective in reducing arterial and venous thrombosis. However, there are
some side effects, which limits their use. Improvement on safety and effectiveness of
antiplatelet drugs is required. In this study, we analyzed the antiplatelet profile of five
new synthetics peptides (PS1045-C, PS1045-D, PS1045-E, PS1045-F and PS1045-H).
Methods: First of all, a theoretical study was performed using Clustal–W, Swiss Model
and Swiss-PDB viewer, and docking programs. This study lead to the design of new cyclic
peptides. Then, an automated bench-top simultaneous multiple solid-phase peptide
synthesizer (PSSM 8 system from Shimadzu) was use for synthesis of all the peptides.
These peptides were tested in human platelet aggregation assays using thrombin
(10nM), ADP (3-4µM) and collagen (2.5µM) as agonists. Discussion and Results: The
peptides PS1045-F and PS1045-H (200µM) are the most potent to inhibit platelet
aggregation (75%) when induced by ADP and thrombin. Additionally, the peptides
PS1045-C and PS1045-E (200µM) were able to inhibit platelet aggregation (50%)
induced by the same agonists. None of the cyclic peptides displayed inhibitory activity
against collagen-induced human platelet aggregation and the peptide PS1045-D does
not inhibit in any agonist. Preliminary results showed that four out of the five
synthesized peptides were able to inhibited platelet aggregation in a concentration
similar to that observed in the literature. Additional studies used in vivo models are
required in other to investigate the antithrombotic profile of these molecules.
Conclusion: Our study demonstrated that peptides might be as a tools to development a
new drug.
Sources of research support:
CAPES, FAPERJ, CNPQ, IMBEB
M7
CRIOFIXAÇÃO DE BIOFILMES DE CANDIDA ALBICANS COM
PRESERVAÇÃO DA MATRIZ EXTRACELULAR POLIMÉRICA OBSERVADO POR
MICROSCOPIA ELETRÔNICA DE VARREDURA
1- FONSECA, B. B.; 1- VILA, T. V. M.; 2 - ISHIDA, K.; 1- DE SOUZA, W.; 1- ROZENTAL, S.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio
de Janeiro; 2- Departamento de Microbiologia, Universidade de São Paulo, São Paulo.
Os biofilmes são comunidades microbianas embebidas em matrizes poliméricas (MEC)
produzidas por elas próprias. Os biofilmes podem desenvolver-se em qualquer
superfície úmida, seja ela biótica ou abiótica e são frequentemente relacionados com
infecções invasivas. Fungos da espécie Candida albicans são importantes formadores de
Página 25
biofilmes, sendo estes relatados por serem mais resistentes a drogas antifúngicas do
que as células planctônicas (FEMS Yeast Res. 6:979-986, 2006). Neste estudo, a
microscopia eletrônica de varredura (MEV) foi utilizada para estudar a morfologia do
biofilme e a preservação da MEC. Em estudos anteriores, com a utilização de fixadores
químicos, não conseguimos obter uma boa preservação da MEC, com isto no presente
trabalho tivemos como objetivo utilizar a criofixação para melhor visualização da MEC.
Realizamos um estudo comparativo de três fixadores químicos diferentes para biofilmes
de fungos: rotina, sacarose e vermelho de rutênio onde observamos uma boa
preservação de células nos biofilmes, no entanto, a MEC foi extraída do biofilme. Assim
propomos uma combinação de congelamento de alta pressão (HPF) com a substituição
a frio (FS) para melhor fixação de biofilmes de C. albicans e para avaliar a sua
capacidade de preservar a MEC. Os resultados mostraram que o método de criofixação
preservou melhor a estrutura geral do biofilme, permitindo a visualização de hifas e
blastoconídeos incorporado em uma matrix densa e bem preservada. Nenhum fixador
químico nos possibilitou o estudo e preservação do biofilme e da MEC, apesar de serem
técnicas bem aceitas para biofilmes bacterianos. O método de criofixação se mostrou o
melhor para a preservação da MEC e do biofilme de C. albicans.
FAPERJ, CNPq, CAPES, INBEB
M8
PHYSALIS ANGULATA INDUCES AUTOPHAGIC PROCESS
DURING DIFFERENTIATION OF MONOCYTES INTO MACROPHAGES
1,2- SILVA, B. J. M, 1,2,3- RODRIGUES, A. P. D, 1,2- FARIAS, L.H.S, 4- NASCIMENTO, J.L.M,
1,2- SILVA, E.O.
1- Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Belém, Brazil; 2- Instituto Nacional de Ciência
e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de
Janeiro, Ilha do Fundão; Brazil 3- Laboratório de Microscopia, Instituto Evandro Chagas,
secretaria de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil; 4Laboratório de Neuroquímica, Instituto de Ciências Biológicas, Universidade Federal do
Pará, Belém, Pará, Brazil.
"Monocytes and macrophages belong to mononuclerar phagocyte system, and are
derived from myeloid precursors in the bone marrow. Monocytes are blood circulation
cells that in response to cytokines and inflammation are induced to migrate into tissues
and differentiation into macrophages and dendritic cells.The process of differentiation
requires activation of autophagy, AKT pathway and caspase-8. After that macrophages
can undergo the activation process and protect the organism against pathogens and for
establishing an effective immune response. This study aim to evaluate the action of an
aqueous extract obtained from Physalis angulata roots (AEPa) in the autophagic process
and cell differentiation of monocytes into macrophages. Bone marrow cells were
obtained by flushing femurs extract, and maintained in cultures treated with AEPa at a
concentration of 100 µg/mL. Ultrasctructural analysis showed that treated cells
increased cytoplasmic volume, numerous cytoplasmic projections, apparent increase in
the number of endoplasmic reticulum and the presence of elongated mitochondria. In
addition, was observed the presence of autophagic vacuoles in the cytoplasm of treated
cells with AEPa. Flow cytometry analysis showed increased labeling LC3 in cells treated
with AEPa, indicating autophagic process. Nitro blue tetrazolium (NBT) cytochemistry
revealed that only cells with small cytoplasm volume showed an increased production
of ROS in the group of cells treated with AEPa. Flow cytometric analysis of CD11b and
F4/80 protein revealed that AEPa induces the differentiation of monocytes into
macrophages.No cytotoxic effect was observed in cells treated with AEPa when
compared to the control. These results demonstrate that AEPa is able to induce
autophagy in bone marrow cells promoting monocyte-macrophage differentiation.
CAPES, CNPq/UFPa, INBEB, FAPERJ, FAPESPA, MCT/CNPq/FNDCT/PROCAD-NF CAPES.
M9
IMMUNOMODULATORY EFFECTS OF KOJIC ACID ON
MONONUCLEAR CELLS OBTAINED OF MURINE BONE MARROW
ALMEIDA, C. M. 1,3; SILVA, B. J. M 1,3; RODRIGUES, A. P. D. 2,3; FARIAS, L.. H. S.
1,3;AZEVEDO, E.P.C.3,4; FOGUEL,D.3,4; SILVA, E. O. 1,3
¹ Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Brasil. 2 Laboratório de Microscopia
Eletrônica, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde do Ministério da
Saúde, Belém, Pará, Brazil 3 Instituto Nacional de Ciência e Tecnologia em Biologia
Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brasil 4
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Ilha do Fundão,
Brasil.
Bone marrow is soft and sponge-like material found inside bones that contains
hematopoietic cells responsible for development and proliferation of peripheral blood
cells. This process occurs through a complex interaction between stromal cells,
cytokines and growth factors that compound the bone marrow microenvironment. The
monocytes proliferation generated in the bone marrow and the differentiation in
macrophages plays a key role in the immune response. This process is important against
pathogens through the production of proteolytic enzymes and reactive oxygen species
(ROS). In this context, the research for drugs that enhance the innate immune response
is needed to restore the homeostasis and the immune response. Kojic acid (KA), a
secondary metabolite, synthesized by some species of fungi from Aspergillus genera,
has several applications as food additive, cosmetics, antitumor agent, macrophage
activator and anti-leishmanial agent. Thus, the aim of this study is to evaluate the
immunomodulatory effects of kojic acid (KA) in the bone marrow cells of mice. These
cells were obtained by flushing femurs, and maintained in cultures treated with KA at
the concentration of 50 and 100 µg/mL for 24-96h of culture. It was observed by optical
microscopy that KA promoted increased cell adhesion, spreading ability and high
number of cytoplasmatic projections and vacuoles in cytoplasm in the mononuclear
cells from bone marrow. To confirm these results, cytometer analysis of surface markers
showed that the KA induced cell differentiation in the bone marrow when these cells
were treated with KA for 96h. In addition, Akt pathway was analyzed by western blot.
KA seems to be able to activate the Akt signaling pathway that has a critical regulatory
role in cellular development, homeostasis and differentiation. Furthermore, no
cytotoxic effects were observed in cells treated with KA when compared to the
untreated bone marrow cells. Thus, KA acts as an immunomodulatory and is able to
induce bone marrow monocyte-macrophage differentiation process.
Supported
by
CAPES,
CNPq/UFPa,
INBEB,
FAPERJ,
FAPESPA,
MCT/CNPq/FNDCT/PROCAD-NF CAPES.
M10
BINUCLEAR COPPER(II) COMPLEXES MODIFIED WITH PYRENE:
DNA CLEAVAGE AND BINDING PROPERTIES
1- SAIBERT, C., 2- DA SILVA, M. P., 2- NEVES, A., 1- TERENZI, H.
1- Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade
Federal de Santa Catarina; 2- Departamento de Química, Universidade Federal de Santa
Catarina
In last years, binuclear complexes of copper(II) have gained attention because of the
large activity as DNA cleavage agents. We report here two new binuclear copper(II)
complexes, derived from the Cu2(L-TCl) (1), that present strong DNA cleavage
properties: Cu2(L-Tdab) (2) and Cu2(L-TN-Mepydab) (3), where L-TCl is 2-chloro-4,6bis(di-2-picolylamino)-1,3,5-triazine;
L-Tdab
is
2-diaminobutane-4,6-bis(di-2-
Página 26
picolylamino)-1,3,5-triazine and L-TN-Mepydab is 2-N-methylpyrenyldiaminobutane4,6-bis(di-2-picolylamino)-1,3,5-triazine. In this study, we compare the activity of the
complexes 2 and 3 towards 1, searching for structure-activity relationships, especially
for the pyrene-derived complex (3) since pyrene is a well know intercalating agent. The
analysis on the cleavage of DNA was made following the conversion of supercoiled form
of pBSK-II plasmid into its cleaved forms using agarose gel. Similarly, the cleavage of a
fluorescent end-labeled hairpin DNA by the complexes was investigated using urea page
polyacrylamide gel electrophoresis. Complexes 2 and 3 showed high activity in terms of
plasmid DNA cleavage, even at mild conditions (pH 7.0, 37°C), low concentration and
incubation time. The substitution of chlorine by butanodiamine and the posterior
addition of a pyrene moiety seem to increase the affinity of the complexes for DNA,
since KM decreases accordingly. Although, changing on the rate of cleavage was not
observed. The reaction mechanism seems to be oxidative and the complexes have
preference for the minor groove of DNA. The high resolution gel analysis suggests that
the complexes cleave DNA in the vicinity of a hairpin structure, mainly for complex 2
that presents a better cleavage pattern on the urea page polyacrylamide gel. The high
activity of 3 in comparison to 1 and 2 by the addition of pyrene motifs represents a good
strategy to increase the cleavage activity of metal complexes. However, the greater
cleavage pattern showed by the complex 2 upon the fluorescent end-labeled hairpin
DNA needs to be more investigated.
CNPq, CAPES, MCT, FINEP, FAPESC, INCT - Biologia Estrutural e Bioimagem
M11
OTIMIZAÇÃO DE HISTOGRAMAS 1D E 2D APLICADO A
RESULTADOS DE SIMULAÇÃO DE DINÂMICA MOLECULAR.
1- IGLESIAS JR, C. F.; 1- FERNANDES, T. V. A.; 1- OLIVEIRA JR, R.; 1 - PASCUTTI, P. G
1 - Instituto de Biofísica Carlos Chagas Filho
Sumariar resultados de simulações de dinâmica molecular em histogramas fornecem
três peças muito importantes de informação sobre as distribuições, como: forma,
localização central e spread. Quando se faz um histograma, se precisa escolher o ótimo
tamanho do bin que será utilizado para evitar os seguintes problemas: escassez de
amostras em cada bin fazendo com que a altura da barra em cada bin sofra uma
flutuação estatistica significativa e a perda de resolução impedindo que o histograma
represente a forma real da distribuição. Ter histogramas otimizados é crucial para
construirmos superficies de energia livre que representam um mapa com um amplo
espectro de conformações, que macromoléculas biológicas normalmente pode acessar
através molecular simulações de dinâmica.Com isso nosso objetivo foi criar um software
que disponibiliza aplicação de métodos e regras para se obter o tamanho ideal de um
bin que resolve estes dois problemas descritos acima.Os métodos Scotts rule e
Freedman-Diaconis rule estão disponíveis para otimização de histogramas em uma
dimensão e Método Shimazaki para otimização de histogramas em duas dimensões. O
software possui a licensa open-source e foi construido utilizando a linguagem de
programação Python. Para ilustrar o potencial do software, nos o utilizamos para
analisar os resultados de simulações de dinâmica molecular da proteina BID e suas
formas ativas tBID e tBID-myr no qual desempenha um papel chave na apoptose e estão
relacionadas a algumas doenças degenerativas. Gerando histogramas otimizandos para
valores de RMSD e Raio de Giro nos podemos criar superfícies de energia livre e
identificar conformações das proteinas BID, tBID e tBID-myr que estão em regiões de
menor energia livre e nas conformações das proteinas tBID e tBID-myr encontramos
cavidades que podem ser exploradas como alvo farmacologico. Ja que tBID e tBID-myr
somente atuam na apoptose e as cavidas encontradas não estão na superficie da
proteina BID.
INBEB, CNPq
M12
SIMULAÇÕES DE DINÂMICA MOLECULAR DE COMPLEXOS DE
CATEPSINA B DE FASCIOLA HEPATICA COM CA074, UM INIBIDOR DIPEPTIDIL NITRILA E
POTENCIAIS INIBIDORES SELETIVOS
1,2- FELICIANO, D.N.; 3-VALIENTE, P.A.; 4-GOMES, D.E.B.; 2- PASCUTTI, P.G.
1- Centro Nacional de Sanidad Agropecuaria; 2- Universidade Federal do Rio de Janeiro;
3- Universidad de la Habana; 4- Instituto Nacional de Metrologia, Qualidade e
Tecnologia
A Catepsina B ( fCatB ), a principal protease secretada a partir da fase juvenil de Fasciola
hepatica, é um alvo relevante para o tratamento quimioterapico da fasciolíase[1] e
também um alvo em potencial para o desenvolvimento de drogas para tratar diversar
enfermidades humanas [2]. No entanto, a estrutura tridimensional (3D ) da fCatB ainda
não foi resolvida experimentalmente. Isto constitui uma desvantagem importante para
o design baseado em estrutura de novos inibidores usando abordagens de triagem
virtual. Inibidores da fCatB revogam o ciclo de vida do parasita e apresentam uma baixa
seletividade para humanos ( hCatB ) e bovinos ( bCatB ), suas proteases homólogas em
seus hospedeiros definitivos.Prevemos inibidores seletivos putativos de fCatB
combinando modelagem comparativa (multiple structures complex template),
Molecular Docking (CA074, um inibidor da dipeptidil nitrila e compostos da bases de
dados Hitfinder-Myabridge) e simulações de dinâmica molecular . Uma previsão de
cavidades, interacções de ligante de ensaio e simulações de dinâmica molecular foram
feitas. Além disso, 13 resíduos potencialmente determinantes na especificidade de
substrato foram identificados podendo sugerir que árvore deles poderia melhorar o
design de inibidores seletivos da catepsina B de Fasciola hepatica em relação às
Catepsinas de mamíferos.
INBEB, CAPES, FAPERJ, CNPq
M13
BEX3, UMA PROTEÍNA NEGLIGENCIADA RELACIONADA AO
CÂNCER E DOENÇAS NEURODEGENERATIVAS, FORMA OLIGÔMEROS QUE PROTEGEM
CONTRA AGREGAÇÃO E PROTEÓLISE
RAYMUNDO, D. P. 1, CABRAL, K. M. S. 1, HILL, L. F.2, SILVA, V. S. 1, CORDEIRO, Y. 3,
CIRAUQUI, N.3 E ALMEIDA, M. S.1.
"1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brazil. 2Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 3Faculdade de
Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil."
"A proteína BEX3 (Brain expressed X-linked 3) é específica de mamíferos placentários e
é codificada por um gene que está em uma região do cromossomo X que contém genes
relacionados com a evolução do neurocórtex.
BEX3 atua nas vias extrínsecas e intrínsecas da apoptose através da interação com
p75NTRDD e Smac, respectivamente. Além destes, outros parceiros de interação com a
BEX3 já foram identificados como DRG-1, 14-3-3e e Hamartina.
Embora a BEX3 esteja envolvida com apoptose e provavelmente com câncer, além de
ser conservada em diversas espécies de mamíferos, muito pouco foi estudado a
respeito da estrutura desta proteína.
Ao longo do presente estudo, realizamos a caracterização estrutural da BEX3. Ensaios de
Ressonância Magnética Nuclear (RMN), Fluorescência Intrínseca do Triptofano (FIT),
Proteinase K (PK) e Dicroísmo Circular (CD) mostraram que esta proteína possui
estrutura secund ria em hélice α e regi es desordenadas no - e C - terminal,
caracterizando esta proteína como parcialmente enovelada. A BEX3 parece formar um
oligômero atra és de uma or o em hélice α. lém disso, e erimentos de modelagem
molecular ajudaram a descrever os resultados experimentais obtidos e ainda
Página 27
levantaram a hipótese de que BEX3 se apresenta na forma de duas subunidades
antiparalelas (coiled-coil). Dados de Microscopia de Força Atômica (AFM), crosslinking,
Espalhamento de Raios-X a baixo ângulo (SAXS), Tioflavina T (ThT) e Gel Filtração (GF)
mostram que BEX3 forma um oligômero solúvel com a formação de um núcleo
hidrofóbico (FIT, Bis-ANS e PK). Além disso, resultados de ensaios de fluorescência por
ThT e eletroforese em gel de poliacrilamida em condições desnaturantes (SDS-PAGE)
sugerem que esta proteína forma agregados que podem ter implicações fisiológicas."
CNPq, INBEB, FAPERJ
M14
CELL AND GENE THERAPY: COMBINING TWO APPROACHS TO
PROMOTE NEUROPROTECTION AND NEUROREGENERATION AFTER A MODEL OF CNS
INJURY.
1- NASCIMENTO-DOS-SANTOS, G.; 1- MESENTIER-LOURO, L.A.; 1- TEIXEIRA-PINHEIRO,
L.C.; 1- SILVA-JUNIOR, A.J.; 1- FIGUEIRÊDO, A.B.P.; 2- TEIXEIRA, C.; 2- TOVAR-MOLL, F.; 1MENDEZ-OTERO, R.; 1- PETRS-SILVA, H.; 1- SANTIAGO, M.F.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto Nacional de Tecnologia de Biologia Estrutural e Bioimagem.
After injury, the axons in the central nervous system (CNS) are not able to regenerate
great distances and permanently lose their connections in the brain. Two promising
approaches to reverse this condition are the cell and gene therapies. In previous work,
we demonstrated that the intravitreous transplantation of bone marrow mononuclear
cells (BMMC) were capable of promoting an increase in retinal ganglion cells (RGCs)
survival and axonal outgrowth 14 days after optic nerve crush. In the present work, we
evaluated the therapeutic potential of mesenchymal stem cell transplantation (MSC)
and pigment epithelium derived factor (PEDF) gene therapy using adeno-associated viral
vectors (AAV) in a model of rat optic nerve crush. In MSC transplantation adult (3-5
months old) Lister rats underwent unilateral optic nerve crush followed by MSC or
vehicle injection into the vitreous body. Sixteen and 28 days after injury, RGC survival
was evaluated assessing the number of Brn3a-positive cells in flat-mounted retinas, and
nerve regeneration was investigated after anterograde labeling of the optic nerve
axons. For gene therapy, adult (2-3 months old) Lister rats underwent unilateral optic
nerve crush 30 days after AAV-PEDF, AAV-GFP or vehicle injection into the vitreous
body. Twenty eight days after injury, RGC survival was also evaluated assessing the
number of Brn3a-positive cells in flat-mounted retinas. Cell therapy with MSC increased
the number of Brn3a positive cells in the retina and the number of axons distal to the
crush site both at 16 and 28 days after optic nerve crush when compared to control. The
PEDF gene therapy using adeno-associated viral vectors also increased the number of
Brn3a positive cells in the retina. Considering the results, our propose is to combine
these two therapies in order to enhance the effects we have obtained so far by
transplantation of the MSC previously transducted with AAV-PEDF.
CAPES, FAPERJ, INBEB, CNPq.
M15
WHAT IS THE BEST WAY TO PROCESS CAT INTESTINE TO
VISUALIZE TOXOPLASMA GONDII?
VERAS, GM1; DUBEY, JP3; DE SOUZA, W1, 2; ATTIAS, M1
1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Brasil. 2Diretoria de Metrologia Aplicada às Ciências da Vida do INMETRO 3United
States Department of Agriculture, Animal Parasitic Diseases Laboratory, EUA
Toxoplasmosis is a worldwide disease caused by the intracellular protozoan Toxoplasma
gondii. This parasite can infect any nucleated cell of any warm-blooded animal. The
transmission can be from the mother to the foetus by tachyzoites, by eating raw or
undercooked meat containing tissue cysts or by ingestion of contaminated food and
water with sporulated oocysts. The sexual cycle occurs only into the felids small
intestine where the oocysts are formed and released with the feces to the environment.
The aim of this work was to visualize the enteroepithelial stages of Toxoplasma gondii in
cat intestinal villus by scanning electron microscopy after cold fracture. For this, cats
were contaminated with tissue cysts, intestine segments or intestinal villi that were
gently scrapped from the ileum were fixed with 2.5% glutaraldehyde and 1%
formaldehyde, post-fixed with 1% of osmium tetroxide (OsO4) and 1.25% of potassium
ferrocyanide in 0.1 M sodium cacodylate buffer, pH 7.2, for 40 minutes. Then, the
intestinal villi were embedded in 10% gelatin with or without 2% of chitosan, re-fixed
with 0.5% glutaraldehyde and 0.5% formaldehyde in 0.1 M sodium cacodylate buffer for
1 hour at 4oC. After this, the samples were cut into small pieces and infiltrated in 2550% glycerol per 1 hour, then frozen by immersion in liquid nitrogen and fractured with
a razor blade. Fracture was followed by thawing and glycerol deinfiltration in water.
Some of the specimens were immediately dehydrated in crescent series of ethanol,
critical point dried or went to maceration protocol, where the samples were placed in
0.1% OsO4 solution in cacodylate buffer for 10 days before dehydration and critical
point drying. Segments of the ileum were post-fixed and dehydrated in crescent series
of ethanol. Then, the samples were frozen by immersion in liquid nitrogen, fractured
with a razor blade, thawed in 100% ethanol and critical point dried. After gold
sputtering, the samples were visualized in a Quanta 250 or in a Quanta 450 FEG
scanning electron microscopes. Cleaved planes showed different, and feline exclusive,
stages of the parasite at the upper portion of intestinal villi. Merozoites, schizonts,
gamonts and oocysts were visualized. Observation of the intestinal villus revealed
moreinformation than the intestine fragments because of the parasite localization,
usually between the nucleus and the microvilli. Either method (cleavage in frozen
ethanol or cleavage of cryoprotected samples, followed by long term maceration)
proved to be adequate to the observation of intraepithelial forms of Toxoplasma gondii.
However, observations made on a FEG-SEM had, as expected, a much higher resolution.
In conclusion, we can recommend fracture of samples frozen in 100% ethanol as a time
saving and reliable method of exposing the inside organization of tissues and cells.
INBEB, FAPERJ, CNPq
M16
CHARACTERIZATION
OF CRYPTOCOCCUS
NEOFORMANS POLYSSACHARIDE CAPSULE BY HIGH-RESOLUTION SCANNING
ELECTRON MICROSCOPY
Glauber R de S. Araújo1,2, Daniel G. I. Vieira1, Kildare Miranda1,2, Thais C. SoutoPadrón3, Danielle Cavalcanti2 , Daniela Leão2, Wanderley de Souza1,2 and Susana
Frases1,2*
1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 2Diretoria
de Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade
e Tecnologia, Rio de Janeiro, Brazil. 3Instituto de Microbiologia Prof. Paulo de Góes,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
The encapsulated fungus Cryptococcus neoformans is a common cause of lifethreatening disease in immunocompromised individuals. Its major virulence
determinant is the polysaccharide (PS) capsule. The native form of polysaccharides
appears to involve association with other molecules and create highly variable
ultrastructural configurations. Thus, the expression of ultrastructural polysaccharide
molecules can determine the progress and effectiveness of the innate immune response
against pathogens. In this scenario, there is a clear need for studies focused on a better
understanding the ultrastructure characterization of C. neoformans capsule. An
unsolved problem in cryptococcal biology is whether the PSs composing the capsule are
Página 28
linear or complex branched polymers, as well as the implications of this structural
composition in pathogenesis. We analyzed the dependence of capsular PS molecular
mass and the radius of gyration and we noticed a strong evidence against a simple linear
PS configuration. Shape factors calculated from dynamic and static light scattering
measurements in solution and viscosity measurements revealed values consistent with
polymer branching. All this physical data showed evidences of ultrastructure
modification during the pathogenic process. For that reason, we decided to use High
Resolution Electron Microscopy to visualize this PS complexity. Our results showed PS
spherical-like structures similar to other branched that was confirmed analyzing the PS
capsule by high resolution scanning electron microscopy and different fiber sizes
forming the PS capsule by atomic force microscopy (AFM).
FAPERJ, CNPq
M17
INVOLVEMENT OF THE TRANSCRIPTION FACTOR NFAT IN
PARKINSON'S DISEASE
1- DE FREITAS, J.A; 1- FOGUEL, D. 2- ROBSS, B.K.
1- Instituto de Biquímica Médica 2- Faculdade de Odontologia de Nova Friburgo,
Universidade Federal Fluminense
Parkinson's disease (PD) is a neurodegenerative disorder that is caused by the death of
midbrain dopaminergic neurons and affects nearly 5 million individuals worldwide.
There are a large number of studies suggesting that the mechanism which leads to
neuronal loss in PD consists by an abnormal accumulation of a protein known as αsynuclein and subsequent formation of intracellular protein aggregates called Lewy
bodies. Studies have shown that α-synuclein aggregates alter membrane fluidity and
increase calcium (Ca+2) influx, rise levels of intracellular calcium lead to the activation of
calcineurin phosphatase (CnA). Although, the main calcineurin target are Nuclear Factor
of Activated T-Cells (NFAT), its contribution to the PD is very poorly understood. NFAT
proteins directly regulate the expression of genes involved in the control of cell death by
apoptosis, as well as genes involved in the inflammatory process. Apoptosis and
inflammation are known to be key events in neurodegeneration, which is triggered
upon a remarkable increase in intracellular Ca+2. Therefore, the main goal of the
present project is to evaluate the involvement of NFAT in the neurodegenerative
process induced by aggregates of α-synuclein. Our initial results show that only
oligomers of α-synuclein were able to mediate increase in LDH release in primary
cultures of dopaminergic neurons and this effect was partially reversed when these
cultures are pretreated with cyclosporin, an inhibitor of calcineurin. In addition we
examined the involvement of monomers, oligomers and fibers in the synaptic events.
We found that only oligomers and fibers showed to be capable of causing a decrease in
Synapsin I, a protein involved in pre-synaptic events. These results suggest that the
NFAT may have a possible role in the process of neurodegeneration mediated by αsynuclein aggregates.
INBEB, CNPq, FAPERJ
M18
ACTION OF ALKALOID (+)-PHYLANTIDINE ON Leishmania
(Leishmania) amazonensis AND THE HOST CELL.
1,2* - MORAES, L. S. M.; 2,3- RODRIGUES, A. P. D.; 1,2 - FARIAS, L.H.S.; 1,2 - SILVA,
B.J.M.; 4 - DONZA, M.R.H.; 4 - GUILHON, G.M.S.P.; 1,2 - SILVA, E. O.
1 - Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, 2 - Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro. 3 Laboratório de Microscopia, Instituto Evandro Chagas, Secretaria de Vigilância em
Saúde, Ministério da Saúde, Belém, Pará. 4- Programa de Pós-graduação em Química,
Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Brazil,
Universidade Federal do Pará.
"Leishmaniasis is antropozoonotic disease caused by parasites of the genus Leishmania.
These parasites proliferate primarily within mammalian macrophages and promote a
diversity of clinical manifestations ranging from self-healing cutaneous lesions to fatal
visceral leishmaniasis. The treatment available is chemotherapy, but is limited by
toxicity and requires a long term treatment. The use of natural products from plants
currently plays an important role and source as antileishmania agent. (+)-Phylantidine, is
an alkaloid extracted from stem of Margaritaria nobilis of the family Phyllanthaceae.
Thus, the aim of this study is evaluated the effects of (+)-phylantidine on promastigotes
forms of Leishmania (Leishmania) amazonensis and host cell. Antiproliferative activity of
promastigotes forms was observed when parasites were treated with 50, 100 e 200
µg/mL, with reduction of 82.50%, 73.75% and 88.75%, respectively when compared
with non-treated parasites. In the period of 96 hours was observed an IC50 of 56.34
µg/mL. As the reference drug amphotericin B was used and observed reduction of 100%
in parasites treated with 0,1 µg/mL for 96 hours. The treatment with 100 and 200 µg/ml
of (+)-phylantidine promoted morphological alterations in the promastigote.
Transmission Electron Microscopy (TEM) analysis showed alterations in the membrane,
flagellar pocket and increases in the numbers of acidocalcisome. No citotoxic effect in
mouse peritoneal macrophages was detected after treatment with phylantidine.
However, ultrastructural analysis by Scanning Electron Microscopy (SEM) revealed
typical activated cell morphology in treated macrophages, with alteration in cell volume,
an increase in cytoplasmic projections and spreading ability. These results
demonstrated that alkaloid seems to be important not only for host cell but also to
promastigotas growth inhibition. Thus, (+)-phylantidine could be an alternative source
for Leishmaniasis treatment.
Keywords: Leishmania (Leishmania) amazonensis; (+)-phylantidine; ultrastructural
alterations; host cell viability."
CAPES, CNPq/UFPa, UFPA, INBEB, FAPERJ, MCT/CNPq/FNDCT/PROCAD-NF
CAPES/FAPERJ.
M19
RELAÇÕES FUNCIONAIS ENTRE PRPC E O SISTEMA CALICREINA
CININAS: (1)- ESTUDOS ENZIMÁTICOS.
1-Vellasco L; 1- Nascimento CR; 2-Vieira T; 2-Lima Silva J; 1-Mariante R; 1-Linden R; 1Scharfstein J
1 Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2 Instituto de
ioquímica Médica Leopoldo de Meis.
O sistema calicreína – cininas (SCC) é uma rota proteolítica iniciada pelo contato do FXII
com polímeros negativamente carregados, como o dextran sulfato (DXS). A forma ativa
do FXII cliva e ativa a calicreína plasmática, protease envolvida na liberação de
bradicinina (BK) através da clivagem do cininogênio de alto peso molecular (HK). A
ativação desta via apresenta impactos trombogênicos por ativação da via intrínseca da
coagulação e inflamatórios, em consequência da liberação de BK, C5a entre outros
mediadores. Polifosfatos e heparina, liberados em grânulos de mastócitos e plaquetas
ativadas são ati adores end genos do FXII. Foi sugerida ainda a ati a o elas
T’s
liberadas or neutr filos. ati a o do CC or T’s sugere um a el dos neutr filos
na regulação positiva de mecanismos tromboinflamatórios. Estudos demonstram a
expressão da proteína príon celular (PrPc) na superfície de neutrófilos, sendo esta
modulada por estímulos inflamatórios (Mariante et al., 2012). Considerando os achados
que mostram a ligação da PrPc purificada à heparina (Vieira TC et al., 2010; e dados não
publicados), hipotetizamos que a PrPc fosse capaz de mitigar a interação de ativadores
da via de contato (poly P, DNA, heparina) com FXII, modulando negativamente a
ativação do SCC. Com este propósito, investigamos o efeito da PrPc na ativação do SCC
Página 29
utilizando substratos fluorescentes que mimetizam o HK. Consistente com nossa
hipótese, observamos menor hidrólise do substrato em resposta ao DXS e heparina na
presença de PrPc purificada. Neutrófilos de animais selvagens versus animais PrPc-KO
foram incubados com DXS-FITC e a ligação da molécula fluorescente à PrPc foi
analisada. As análises de citometria não indicaram diferenças na endocitose de DXS-FITC
entre as células selvagem e PrPc-KO. Novos abordagens serão realizadas para
determinar se PrPc pode mitigar o impacto da ativação intravascular do SCC sobre o
perfil funcional de neutrófilos.
CNPq, FAPERJ, INBEB
M20
-
INVESTIGATION ON THE BEHAVIOR OF TRICHOMONAS TENAX
1,2- RIBEIRO, L. C. S.; 3- SANTOS, C.; 2-BENCHIMOL, M.
1- Laboratório de ultraestrutura celular, Universidade Santa Úrsula; 2- Instituto de
Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 3- Instituto
Nacional de Metrologia, Qualidade e Tecnologia, INMETRO
T. tenax has been considered a commensal of the human mouth found under conditions
of poor oral hygiene or associated with periodontal diseases. There are some
controversies concerning the pathogenicity of this protozoan. T. tenax is very similar to
Trichomonas vaginalis, a parasite that inhabits the human genitourinary tract and
known to induce damage to various mammalian cells. Currently, there is a discussion
whether T. tenax would be a genetic variant of T. vaginalis. The aim of this study was to
investigate the capacity of T. tenax to provoke damage to mammalian cells and also to
compare their cytotoxicity with T. vaginalis. For this, interaction assays of both protozoa
with host cells, such as MDCK, HeLa and 3D spheroids were performed with the ratio of
5:1 in different periods of time. The samples were examined by scanning electron
microscope (SEM) and the cytotoxic effects of the protozoa were analyzed by viability
assays such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
and Trypan blue. Our results indicated some similarities between both parasites, such as
(1) during the interaction T. tenax showed different forms; (2) T. tenax and T. vaginalis
were able to adhere on mammalian cells and both provoked injury; (3) both protozoa
presented cytotoxic effects on monolayer cells, as seen by MTT and Trypan blue
experiments. Interestingly, T. tenax provoked damage to 3D spheroids of oral cells. Take
together, these results indicated that T. tenax could be a parasite in vitro, not a
commensal, and further experiments are necessary to better confirm its pathogenicity
in vivo.
CNPq, FAPERJ, AUSU, PRONEX, INBEB
M21
G
EFEITO DAS MICROPARTÍCULAS DE EPIGALOCATEQUINA-3(EG G) N GREG
α-SINUCLEINA
1-FERNANDES, L.;1-BRAGA, C.;1-PALHANO,F.;1-FOGUEL,D.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
A Doença de Parkinson (DP) é caracterizada pela morte de neurônios dopaminérgicos na
substância nigra e pela presença dos Corpos de Lewy que são inclusões protéicas
intracelulares, majoritariamente, compostos por agregados amil ides de α-sinucleína
α-sin). Esses agregados são caracterizados pelo alto conteúdo de folhas-β e or serem
insolúveis. As terapias utilizadas no tratamento da DP, atualmente, são apenas
paleativas e alguns compostos têm sido testados na tentativa intervir no curso da
doença. Dentre os compostos estudados como estratégia para prevenir e tratar a DP
temos o epigalocatequina-3-galato (EGCG). EGCG é uma catequina, sendo o composto
bioativo mais abundante na folha do chá verde. Essa molécula é um antioxidante que
quando oxidado transforma-se em quinonas capazes de reagir com outras quinonas,
formando polímeros de EGCG. Interessantemente, sob condições controladas in vitro, o
EGCG polimeriza na forma de micropartículas homogêneas com 1-2um de diâmetro
(EGCG bead). Foi demonstrado que o EGCG possui um potente efeito inibidor na
agregação de proteínas amilóides, sendo capaz de interferir no processo de formação
desses agregados. O mecanismo de ação do EGCG ainda não é conhecido, no entanto,
sugere-se que há necessidade do EGCG sofrer oxidação e auto-polimerizar para exercer
sua ação. Neste trabalho, nosso objetivo foi avaliar o efeito dessas micropartículas de
GCG GCG bead , o idado, em inibir a agrega o da α-sin. Quando incubada sob
condições de agrega o na resen a do GCG bead, a roteina α-sin permanece com
estrutura secundária randômica, enquanto a amostra sem EGCG bead apresentou alto
conteúdo de folhas-β.
s centrifuga o e an lise or
-PAGE, encontramos a maior
arte da α-sin na fração solúvel quando incubada com EGCG bead, enquanto o controle
se mostrou agregado. sses resultados sugerem ue GCG bead é ca a de ligar α-sin
e inibir a formação de agregados amilóides.
INBEB,CAPES
M22
INTRACELLULAR AGGREGATION OF P53 PROTEIN IN HUMAN
GLIOBLASTOMAS
1,2-PEDROTE, M.M.;1,2-COSTA, D.C.F.; 1,2--OLIVEIRA, G.A.P.; 3-LIMA, F.R.S.; 1,2-SILVA,
J.L.
1- Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de
Janeiro; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e
Bioimagem, Universidade Federal do Rio de Janeiro; 3 - Instituto de Ciências
Biomédicas, Universidade Federal do Rio de Janeiro.
Introduction:
Glioblastoma Multiforme (GBM) is the most common and aggressive brain tumor in
humans. According to World Health Organization (WHO), it is classified as a grade IV
glioma. This feature is correlated to a high invasiveness from the tumor to the adjacent
normal nervous tissue. p53 is a tumor suppressor protein which plays an essential role
in preventing the development of cancer by inducing cell cycle arrest or apoptosis in
response to DNA damage. Mutations in TP53 gene is frequently associated to a high
probability of cancer development. Therefore it is a good target against cancer.
Mutations in p53 core domain normally affect protein stability, which may trigger to a
strong tendency toward aggregation. Studies from our group have show that p53
aggregation in a mixture of oligomers and fibrils sequestrates the native protein into an
inactive conformation, typical of a prionoid behavior. Furthermore, these aggregates
are present in tissue biopsies of breast cancer especially in more aggressive ones.
Objective: The main goal of this study is to evaluate whether p53 aggregates may be
present in human glioblastoma cells and participate during tumorigenic process.
Material and Methods: Primary GBM cultures obtained from deceased patients and
T98G and U87MG cell lines were evaluated against p53, oligomer and fiber antibodies
and imaged by confocal fluorescence microscopy. Results and Discussion: Our
preliminary data shows greater colocalization between p53 and amyloid fibers as
compared to oligomers. This behavior was observed in three GBM primary cultures. We
observed a major distribution of p53-oligomer colocalization in perinuclear regions and
a diffuse and citosolic distribution for p53-fiber colocalization. These cells also revealed
a strong labeling of p53 in nucleolus and weak nuclei localization. Conclusions: In
conclusion, our data suggest the presence of p53 oligomers and amyloid fibers in
human glioblastoma cells. Further investigation will be conducted to address the
participation of these p53 aggregates in other cell lines and to the tumor progression
and aggressiveness.
INBEB, Faperj and CNPq
Página 30
M23
HEME STIMULATES NA+/K+ ATPASE ACTIVITY TROUGH
HYDROGEN PEROXIDE GENERATION IN LEISHMANIA AMAZONENSIS
1,2-ROCCO-MACHADO, N. 1,2-COSENTINO-GOMES, D. AND ROCCO-MACHADO, N.
COSENTINO-GOMES, D. AND 1,2-MEYER-FERNANDES, J.R.
1-Instituto de Bioquímica Medica, Centro de Ciências da Saúde, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2-Instituto Nacional de Ciência e Tecnologia em
Biologia Estrutural e Bioimagens, Centro de Ciências da Saúde, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Introduction: A recent work of our group has shown the activation of a Na+/K+ ATPase
in L. amazonensis, through a signal transduction cascade involving the presence of heme
and PKC activity. Heme is an important biomolecule with a pro-oxidant and signaling
capacity. Recently, hydrogen peroxide (H2O2) has been considered an important second
messenger, being able to stimulate PKC activity in several models. Our goal in this work
is to investigate the role of heme-dependent hydrogen peroxide generation on Na+/K+
ATPase activity of L. amazonensis. Materials and methods: H2O2 released by L..
amazonensis intact cells was determined by the Amplex red oxidation method; The
Na+/K+ activity was calculated as the difference between 32Pi released in the absence,
and in the presence, of 1mM ouabain; For PKC assay was used the Kinase-Glo
luminescent kit and the PKC specific substrate neurogranin. Results and discussion: Our
results shows that increased concentrations of heme stimulated H2O2 generation in a
dose dependent manner, reaching its maximum at 2,5 μM and Na+/K+ ATPase was
stimulated by increasing concentrations of H2O2, reaching its maximum at 0.1 µM. In
order to investigate the role of PKC in this signaling pathway, we verified the production
of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and
inhibitor, calphostin C. Both had no effect on the generation of H2O2. Furthermore, we
found that PKC activity is increased in the presence of H2O2 and in the presence of
calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. Conclusion: The results
suggest that PKC is activated by H2O2 generated in the presence of heme, thus
activating the Na+/K+ ATPase. We are now investigating where this H2O2 is produced.
INBEB, FAPERJ, CAPES, CNPQ
M24
EFFECTS OF KOJIC ACID ON HUMAN NEUTROPHILS AND
DURING INTERACTION WITH LEISHMANIA (LEISHMANIA) AMAZONENSIS IN VITRO
1,2-FRADE, P. C. R.; 1,2-COSTA, J. P.; 2,3-RODRIGUES, A. P. D.; 1,2-FARIAS, L.H.S.; 1,2SILVA, E.O.
1-Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências
Biológicas, Universidade Federal do Pará; 2- Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem , Universidade Federal do Rio de Janeiro; 3Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Secretaria de
Vigilância em Saúde do Ministério da Saúde.
Neutrophils are phagocytes involved in the primary immune responses to eliminate
pathogens. Leishmania is a protozoan responsible for the one of the most important
infectious diseases in the world and chemotherapy is potentially useful, but besides the
high cost, these drugs are toxic and require a long period of treatment. Therefore the
search for new drugs is of great interest. In this work, we have used kojic acid (KA), a
secondary metabolite synthesized by some species of fungi from Aspergillus, Penicillium
and Acetobacter genera. KA has several applications, being used as a food additive,
cosmetics, antitumor agent, macrophage activator and anti-leishmanial agent. Thus, this
study was designed to determine the effects of KA- induced human neutrophils
activation and interaction with Leishmania (Leishmania) amazonensis.
Human
neutrophils were obtained from buffy coats donated from Hemocenter Fundation of
Para State. Cells isolation were performed using HISTOPAQUE® 1077-density-gradient.
Neutrophils were treated for 1 hour with 50 μg/mL of KA. No cytotoxic effect was
observed on the cells treated with 10-200 μg/mL of KA for 1 hour and 1-24 hours with
50 µg/mL of KA by colorimetric MTT assays. Treated neutrophils had a higher capacity
to phagocytose Leishmania parasites and decreased the number of intracellular
parasites a long time. Subsequently, was assessed neutrophil oxidative burst induced by
KA (reactive oxygen species-ROS, myeloperoxidase activity and nitric oxide-NO), which
are important leishmanicidal agents. Treated cells demonstrated microbicide activity
that occurred through ROS production, detected by Nitro Blue Tetrazolium (NBT) assay
and CellROX® green dye; increase myeloperoxidase (MPO) activity, however NO
production was not detected. In conclusion, these results show that KA is involved in
neutrophil activation might be via ROS production suggesting a possible control
mechanism for Leishmania infected neutrophils.
INBEB, CNPq, Capes.
M25
AÇÃO PARÁCRINA DAS CÉLULAS-TRONCO MONONUCLEARES
DERIVADAS DA MEDULA ÓSSEA (CDMO) MEDIADA POR ÁCIDO LISOFOSFATÍDICO
(LPA) NA PREVENÇÃO DA I/R RENAL
MATTOS, P.; GONSALEZ,S.R.; VIEYRA,A.; LARA,L.S.; EINICKER-LAMAS,M.
1 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 Instituto de Ciências Biomédicas
Devido a sua capacidade de diferenciação, as CDMO são elementos-chave para a terapia
regenerativa. No entanto, seus efeitos benéficos não são restritos a recuperação
tecidual, mas também por seus efeitos parácrinos mediados pelo LPA. O objetivo deste
estudo é caracterizar o efeito do tratamento com CDMO no modelo de I/R renal
avaliando a modulação sobre a autotaxina (enzima produtora de LPA) e sobre os
receptores de LPA. Ratos Wistar machos (~260 g) foram divididos nos grupos controle,
I/R e I/R+CDMO (n=5). Após a coleta de sangue e urina para a avaliação da função renal,
os animais foram eutanasiados e os rins removidos para os ensaios bioquímicos. Em
relação à função renal foi observado nos grupos I/R e I/R+CDMO: (1) aumento do
volume urinário, sem alteração no peso e no consumo de água; (2) a diminuição do
ritmo de filtração glomerular e da carga filtrada de Na+; (3) diminuição da concentração
sérica de K+. No entanto, o tratamento com CDMO preveniu o aumento do nitrogênio
uréico plasmático e da proteinúria e promoveu o aumento da excreção urinária de K+. A
concentração sérica de Na+ e o pH urinário não foram modificados nos três grupos. A
análise por Western blot detectou que o tratamento com CDMO preveniu o aumento
do conteúdo proteico dos receptores de LPA1 e LPA3 promovido pela I/R e diminuiu a
abundancia de autotaxina. Estes dados indicam que o tratamento com CDMO previne
parcialmente a perda da função renal através de sua ação tubular e não glomerular.
Este evento pode ser mediado pela normalização dos níveis proteicos do receptor de
LPA e pela queda da produção local deste lipídio pela diminuição do conteúdo de
autotaxina.
CAPES-PROBITEC,FAPERJ, CNPq-DECIT
M26
ESTUDO DA RELAÇÃO ENTRE O CICLO DE VIDA E O FENÓTIPO
MAGNÉTICO NA ESPÉCIE DE BACTÉRIA MAGNETOTÁTICA MAGNETOVIBRIO
BLAKEMOREI
1- LEÃO,P.E.; 1- ABREU, F.; 2- BAZYLINKI, D.A.; 1- LINS, U.
1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2School of Life Sciences, University of Nevada Las Vegas, USA
"Bactérias magnetotáticas são capazes de se alinhar as linhas do campo geomagnético
(CGM) utilizando-as para se guiar no gradiente de oxigênio encontrado em ambientes
aquáticos. Esta propriedade é possível devido à biomineralização de nanopartículas
Página 31
magnéticas no interior da célula chamadas magnetossomos. Os magnetossomos se
alinham em cadeia na porção central da célula. Neste arranjo ocorre à soma dos
momentos magnéticos das partículas e a cadeia funciona como um dipolo magnético:
norte e sul. Este dipolo está fixo no corpo celular por interações proteicas, assim o
torque exercido sobre a cadeia pelo CGM é transferido para a bactéria magnetotática,
fazendo com que ela fique orientada em relação ao CGM. O mecanismo que associa
esta orientação com a aerotaxia, que guiará o sentido de natação bacteriano, chama-se
magneto-aerotaxia.
Para o funcionamento do modelo de magneto-aerotaxia é preciso existir a diferenciação
entre dois fenótipos baseados na orientação do dipolo magnético: Tipo sul (TS), onde o
momento magnético está localizado de forma antiparalela ao flagelo celular e direção
de nado e Tipo norte (TN), onde o momento magnético está localizado paralelamente a
direção de ando. Segundo o modelo e a observação de amostras ambientais, o TS
predomina no HS e o TN no HN. Neste trabalho estudamos como este fenótipo é
preservado ao longo das gerações utilizando a bactéria magnetotática Magnetovibrio
blakemorei como modelo. O inóculo contendo células sem magnetossomos foi
adquirido após o cultivo em meio sem ferro. Com o inóculo, foram feitos três cultivos:
dois sob a influência de um campo magnético artificial (CMA) de mesma intensidade e
direção, porém sentidos opostos em cada condição, e outra condição controle, onde
não houve influência de um CMA. Realizamos a quantificação de cada fenótipo nos
tempos:10min/1h/3h/6h/12h/24h/36h/48h através de filtração e microscopia de
fluorescência. A microscopia eletrônica de varredura será utilizada para observação da
cadeia de magnetossomos e do flagelo no momento da divisão celular. Resultados
preliminares sugerem que os cristais sintetizados de novo já tem uma polaridade prédefinida e que o ciclo celular bacteriano auxilia na preservação do fenótipo dominante
na população."
FAPERJ, CNPq, CAPES
M27
ESTUDO DOS EFEITOS DAS MUTAÇÕES L81N, I88N, L81N-I88N
E L50S NA ESTABILIDADE DA PROTEÍNA CAPSÍDICA POR FLUORIMETRIA E DINÂMICA
MOLECULAR
1,2-Stoque, R.M.; 1,2-Gomes, D.E.B.; 1- Pascutti, P.G.; 1-Borges, R.S.M.
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Divisão de Metrologia Aplicada às Ciencias da Vida, Instituto Nacional de Metrologia e
Qualidade
"A dengue é uma arbovirose reemergente, transmitida ao hospedeiro humano pela
picada de fêmeas do mosquito Aedes aegypti. Estimativas da OMS apontam para cerca
de 50 a 100 milhões de casos de infecção por dengue a cada ano e 2,5 bilhões de
pessoas vivam em áreas com risco de infecção. No Brasil segundo o Ministério da Saúde
houve um aumento de 521.830 casos. Devido à inexistência de uma vacina e de drogas
antivirais, a principal forma de combate é o controle populacional do vetor, que tem se
mostrado ineficiente, tornando-se necessário um estudo mais aprofundado do vírus
para um melhor controle da doença.
O vírus da dengue é formado por um envelope composto pela proteína do envelope (E),
de membrana (M) e a bicamada lipídica, e um capsídeo formado pela proteína capsídica
(C). Quando madura, essa proteína possui 100 resíduos de aminoácidos, positivos e
hidrof bicos em sua maioria. la é formada or uatro α-hélices ligadas por pequenos
loops, com massa molecular de 12kDa que prontamente se dimeriza em solução, sendo
sugerido que essa seja a unidade funcional do capsídeo.
No presente trabalho, as proteínas contendo mutações simples, já descritas como
comprometedoras para a funcionalidade da proteína, foram expressas com sucesso em
E. coli. Os dímeros formados por proteínas contendo mutações simples (L50S e I88N)
apresentaram menor estabilidade se comparados àqueles formados pela proteína C
selvagem, sugerindo um papel importante destes resíduos hidrofóbicos para a
estabilidade do dímero. Simulações de Dinâmica Molecular foram então realizadas com
as proteínas mutantes L81N, I88N, L81N-I88N, e L50S, com a finalidade de entender o
efeito destas mutações na estrutura e estabilidade das proteínas. Os resultados
mostraram que as mutações propiciam a entrada de água no core hidrofóbico do
dímero, sem alterar significativamente a estrutura secundária dos monômeros, mas sim
afetando suas estrutura terciária e quaternária."
FAPERJ, CNPq, CAPES
M28
ESTUDO DE COMPLEXOS DAS CISTEÍNO-PROTEASES
PLASMODIAIS FALCIPAÍNA-2 E FALCIPAÍNA-3 PARA O DESENHO DE FÁRMACOS
ANTIMALÁRICOS
LEITE, R. G. G.1,2; PASCUTTI P. G.1,2
"1- Laboratório de Biologia Computacional, Diretoria de Metrologia Aplicada às Ciências
da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia - INMETRO; 2Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro - UFRJ."
"A malária é considerada um dos mais graves problemas de saúde pública mundial e sua
distribuição depende de fatores como temperatura, umidade e chuva. Os parasitas
causadores da doença são protozoários pertencentes ao gênero Plasmodium sp. No
Brasil as espécies P. vivax e P. falciparum são as responsáveis por, respectivamente, 15%
e 85% dos casos, e esta última pela maioria das mortes. O parasita é altamente
adaptável resultando na crescente resistência à terapia combinada à base de artemisina
(tratamento de primeira linha), porém esta estratégia está ameaçada devido ao
aparecimento de parasitas resistentes no sudeste asiático.
Através da conclusão do genoma do P. falciparum, quatro cisteíno-proteases do tipo
papaína puderam ser identificadas, caracterizadas e isoladas. Porém, as falcipaínas 2 e 3
(FP2/FP3) foram descritas como as principais hemoglobinases presentes no vacúolo
digestivo do parasita. Isso se comprova interrompendo o gene da FP2 que leva a um
acúmulo de hemoglobina não degradada.
Neste trabalho, estamos estudando dois inibidores diferentes de falcipaínas,
selecionados a partir dos resultados obtidos de trabalhos anteriores, que exibiram
melhores valores de área de contato, ligações de hidrogênio e energia livre das ligações
com cada enzima. Foi encontrado um terceiro inibidor promissor contra a FP3 devido ao
seu baixo valor de energia livre de ligação, ligando-se fortemente a FP3.
Simulações de Dinâmica Molecular indicaram uma grande flexibilidade das enzimas.
Como perspectivas, novas simulações estão em curso, assim como o ancoramento dos
inibidores nas enzimas para avaliarmos suas interações, focalizando-se no sítio ativo.
Também estão sendo realizadas alterações nas estruturas dos inibidores a fim de
desenvolvermos compostos mais seletivos e específicos para as falcipaínas."
PRONAMETRO, CAPES.
M29
INVOLVEMENT OF TRANSCRIPTION FATOR NFAT IN
Z EI ER’S ISE SE
1 - REQUIÃO, R.D.; 1 - FERREIRA, S.; 2 - ROBBS, B.K; 1 - FOGUEL, D.
1 – Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro. 2 –
Faculdade de Odontologia de Nova Friburgo, Universidade Federal Fluminense (FOUFF),
Nova Friburgo.
Alzheimer's disease (AD) is a neurodegenerative disease and the primary cause of
dementia in elderly. It is characterized by a progressive loss of cognitive function and it's
main symptom is the loss of recent memory. The responsible factor for the clinical
Página 32
manifestation of the disease is the progressive loss of synapses and neurons, especially
in the hippocampus region. An important feature of AD is the presence of amyloid
plaques, found in the extracellular region of patients' brains, composed mostly of the
peptide beta-amyloid (Aβ). Exposed neurons to Aβ display higher levels of intracellular
calcium, which could lead to the activation calcineurin A (CnA). CnA is a
Ca2+/calmodulin dependent phosphatase and one of it's main targets is the
transcription factor NFAT. Inhibition of NFAT leads to a prevention of both spine loss
and dendritic simplification. The primary objective of the project is to define the
molecular mechanisms of gene control by the transcription factor NFAT that may be
involved in the AD. We already demonstrated that mice primary neuron culture treated
with Aβ oligomers display NFAT translocation to the nucleus that can be completely
reversed by ciclosporin A (CsA), an inhibitory drug of the NFAT activation pathway,
suggesting an activation of this transcription factor. We also have evidence that these
primary cultures treated with Aβ peptides have a smaller amount of dendritic spines
and pre-synaptic terminals (in relation to the post-synaptic terminals), suggesting a
decrease of synapses, which is also reversed by CsA. Preliminary Real Time PCR results
revealed that cultures treated with Aβ oligomers show higher levels of PAK7 expression,
a gene related to cytoskeleton organization, and this expression is reversed by CsA.
Based on these results, we postulate that Aβ peptides are promoting a decrease of
spines and synapses through activation of the calcineurin/NFAT pathway through a
PAK7 overexpression.
CNPq
M30
-
CARACTERIZAÇÃO ESTRUTURAL DA PROTEÍNA TWA1
1-ARAUJO, T. S.; 2-CIRAUQUI, N.; 1- ALMEIDA, M. S.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Faculdade
de Farmácia, Universidade Federal do Rio de Janeiro.
Este trabalho tem como objetivo a determinação da estrutura tridimensional da
proteína Twa1, uma proteína nuclear de 228 aminoácidos, identificada em 2003,
através de um sistema de duplo-híbrido em levedura tendo como "isca" a proteína
RanBPM. A análise da sequência primária da proteína Twa1 mostra que a mesma possui
três domínios conhecidos: o domínio LisH, o domínio CTLH e o domínio CRA, dos quais
os dois últimos permanecem com função e estrutura desconhecidas. O domínio LisH é
frequentemente encontrado em proteínas envolvidas na segregação de cromossomos e
migração celular. Além disso, estudos mostram que este domínio está envolvido na
formação de homodímero. A Twa1 e RanBPM formam, em conjunto com outras
proteínas, um complexo denominado Complexo CTLH, que apresenta atividade
enzimática E3 ubiquitina ligase. A ubiquitinação de proteínas é uma importante forma
de modificação covalente que regula diversos processos biológicos. Para realização
deste trabalho três diferentes construções da proteína Twa1 foram produzidas em
Escherichia coli sendo elas: Twa1 selvagem, Twa 1-212 (ausência de 16 aminoácidos do
c-terminal que são sensíveis a proteinase K) e Twa1 66-209 (compreende os domínios
CTLH e CRA). Neste estudo, realizamos a caracterização estrutural das diferentes
construções através de ensaios de Gel Filtração, Dicroísmo Circular, Fluorescência
Intrínseca do Triptofano, Proteinase K e Ressonância Magnética Nuclear. Os resultados
obtidos mostram que as proteínas produzidas apresentam um enovelamento parcial e
com conte do de estrutura secund ria de α-hélice. No momento estamos buscando
melhorar as condições de produção da proteína recombinante Twa1 para que possamos
determinar sua estrutura tridimensional por RMN em solução ou cristalografia por
raios-X.
M31
INVESTIGAÇÃO IN SILICO DO MECANISMO DE INIBIÇÃO DA
PROSTAGLANDINA E MICROSSOMAL SINTETASE I (MPGES-1).
1-FROES, T.Q. (PG), 1- SOARES, D.M.(PQ), 1- CASTILHO, M.S.(PQ)
1- Facculdade de Farmácia, Universidade Federal da Bahia
A prostaglandina E microssomal sintetase I (mPGES-1) é a última enzima responsável
pela síntese de PGE2, principal prostanoíde envolvido na resposta inflamatória, a partir
de PGH2 usando glutationa como cofator. Dessa forma ela é considerada um alvo
promissor para o desenvolvimento de antiinflamatórios mais seletivos e seguros que os
AINEs e os inibidores seletivos de COX-2. Embora dezenas de inibidores de mPGES-1
tenham sido descritos, não existem dados estruturais que comprovem o modo de
ligação para nenhum deles. Por essa razão, empregamos técnicas de similaridade
química e modelos farmacofóricos para determinar o provável modo de interação de 5
classes de inibidores na estrutura cristalográfica de mPGES-1. Inicialmente 127
inibidores da mPGES-1 foram sobrepostos na bis-fenil-glutationa (análogo do cofator)
-octil glucosídeo (análogo do substrato) complexados com a mPGES-1 (PDB:
4AL1). A seguir, as 5 moléculas de melhor sobreposição foram utilizadas para gerar
modelos farmacofóricos, os quais foram avaliados quanto a capacidade de diferenciar
inibidores verdadeiros de falsos positivos. Em ambos os casos nenhum falso positivo foi
reconhecido, mas o modelo gerado a partir da bis-fenil-glutationa recupera um número
-octil glucosídeo
(108). Esses dados sugerem que a maior parte dos inibidores podem se ligar tanto no
sítio da gluationa quanto no sítio do substrato, o que está de acordo com dados
recentes da literatura. Visando corroborar essa hipótese realizamos o acoplamento
molecular de inibidores rígidos da mPGES-1 (derivados de fenantreno) na cavidade que
seria ocupada pelo cofator e o substrato. Os 5 inibidores de maior pontuação foram
utilizados para a construção de um novo modelo farmacofórico, o qual apresenta 2
doadores ligação de hidrogênio, 1 aceptor de ligação de hidrogênio e 5 grupos
hidrofóbicos ou aromáticos. Esse modelo é superior aos anteriores pois reconhece 124
inibidores.
INBEB, CAPES, CNPq.
M32
EFFECT OF EXTRACTS SCHINUS TEREBINTHIFOLIUS AND
PUNICA GRANATUM ON MAYARO VIRUS REPLICATION.
1,3- SALLES, T. S.; 1- MENESES, M. D. F.; 1- GUIMARÃES, T. ;2- KUSTER, R. M.; 3SOARES, M. R.; 1- FERREIRA D. F.*
1- Laboratório de Interação Vírus Célula, Departamento de Virologia, Instituto
Microbiologia Prof. Paulo de Góes. Centro de Ciência e Saúde, Universidade Federal do
Rio de Janeiro; 2- Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da
Saúde, Universidade Federal do Rio de Janeiro; 3- LAMMP, Departamento de
Bioquímica, Instituto de Química, Centro Tecnológico, Universidade Federal do Rio de
Janeiro. *[email protected]
Mayaro virus (MAYV) is an arbovirus belonging to the genus Alphavirus, Togaviridae
family which causes a disease known as Mayaro fever presenting symptoms similar to
the classic dengue fever. MAYV is endemic in tropical and subtropical regions on the
borders of the Amazon Basin. The Brazilian popular medicine explores the antiinflamatory properties of four substances extracted from the plants Schinus
terebinthifolius (known as mastic) and two substances Punica granatum (known as
Pomegranate). In this work we tested the antiviral effect of these substances against the
Alphavirus MAYV. The toxicity of these substances in VERO cells was tested by the
incorporation of neutral red after 24h of treatment. The CC50 of each substance was
determined. For evaluation of the antiviral effect, cells were infected for 1 h with
Mayaro virus using a multiplicity of infection of 0,1. Treatment of cells was carried out
Página 33
for 24 hours with increasing concentrations. The viral production was quantified by
TCID50 method and it was observed that the substances exhibit antiviral activity. The
selectivity index (ratio CC50/IC50), was determined and the values were greater than 7
for all six substances. We also tested the substances for virucide properties, adsorption
impairment, pretreatment, immunofluorescence and transmission electron microscopy
(TEM). Our results showed more than 95% virucidal effect for the partitions acetate,
flavonoids mastic and Crude Extract of pomegranate. Mastic oil and acetate of
pomegranate did not present virucidal effect. Virucidal effect was also visible by
immunofluorescence images. Was also observed in the TEM images damage in viral
particles treated with substances. We conclude that some of the partitions in our
substances act directly on the virus particle, and we are now assaying for this
interaction at the molecular level.
INBEB, FAPERJ, CAPES.
M33
EXPLORING BIOTECHNOLOGICAL FEATURES OF HYDROLYTIC
XANTHOMONAS AXONOPODIS PV. CITRI ENZYMES
1- QUEIROZ, V.L.; 1- SCHULTZ, L.G.; 1- MARIÑO, M.; 1- CYPRIANO, D.Z.; 1- LEITE, H.C.; 1TASIC, L.
1- Laboratório de Química Biológica, Instituto de Química, Universidade Estadual de
Campinas
"The search for renewable energy resources has become increasingly important as a
sustainable alternative to petroleum derived fuels. Brazil is the biggest producer of
oranges in the world, with 50% of the orange fruit used for obtaining juice and the other
50%, comprising bagasse, is currently dried and processed into pellets for livestock feed.
Orange bagasse has high levels of carbohydrates, high susceptibility to enzymatic
hydrolysis and low costs; moreover, its use does not compete with food production.
Such features make the orange bagasse a promising carbon source for secondgeneration ethanol production.
Previous studies have shown that whole enzymatic extract from Xanthomonas
axonopodis pv. citri (Xac), a potent pathogen responsible for the citrus canker disease,
was capable of hydrolyzing orange bagasse that was later fermented to produce
second-generation ethanol.
The aim of this work is to improve the efficiency of hydrolysis of orange bagasse for the
production of second-generation ethanol through the study of hydrolases purified from
Xanthomonas axonopodis pv. citri. Herein, we compared the structures of our target
enzymes to the ones already crystallized and combined these results with peptide-signal
prediction tools to construct primers for cloning of these enzymes. Physicochemical
characterization and substrate kinetics will be done after successful enzyme
purification. We expect that cocktail of purified Xac enzymes can provide higher yields
of fermentable sugars from orange bagasse and, consequently, higher yields of ethanol2G in fermentation processes.
INBEB
M34
MOLECULAR CLONING AND EXPRESSION OF DISINTEGRINS
FROM THE VENOM OF BOTHROPS JARARACA
1 - DAVID, V.; 1 - GUERRERO, T.; 1 - GERALDO, R. B.; 3 - ALBANO, R. M.; 2 WERMELINGER, L. S.; 1 - ZINGALI, R. B.
1Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, RJ, Brazil; 2 Departamento de
Análises Clínicas e Toxicológicas, UFRJ, RJ, Brazil; 3 Departamento de Bioquímica, UERJ,
RJ, Brazil;
Introduction: Snake venoms contain several components, including disintegrins that are
a family of small peptides able to bind to integrin receptors. These integrins are
glycoproteins that play a fundamental role in regulating physiological and
pathophysiological processes, such as in arterial thrombosis. Disintegrins have been
used to develop new drugs some are now used in treatment of human diseases. Two
disintegrins isolated from Bothrops jararaca, in our laboratory, called jarastatin (JARC)
and jararacin (JAST), have the ability to inhibit platelet aggregation. In this study, we aim
to clone the rJARC and rJAST disintegrins from the venom of Bothrops jararaca using the
vector pET-15b, aiming to have enough amount of peptide to structural studies, once
the last vector pET-32a was unsuccessful. Materials and Methods: The disintegrins were
amplified from the cDNA library using Bothrops jararaca gland. Then, an agarose gel
1.5% was performed and the DNAs were extracted and digested with restriction
enzymes. The products of these reactions were subcloned into Pet-15b and transformed
in bacteria Escherichia coli BL21. After that, E. coli was coated on agar, containing
ampicillin and chloramphenicol, and incubated at 37°C for 16h. The colonies obtained
were grown in circle growth medium to verify the integrity of the plasmid by enzymatic
digestion and the expression. Results and Discussion: Amplification of the cDNA was
confirmed by the profile of agarose gel, showing bands corresponding to 243 bp of
inserts of disintegrins. After transfection of the plasmid the bacteria growth was
observed as colonies on the plates. In addition, the integrity of the plasmid was
confirmed to rJAST through the agarose profile gel that showed bands of 243 bp and the
expression was successful, while rJARC showed several random bands. Conclusions: It
was possible to clone the rJAST successfully. New trials in different conditions are
required for cloning of rJARC.
FAPERJ, CNPq and Capes
Página 34
Doutorandos
D1
BONE MARROW MONONUCLEAR CELL THERAPY AFTER
GLOBAL CEREBRAL ISCHEMIA IN RATS
1- RAMOS, AB., 1- BARRIA, AC., 2- NEVES, G., 2- CINTRA, WM., 1- MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro.
"Introduction: The pyramidal neurons of the hippocampal CA1 layer are essential for
cognitive functions and selectively destroyed after global cerebral ischemia. Bone
marrow mononuclear cell (BMMC) therapy has shown positive results in preclinical
models of focal cerebral ischemia, but has not been studied in transient global ischemia.
Aim: The aim of our study was to investigate whether BMMC treatment could reduce
the neurodegeneration and the inflammation observed in CA1 layer of ischemic
animals.
Methods: Transient forebrain ischemia was performed by four vessels occlusion
method. To establish the time course of CA1 neurodegeneration CA1, the rats were
transcardially perfused 3, 7, 14, 21 and 90 days after ischemia (DAI). To analyze the
effect of BMMC therapy in neurodegeneration, neuronal survival and reactive
microgliosis in CA1 layer, ischemic animals received 3x107 BMMC 3 DAI, in the left
carotid, and were sacrificed different days after ischemia. For quantification of neuronal
degeneration, survival and reactive microgliosis, we counted Fluoro-Jade C, NeuN and
ED-1 positive cells in CA1 layer, respectively.
Results: We observed a greater number of FJC positive cells in CA1 layer of ischemic
animals 7 DAI when compared with the others days. The time course of neuronal
survival shows a reduction of pyramidal neurons 7 DAI and in the days after. In the
analysis of the effect of BMMC in neurodegeneration and neuronal survival, we
observed a significant number reduction of FJC positive cells and increase of NeuN
positive cells in CA1 of animals injected with BMMC and sacrificed 7 DAI. Furthermore,
we observed a lower number of ED-1 positive cells in ischemic animals treated with
BMMCs compared with ischemic animals that received only saline.
Conclusion: We suggest that the BMMC therapy in transient global ischemia has a
neuroprotective effect in CA1 layer that was followed by reduction of microgliosis in this
region."
CNPq, Pibic, FAPERJ
D2
TRATAMENTO SISTÊMICO COM GUANOSINA EM UM MODELO
DE LESÃO NO NERVO ÓPTICO
SILVA-JUNIOR, A.J.; MESENTIER-LOURO, L.A.; ZAVERUCHA-DO-VALLE, C.; MENDEZOTERO, R.; SANTIAGO, M.F.
Instituto de Biofísica Carlos Chagas Filho
Optic nerve lesions can lead from partially loss of vision to complete blindness. These
types of lesions affect especially the retinal ganglion cells (RGCs). One potential therapy
for this case is the administration of the nucleoside guanosine. The guanosine treatment
has demonstrated neuroprotective effects in several models. This work invesgated the
putative neuroprotective effect of systemic administrations of guanosine in a model of
optic nerve lesion. Lister-Hooded rats were submitted to crushing of the optic nerve,
followed by intraperitoneal injection of guanosine or vehicle. The treatment was
evaluated in three distinct protocols. In Protocol 1the groups received just only one
guanosine injection (7,5 mg/kg). In Protocol 2, the animals received a daily injection of
guanosine for seven days (7,5 mg/kg). In Protocol 3, the animals received 1 injection
every 12 hours for seven days (60 mg/kg). The experimental groups used in Protocols 1
and 2 were evaluated fourteen days after surgery, while the groups were analyzed after
seven days. The RGC survival was assessed by Brn3a immunostaining in whole mount
retinas. The number of RGCs in the fellow contralateral eye was used as control. There
was no significant difference in the number of RGCs in guanosine groups when
compared to the vehicle groups in all experimental protocols tested. The number of
extended axons beyond the lesion site in guanosine-treated animals was similar to the
vehicle-treated ones. We conclude that the guanosine systemic treatment using these
doses and therapeutic windows was not effective to promote the survival and axonal
extension in RGCs after optic nerve lesion.
D3
STRUCTURAL STABILITY OF MS2 VIRUS-LIKE PARTICLES
DISPLAYING FOREIGN PEPTIDES.
1- VICENTE, A.C.S.; 1- BARROSO, S.P.C.; 1- OLIVEIRA, E.G.; 1- OLIVEIRA, G.A.P.; 2PEABODY, D.S.; 3- FERREIRA, D.F.; 4- CORDEIRO, Y.; 5- DE MESQUITA, J.F.; 1- SILVA, J.L.
& 1- OLIVEIRA, A.C.
1- Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis,
Universidade Federal do Rio de Janeiro; 2- Department of Molecular Genetics and
Microbiology, University of New Mexico; 3- Instituto de Microbiologia Prof. Paulo de
Góes, UFRJ; 4- Faculdade de Farmácia, UFRJ; 5- Universidade Federal do Estado do Rio
de Janeiro.
Virus-like particles (VLPs) can be considered as dense arrays of one or more repetitive
subunits of a protein and this characteristic confers highly advantageous properties for
their use as vaccines platforms. The stability of a virus-like particle (VLP) is an important
consideration for its use in nanobiotechnology. Here, we describe a platform for vaccine
development based on the VLPs of bacteriophage MS2. Peptides representing the V3
loop of HIV gp120, the ECL2 loop of the HIV coreceptor, CCR5, and the epitope Flag
were inserted into a surface loop of MS2 coat protein. The effects of insertion of these
peptides on the structural stability of MS2 VLPs were assessed by dynamic light
scattering, small-angle x-ray scattering (SAXS), intrinsic and extrinsic fluorescence and
circular dichroism (CD). We analyzed the morphology of VLPs by transmission electron
microscopy. In addition, we predicted the structure of the coat protein containing the
different inserts. The VLPs displaying the Flag, V3 and ECL2 peptides on their surfaces
showed a different structure and lower stability than VLP formed by the native coat
protein. CD measurements indicate no significant changes in secondary structure
between VLPs under high concentration of urea. SAXS data show that the effect of 3
hours of pressurization was not able to promote the VLPs disassembly. Our results
demonstrate that the VLPs assembled from coat protein containing peptides insertions
behave slightly differently from the ones assembled from native coat protein, however
they showed structural stability under most of the conditions used, suggesting that
these particles are very promising for application as a vaccine platform.
CAPES-FAPERJ-CNPq-PRONEX-INBEB
D4
ENVOLVIMENTO DA VIA AMPK-eNOS-N κB N
P PE
NEUROPROTETOR DO SILDENAFIL (VIAGRA®) EM MODELO DESMIELINIZAÇÃO
1- NUNES, A.K.S; 2-RAPÔSO C; 1 - OLIVEIRA W.H; 1-BARBOSA K.P; 1-ROCHA S.W.S, 2CRUZ-HOFLING M.A; 1-PEIXOTO C.A.
1-Laboratório de Ultraestrutura - Instituto de Pesquisas Aggeu Magalhães - FIOCRUZ; 2Departamento de Histologia e Embriologia, Universidade Estadual de Campinas –
UNICAMP
Página 35
Recentemente, foi demonstrado que o sildenafil (Viagra®) tem papel neuroprotetor,
inibindo a inflamação e desmielinização no cerebelo. No entanto, os mecanismos de
neuroproteção ainda são desconhecidos. O AMPK é uma proteína reguladora do
metabolismo lipídico e glicídico e sua ativação modula processos inflamatórios,
possivelmente através da ativação da eNOS. O fator de transcrição nuclear NFkB
desempenha um papel importante na regulação da expressão de mediadores
inflamatórios. Estudos têm mostrado que a sinalização através da AMPK pode suprimir a
ativação do NFkB, controlando a inflamação. O presente trabalho utilizou ressonância
magnética (RM) e investigou se a via AMPK-eNOS-NFkB está envolvida na
neuroproteção, em modelo de desmielinização induzido por cuprizona. Cinco
camundongos machos (C57BL/6) foram separados por grupo e tratados por quatro
semanas: cuprizona (CPZ) 0.2% misturada na ração; CPZ na ração associada à
administração de sildenafil (25 mg/kg) por via oral, começando concomitantemente
(sild-T0) ou 15 dias (sild-T15) após o início da CPZ. Os controles receberam ração e água
puras. Os animais foram submetidos a análises por RM e os cerebelos foram
processados para western blotting. A análise por RM mostrou que, após a ingestão de
CPZ, os animais apresentaram redução do corpo caloso e dilatação dos ventrículos
cerebrais, indicando perda de estrutura. Os animais do grupo sild-T0 apresentaram
recuperação parcial da estrutura do corpo caloso, bem como diminuição da dilatação
dos ventrículos, o que não foi observado no grupo T15. A expressão de AMPK inativa
aumentou e os níveis de eNOS diminuíram após a administração de CPZ. Apenas o
tratamento com sildenafil (T0) reverteu esses efeitos, aumentando significativamente a
expressão da eNOS e diminuindo os níveis de AMPK. De forma semelhante, a expressão
de NFkB total foi aumentada após a ingestão de CPZ. Sildenafil (T0 e T15) diminuiu
significativamente os níveis de NFkB. Esses dados indicam que os efeitos
neuroprotetores do sildenafil envolvem a via AMPK-eNOS- FκB, sendo esta droga
potencialmente útil no tratamento de doenças desmielinizantes.
INBEB, PAPES, FACEPE, CNPq, CAPES
D5
A TIME-DEPENDENT NEUROINFLAMMATORY RESPONSE
AFTER ACUTE RESTRAINT STRESS
Oliveira-Poleto, A.L.; Guercio, G.D.; Panizzutti, R.
1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro
"Stress comprises a series of physiological events that prepare the organism for a
challenge. Acute stress induces cognitive deficits, which is accompanied by
inflammatory response in some brain structures. In a previous study we found that
acute stress decreases the levels of the neuromodulator D-serine in the frontal cortex
but not in the hippocampus. Therefore, our aim was to investigate whether the
inflammatory response is important for the stressinduced decrease in D-serine levels. To this end, we used imunohistochemistry to
evaluate microglial activation and astrogliosis in after 90 or 180 min of acute restraint
stress protocol in C57Bl/6 male adult mice. We observed increased microglial activation,
in the frontal cortex but not in the hippocampus, after 90 min of stress. This increase in
microglial activation in the frontal cortex was not observed after 180 min of stress. The
astrogliosis measured using GFAP in the frontal cortex was not affect after 90 min of
stress but it was reduced after 180 min of the stress. In the hippocampus astrogliosis
was reduced after 90 min of stress and increased after 180 min of stress. These results
indicate that our experimental model is capable to induce different profiles of
inflammatory response in frontal cortex and hippocampus. Further analyses must be
made to understand if this process is involved in the stress-induced decrease in D-serine
levels, and if anti-inflammatory interventions could ameliorate the cognitive deficits
induced by acute stress."
CNPq, Faperj
D6
MODULATION OF THE ECTO-3’NU E I SE
I I Y
EIS
NI
Z NENSIS BY 5’NU E I ES.
1-FREITAS-MESQUITA, A. L., 1- GOMES, M.T., 2-VIEIRA, D. P., 2-LOPES, A. H. C. S., 1MEYER-FERNANDES, J. R.
1- Instituto de Biquímica Médica Leopoldo de Meis, Universidade Federal do Rio de
Janeiro; 2- Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do
Rio de Janeiro
INTRODUCTION: Leishmania amazonensis parasites are intracellular protozoa and the
etiological agent of cutaneous leishmaniasis. The flagellated metacyclic promastigote
forms are transmitted to vertebrate hosts by sandfly bites and develop into amastigotes
inside macrophages. During this lifecycle, membrane interactions between parasites
and hosts are crucial for its survival, from both physiological and immunological
viewpoints. Cytoplasmic membranes contain enzymes whose active sites face the
external medium rather than the cytoplasm. The activities of these enzymes, referred to
as ecto-enzymes, can be measured using intact living cells. Leishmania species present
an ecto-en yme that hydroly es both e tracellular 3’-nucleotides and nucleic acids. The
hydrolysis of 3’ MP generates adenosine that lays im ortant roles in immunological
events and in the acquisition of purines, since trypanosomatids are unable to synthesize
purines de novo. This work aimed to investigate and characterize the modulation of
ecto-3’nucleotidase acti ity from . ama onensis by 5’nucleotides. M TERIAL E
METHODS: Ecto-3’nucleotidase acti ity was determined colorimetrically by measuring
the content of generated phosphate after one hour of reaction. The reaction medium
contained buffer solution, 3 mM 3’ MP, . ama onensis romastigotes and increasing
concentrations of different 5’nucleotides. R U T
I CU I : Micromolar
concentrations of guanosine, 5’GMP, G P and GTP inhibited ecto-3’nucleotidase
activity, which was not observed when other nucleotides were tested. The analysis of
kinetic parameters indicated that in the resence of 5’GMP the a arent Km and Vma
for 3’ MP hydrolysis were lower than control alues, which suggests the occurrence of
an uncom etiti e inhibition. It is already known that 3’ MP increases macro hage
infection by parasites. We obser ed that 5’GMP was able to attenuate the increase of
interaction romoted by 3’ MP. C C U I : tracellular nucleotides are intimately
involved in the regulation of ecto-3’nucleotidase acti ity. The determination of these
regulatory mechanisms is very important, since this enzyme seems to play an important
role in the establishment of Leishmania infection.
CNPq, CAPES and FAPERJ (INCT – INBEB)
D7
INTERFERENCE OF MYOSIN VA OR VB FUNCTION DISRUPTS
HUMAN RHINOVIRUS 14 REPLICATION
1,2- Real-Hohn, A.; 3- Provance Jr., D.W.; 2- Salerno, V.P.; 1- Gomes, A.M.O.
1- Instituto de Bio u mica Médica eo oldo e Meis, UFRJ - scola de duca o F sica
e Desportos, UFRJ; 3- Centro de Desenvolvimento Tecnológico em Saúde, Fiocruz
Human rhinovirus infections are the major cause of the common cold. Following cell
entry, the virus releases its RNA genome into the cytoplasm and commandeers the
resources of the cell for replication. The mechanisms for translocation of the virus
genome to the viral replication center in the perinuclear region remain unknown.
ndogenous myosin V’s are e cellent candidates since they are in ol ed in both R
transport and intracellular trafficking from the periphery to the cell center. Dominant
negative expression vectors, immunofluorescent localization and immunoprecipitation
studies all suggest that both myosin Va and Vb function contribute to viral replication.
Furthermore, the results suggest that each participates at different stages in the cycle
Página 36
with myosin Va early and myosin Vb late. Together, the data provide greater insight into
the cellular resources utilized by HRV-14 to propagate, which have implications in the
processes involved with Poliovirus, Hepatitis A virus and Foot-and-mouth disease virus.
INBEB, FAPERJ, CNPq, Capes, FINEP, PRONEX
D8
NMR STRUCTURE OF Q4D059, A CONSERVED AND SPECIFIC
HYPOTHETICAL PROTEIN FROM KINETOPLASTIDS
LOPEZ-CASTILLA, A.; D' ANDREA, E.; MENEZES, R.; PIRES,. JR
Instituto de Bioquímica Médica Leopoldo de Meis
In a structural genomics context, here we report the expression, purification and
structural characterization of Q4D059 (Uniprot entry), an 86 residue-hypothetical
protein from Trypanosoma cruzi, essential in Trypanosoma brucei and also conserved in
Leishmania major, protozoan parasites of the kinetoplastid order. These parasites are
the causal agents of the neglected diseases: Chagas, Sleeping Sickness and
Leishmaniases respectively and had recently their genomes fully sequenced. Currently,
there are no vaccines to prevent or treat these infections and available drugs are
inadequate because of resistance and toxicity. Q4D059 shows low-sequence homology
with mammal proteins, and because of its essentiality demonstrated in T. brucei, it is a
potential target for anti-parasitic drugs. NMR and structural data is desirable facilitating
drug screening and providing insights on Q4D059 function, since primary sequence
homology searches only match trypanosome hypothetical proteins of unknown
function. We solved the structure of Q4D059 by standard solution NMR techniques. It is
composed by a parallel/anti-paralell three-stranded -sheet packed against two almost
parallel oriented -helices. The structure is well defined by ca. 9 NOEs per residue and a
backbone RMSD of 0.45 ± 0.19 Å was obtained for the 15 lowest-energy structures out
of 200 calculated. We also investigated the dynamics by 15N R1, R2 relaxation rates and
1H-15N NOE measurements. The structure is overall rigid except for residues from 9 to
11 undergoing rapid motion in the ps-ns timescale, and V78 with motion in the µs-ms
timescale. A search for structural homologs using the program DALI identified the
subdomain IIB of the human heat shock protein 70kDa with the best Z-score (2.9);
suggesting an evolutionary relationship between Q4D059 and an ATP-binding domain.
CAPES, CNPq, FAPERJ – Brazil
D9
-
LIPIDOMIC PROFILING OF THE AMAZONIAN FISH
1- BARBOSA, B. S.; 2- VAL, A. L.; 1- TASIC, L.
1- Laboratório de Química Biológica, Universidade Estadual de Campinas; 2- Laboratório
de Ecofisiologia e Evolução Molecular, Instituto Nacional de Pesquisas da Amazônia
"The importance of phospholipids (PL) to human health is mainly related to the
significant quantities of fatty omega-3 acids in their structures. Characterization of the
lipids in their intact form is scarce and remains challenging due to the complexity and
specificity of these biomolecules. Opposed to the proteomics and genomics, lipidomics
is not based on information that can predict the number and composition of individual
lipid molecules present in an organism. Also, quantity and type of lipids may vary due to
many factors, such as alimentation habits, still poorly understood.
Thus, this work involves the investigation of lipids and phospholipids (PL) isolated from
the dorsal muscle and/or liver from nine Amazonian fish species. All fish samples were
collected during two distinct periods of the Amazon River in 2013, flood and drought,
and belong to groups with different feeding habits. At least three samples from the
same specie were analyzed.
Using classical Bligh-Dyer technique, total lipids were isolated, then fractionated and
studied applying liquid nuclear magnetic resonance spectroscopy.
The obtained results are very interesting and contribute to better understanding of
biosynthetic pathways for Amazonian fish phospholipids (PL), whose structures and
types of linked fatty acids are very different compared to other fish lipidomics. We also
expect that clinical trials and subsequent inclusion into the human diet that are
underway (collaborative work with the Faculty of Medicine, UNIFESP) can bring new
insights in treating neurodegenerati e diseases such as Parkinson’s, l heimer’s, and/or
untington’s."
INBEB, FAPESP, PRP- UNICAMP
D10
DEVELOPMENT OF NANORADIOPHARMACEUTICAL FOR BONE
METASTASIS
1,2- PATRICIO, B. F. D. C.; 2- SANTOS-OLIVEIRA, R.; 2- WEISSMÜLLER, G.
1- Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Centro de
Ciências da Saúde, Universidade Federal do Rio de Janeiro. 2- Laboratório de
Nanorradiofármaco, Instituto de Engenharia Nuclear, Rio de Janeiro.
"Introduction: Cancer is a problem of worldwide concern and future perspectives are
worrisome. After non-melanoma skin cancer, the two types of highest incidence are the
breast and prostate. This ones lead with high frequency to bone metastasis. The
treatment with radiopharmaceutical stands out a palliative treatments witch improves
the quality of life and increase survival time. The 153Sm-EDTMP shows 70-80% of
patients with a clear improvement. The major disadvantage of this radiopharmaceutical
is the requirement of multiple doses. The nanopharmacology is a growing branch
promising properties such as controlled, prolonged and sustained release of the active
ingredient, reduction of the required dose for therapeutic effect and the toxic effects.
Objectives: In order to circumvent the deficiencies of the radiopharmaceutical 153SmEDTMP, this work has the objective to enclosure the EDTMP in polymeric nanocapsules
of PLA/PVA.
Methods: Nanoparticles: They were produced by the method of double emulsion using
poly-lactic acid (PLA) and poly-vinyl alcohol (PVA) as polymers and EDTMP as the drug.
Characterization of the nanoparticles: The morphology and dimension of the
nanoparticles were obtained by atomic force microscopy (AFM) using Peak Force
Tapping Mode (PFQNM)®. Results: The nanoparticles showed a medium dimension
250nm, spherical shapes and a heterogeneous surface showed only in the adhesion and
elastic images obtained with PFQNM. Conclusion: The nanoparticles were successfully
manufactured by the double emulsion methodology and this new mode of analysis by
AFM allows the evaluation of the nanoparticles physical properties."
INBEB, CNPq
D11
PATHOLOGICAL
IMPLICATIONS
OF
NUCLEIC
ACIDS
INTERACTIONS WITH PRION PROTEINS: IN VITRO AND IN VIVO APPROACHES
Bruno Macedo¹, Mariana P. B. Gomes¹, Natália C. Ferreira1, Matheus Tempone¹, Priscila
S. Ferreira², Felipe Campos², Fernanda Neves¹, Julia Clarke², Jerson L. Silva², Claudia P.
Figueiredo1 e Yraima Cordeiro¹
1 - Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brazil; 2 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brazil
INTRODUCTION: Prion proteins (PrPs) are involved in lethal neurodegenerative diseases
that affect humans and other animals. So far, many issues remain unclear about PrP
physiopathological role. The misfolding of its normal cellular form (PrPC) into an
abnormal form (PrPSc) characterizes the central event of prion diseases. Some studies
have suggested nuclear localization for PrPC and functions derived from its interaction
with nuclear components. Having a common place to cross-talk, nucleic acids (NAs) are
Página 37
intriguing PrP molecular partners, and have been proposed as cofactors in the protein
misfolding process. OBJECTIVES: Clarify the biological relevance of PrP-NA interactions.
MATERIALS AND METHODS: We investigated the binding of several oligonucleotides
(RNA and DNA) with the recombinant murine PrP (rPrP) through isothermal titration
calorimetry (ITC) and spectroscopic assays. We performed different cellular assays to
evaluate cytotoxicity and cell viability, working with two cell lineages (neuroblastoma N2a - and kidney cells – HK2). We also investigated interaction of a well-known
neurotoxic human prion peptide (PrP106-126) with the oligonucleotides. RESULTS AND
DISCUSSION: We found that interactions of rPrP or PrP106-126 with NAs can lead to
different aggregates and neurotoxic species, depending on the NA molecule.
Interestingly, the N2a cells morphology changed in the presence of the cytotoxic PrP-NA
complexes, indicating that they are affecting cell signaling. The preliminary in vivo
results of the intracerebral inoculation of a rPrP-DNA complex in healthy mice (C57BL/6)
indicate significant impairment of memory consolidation, although there was no
apparent locomotor dysfunction in these mice through extensive behavioral tests.
CONCLUSIONS: Our results show the NA ability of binding to rPrP, induce
conformational changes, modulate protein aggregation and cytotoxicity in vitro and to
induce neurological damages in vivo. Understanding the structural, cellular and in vivo
effects observed for PrP and NAs interactions may enlighten the still obscure prion
physiopathology.
FAPERJ, CAPES, CNPq
D12
VÍRUS DA INFLUENZA HUMANA INATIVADO POR PRESSÃO
HIDROSTÁTICA: INVESTIGANDO UM CANDIDATO PARA VACINA UNIVERSAL
1-DUMARD, C.H.; 1-Félix, A.L.; 1-SOUZA-SANTOS, P.; 1-BARROSO, S.P.C.; 2-NICO, D. 1SILVA, J.L.
1-Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber,
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto de
Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro
A gripe é uma doença inflamatória causada pelo vírus da influenza, que se divide nos
gêneros A, B e C. Estima-se que anualmente ocorram de 3 a 5 milhões de casos de
doença grave, e entre 300 a 500 mil mortes. A vacinação é a forma mais efetiva para
prevenção da doença e suas complicações. Os principais modelos de vacina se baseiam
em proteínas virais purificadas ou em vírus atenuados. As vacinas baseadas em
proteínas purificadas raramente geram complicações, mas também geram uma
resposta imunológica mais pobre oferecendo pouca proteção contra os diversos
subtipos. A vacina atenuada gera uma resposta imunológica mais eficiente, com
produção de anticorpos de mucosa, no entanto pode gerar mais reações adversas e
apresenta restrições para ser administrada em grupos de risco. Em nosso trabalho
testamos um novo modelo de vacina baseado na inativação por pressão do vírus da
influenza humana H3N2. Camundongos apresentaram resposta imunológica serológica
e de mucosa ao receberem duas doses de vacina. Além disso, estes animais se
mostraram eficientes ao produzirem citocinas após a vacinação. Também observamos
resposta de anticorpos de memória nos animais avaliados 3 meses após vacinação.
Animais situados nos grupos de risco também foram capazes de gerar anticorpos.
Resposta de anticorpos in vitro contra a hemaglutinina (principal glicoproteína viral) foi
induzida contra diversos subtipos de antígenos virais, indicando um largo espectro de
proteção. A vacina inativada por pressão hidrostática mantém as atividades das
glicoproteínas virais sendo capaz de se ligar e entrar na célula. Por apresentar muitas
das etapas do ciclo infeccioso preservada sem, no entanto apresentar infecciosidade,
este modelo pode ser a base de um modelo de vacina capaz de gerar resposta
imunológica contra os diversos subtipos sendo ao mesmo tempo segura para indivíduos
em grupos de risco.
INBEB, FAPERJ, CAPES, CNPq
D13
ANALYSIS OF CARBOHYDRATE EXPRESSION ON THE SURFACE
OF INTESTINAL EPITHELIUM OF Lutzomyia longipalpis and Lutzomyia antunesi
(DIPTERA, PISYCODIDAE) OF PARA STATE.
1-OLIVEIRA, D. M. S.; 1,2-FARIAS, L. H. S; 1,2-SILVA, B. J. M ;1-SILVA, E. O1.
1-Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Brazil.; 2-Instituto Nacional de Ciência e
Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro,
Ilha do Fundão, Brasil
Leishmaniasis are worldwide diseases that occur in 98 countries. In Brazil cases of
American Visceral Leishmaniasis (AVL) and Cutaneous Leishmaniasis (CL) has been
registrated in all State Federation including Pará State. Lutzomyia (Lutzomyia)
longipalpis is the putative vector of Leishania infantum chagasi the etiological agent of
AVL while Lutzomtya (Nyssomyia) antunesi is the proven vector of Leishmania (Viannia)
lindenbergi suspected of causing CL. The specificity parasite-sandflies interaction is
mediated by leishmania surface molecules, as Lipophosphoglycan (LPG), which is
recognized by receptors of midgut epithelium. However in permissive sand flies as L.
longipalpis the interaction is mediated by a lectin-like activity on the parasite surface
and the presence of glycoproteins containing N-acetyl- galactosamine (GalNAc) of
epithelial insect midgut. Thus, the aim of this work was to compare the expression of
glycoprotein containing GalNAc in the midgut of L. longipalpis obtained from colony and
forest and glycoprotein of the midgut of L. antunesi collected from forest area of
Cametá Municipality of Pará. Phlebotomines were dissected in paraformaldehyde 4%
and each midgut was sectioned longitudinally. After that, incubated with FITC
conjugated Helix pomatia lectin (HPA-FITC) and Concanavalin A (Con-A-FITC) which are
specific for GalNAc and mannose, respectively. Lysates of seven midguts of each
phlebotomine species from colony and forest were analyzed by SDS-PAGE followed by
western blotting with HPA and Con-A . All midgut lysates from both samples displayed
peptides ranging from 35 to 75 kDa and 51 kDa. These bands react with HPA and CON-A,
respectively, suggesting presence of GalNAc and mannose residues in the midgut of the
sand flies. These results indicated that glycoprotein containing GalNAc and mannose
residues occurs in both species studied indicating the presence of permissive sand flies
to the development of different leishmanias species in endemic areas for AVL and CL of
Para state
CAPES, CNPq/UFPa, INBEB/FAPERJ, MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ and
Evandro Chagas Institute.
D14
ANTI-INFLAMMATORY EFFECTS OF DIETHYLCARBAMAZINE ON
LUNG INJURY INDUCED BY MONOCROTALINA IN MICE
1,3-RIBEIRO, E.L.;1,3- FRAGOSO, I.T.; 1,3-SILVA, A.K.S; 3,4-DONATO, M.A.M.; 1,3;
GOMES, F. O.S.; 2,3-OLIVEIRA, A.C.; 3- SILVA, B.S.; 1,3 PEIXOTO, C. A.
1.PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE
PERNAMBUCO, RECIFE - PE - BRASIL; 2.GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS,
UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL; 3.LABORATÓRIO DE
ULTRAESTRUTURA, INSTITUTO AGGEU MAGALHÃES, FIOCRUZ, RECIFE - PE - BRASIL. 4.
FACULDADE INTEGRADA DE PERNAMBUCO, RECIFE-PE-BRASIL;
Background: Monocrotaline (MCT) is a pyrrolizidine alkaloid produced by Crotalaria
genus, which causes liver damage, modest pulmonary fibrosis and immunotoxicity
effects in animals and mainly employed to establish a model of pulmonary dysfunction.
Página 38
Clinical reports have described favorable results with the use of diethylcarbamazine
(DEC) in bronchial asthma. This drug has an important anti-inflammatory role since it
interferes with arachidonic acid metabolism. In the present study, we investigated the
efficacy of oral DEC treatment in mice model of injury pulmonary induced
monocrotaline. Methods and Results: C57/BL6 male mice, were used in all experiments.
MCT solution intraperitoneal injection (600mg/kg) was administered once per week (7,
14 or 21 days). Six groups (n=10): control; MCT7; MCT14; MCT21, MCT14/DEC14.
(50mg/Kg per day of DEC from 1 to day 14), MCT21/DEC21 (from 1 to day 21).
Bronchoalveolar lavage fluid were collected for imunoassay (Griess reaction) and lung
tissues for light microscopy (H.E.), immunohistochemistry (COX-2, MCP-1, CD24, F4/80,
IL-6, α smooth muscle actin and eNOS) and western blot (COX-2, iNOS and NFkB).
Pulmonary sections of the group control exhibited preserved morphological
characteristics. MCT7 group revealed alveolar exudates, interstitial edema with
thickening of the alveolar septae, perivascular edema, and inflammatory cellular
Infiltrates; MCT14 group presented had an inflammatory infiltrate such as macrophages
in plexiform lesion in the pulmonary arteries and emphysema; and MCT21 group
showed a decrease of injury and the infiltration of PMNs. Nitrite and nitrate levels, were
significantly increased in MCT7, MCT14 and MCT21 BALF comparing to the control
group. In contrast, MCT14/DEC and MCT21/DEC groups showed significantly reduction
of the nitrite and nitrate levels. MCT7, MCT14 and MCT21 groups revealed significant
COX-2, MCP-1, CD24, F4/80, IL-6, iNOS and NFkB higher expression, whereas these
expression were significantly reduced after treatement with DEC (p<0,05). On the other
hand, MCT7, MCT14 and MCT21 groups presented a reduced expression of eNOS and α
smooth muscle actin , after treatment with DEC the expression of these proteins
returned to normal levels (p<0,05). Conclusion: In this study, treatment with DEC
attenuated MCT-induced pulmonary dysfunction, reducing the lung damage.
FACEPE, INBEB e CNPq
D15
SÍNTESE E AVALIAÇÃO DE GUANIL HIDRAZONAS E OXIMAS
AROMÁTICAS COMO INIBIDORAS E REATIVADORAS DA ACETILCOLINESTERASE
1 - PETRONILHO, E. C.; 1- KITAGAWA, D. A. S.; 2- CASTRO, N. G.; 2- SILVA, F. M. R.; 1PINTO, A. C.; 1- FIGUEROA-VILLAR, J. D.
1- Grupo de Química Medicinal, Departamento de Química, Instituto Militar de
Engenharia, Praça Gen. Tibúrcio, 80, Praia Vermelha, 22.290-070, Rio de Janeiro-RJ,
Brasil; 2- Instituto de Ciências Biomédicas, Laboratório de Farmacologia Molecular,
Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 – CCS, Ilha do
Fundão, 21941-902, Rio de Janeiro-RJ, Brasil.
Os agentes químicos representam uma grave ameaça ao mundo moderno. Dentre eles
se destacam os organofosforados (OP) neurotóxicos, devido a sua grande
periculosidade, baixo custo e fácil manufatura. Os OP inibem a acetilcolinesterase
(AChE), uma importante enzima responsável pela hidrólise do neurotransmissor
acetilcolina (ACh), causando uma síndrome colinérgica e morte por insuficiência
respiratória. Para reativar a AChE são usadas as oximas catiônicas, mas nenhuma delas é
eficaz contra todos os agentes OP, demonstrando a necessidade de desenvolver novos
reativadores que sejam potentes e com baixa toxicidade. Alguns estudos sugerem a
avaliação de diferentes nucleófilos, como as guanil hidrazonas e a dependência da
atividade biológica com a acidez. A AChE também está envolvida na Doença de
Alzheimer (DA), onde un tratamento é a inibição da AChE, o que permite aumentar a
quantidade de ACh, permitindo melhorar os impulsos nervosos e memória. Com esse
objetivo, neste trabalho foram sintetizadas uma série de guanil hidrazonas e oximas
aromáticas que possuem grupos eletroatratores, para avaliar o efeito destes grupos no
processo de reativação e inibição da AChE. Estes compostos foram testados como
inibidores e reativadores da AChE de Electrophorus electricus inibida com etilparaoxon,
utilizando como referência positiva a pralidoxima (2-PAM), através do método de
Ellman e RMN. Os compostos obtidos somente mostraram boa ação inibitória da AChE.
INBEB, CNPq, CAPES, FAPERJ, MINISTÉRIO DA DEFESA
D16
MODULATION OF HEPATIC CU(I)-ATPASE (ATP7B) ACTIVITY BY
INSULIN AND GLUCAGON
1,2- HILÁRIO-SOUZA, E.; 3- CUILLEL, M.; 3- CHARBONNIER, P.; 3- MINTZ, E.; 1,2- VIEYRA,
A.; 1,2- LOWE, J.
1- Instituto de Biofísica Carlos Chagas Filho (IBCCF), UFRJ, Rio de Janeiro, Brazil. 2Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem (INBEB),
UFRJ, Rio de Janeiro, Brazil. 3- Comissariat l’ nergie tomi ue C , Grenoble,
France.
"Molecular studies of copper active transport are essential to understand the copperrelated diseases (Menkes and Wilson diseases). A number of studies suggest the
involvement of insulin in copper balance, although the cellular and molecular
mechanisms are not yet established. The main objective of this study was to investigate
whether insulin or glucagon modulates the Cu(I)-ATPase (ATP7B) activity and
intracellular localization in hepatocytes.
In HepG2 cells, ATP7B activity was around 40 nmol Pi x mg-1 x min-1. When they were
treated with 0.1 µM insulin, ATP7B activity was increased by 100% and the treatement
with 0.1 nM glucagon, ATP7B activity was inhibited by 90%. The maximum effect was
observed at 5 minutes for both hormones. After treatment with 0.1 µM insulin and 10
µM H89, a permeable PKA inhibitor, ATP7B activity was similar to that in the presence
of insulin alone. In the presence of 0.1 nM glucagon and 10 µM H89, ATP7B activity was
the same as in the control condition. Therefore insulin inhibited and glucagon
stimulated PKA signaling pathway, leading to the modulation of ATP7B activity.
To analyze whether these hormones also modulates ATP7B intracellular localization,
WIF-B9 cells were used because they have a well-defined bile canaliculi. ATP7B was
localized in the Golgi complex and it was not observed any difference after 24 h
treatment with 0.1 µM insulin or 0.1 nM glucagon. When ATP7B was already at the
canaliculus (treatment with 1 µM CuCl2 for 2 h) and WIF-B9 cells were treated with
different concentrations of insulin, it was observed a change in the localization of ATP7B
from the canaliculi towards the Golgi complex.
For the first time it was shown that hormones which are essential for the energetic
metabolism could regulate the activity of ATP7B, but they do not have any influence in
the protein localization."
CAPES, CNPq, FAPERJ
D17
BRADYKININ DOWN-MODULATES HUMAN IL-6 PRODUCTION
BY P.GINGIVALIS INFECTED GINGIVAL FIBROBLASTS: AN INNATE RESPONSE PATHWAY
REGULATING TH17/TH1-POLARIZATION IN A BUCCAL MODEL OF PERIODONTIS.
1- Ramos-Junior, E.S., 1- Nascimento,C.R. , 1- Morandini, A.C. , 1- Scharfstein, J
1-INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO - Universidade Federal do Rio de
Janeiro
Recently we demonstrated that Porphyromonas gingivalis, a Gram-negative bacterium
that causes periodontitis, activates the kinin system via the cysteine protease gingipain.
Considering the role of the kinin B2 receptor (BK2R) in inflammation and healing, the
purpose of this study was to evaluate the contribution of BK2R to the pathogenesis of
periodontitis. Using an in vitro model with human gingival fibroblasts (HGF) here we
showed that BK down-modulates ERK/1/2 and IL-6 production in P.gingivalis-infected
HGF in BK2R-dependent manner. Consistent with this, we found that addition of
Página 39
purified High Molecular Weight Kininogen (HMWK) down-regulated IL-6 in P.gingivalisinfected HGF. Using a model of buccal P. gingivalis infection we then studied whether
TH-cell responses (anti-P.gingivalis) were modulated by BK2R. Recall assays performed
with submandibular T cells (7 d p.i.) from WT infected mice showed that the bacteriainduced profile of secreted cytokines was dominated by INF-G, accompanied by
significant IL-17 responses. Interestingly, TH1/TH17 cytokines were upregulated by
bacteria-specific T cells of infected BK2R-KO mice. Considering that the IL-6/IL-17 axis is
involved in periodontal pathology, the findings that BK2R dampens IL-6 production by
HGF suggest that TH1/TH17 responses might be modulated by kinins proteolytically
released in the infected oral mucosa.
INBEB, FAPERJ, CNPq
D18
AMYLOID FIBRILS TRIGGER THE RELEASE OF NEUTROPHIL
EXTRACELLULAR TRAPS AND ARE DIGESTED BY ELASTASE
1-ESTEFANIA AZEVEDO, 2-ANDERSON GUIMARAES-COSTA; 1,4-CAROLINA BRAGA ; 3JEFFERY KELLY, 2-ELVIRA SARAIVA; 1-FERNANDO PALHANO; 1-DEBORA FOGUEL.
1-Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brazil; 2-Instituto de
Microbiologia Paulo de Góes, UFRJ, Brazil; and 3-The Scripps Research Institute,
California, USA, 4-Polo de Xerem, Caxias, Rio de Janeiro, Brazil.
Amyloidoses are a group of diseases characterized by accumulation of amyloid fibrils in
different tissues, which are produced by the aggregation of soluble proteins. We asked
whether neutrophil extracellular traps (NETs) could play a role on this disease since
neutrophil elastase was observed on amyloid deposits of different systemic
amyloidoses. Initially, we tested NET induction incubating human blood neutrophils
from healthy donors with three different amyloid fibrils and their soluble proteins
(alpha-synuclein, Sup35 and transthyretin). NET-DNA was quantified in culture
supernatants with the Picogreen dsDNA kit. We demonstrate that amyloid fibrils, but
not soluble proteins, induce NETs release, and transthyretin induces NETs formation in a
dose dependent manner. Immunofluorescence staining with anti-elastase antibody and
DAPI confirmed these results. Importantly, we show that NET-associated elastase
digested amyloid fibrils into oligomeric species, which were toxic for BHK-21 cells, as
evidenced by the staining with live/dead reagents (calcein/ethidium homodimer).
Immunohistochemical analyses of amyloidotic human tissues revealed the presence of
NETs, strengthening the evidence for the participation of neutrophils in amyloid
diseases. Our results reveal that amyloid fibrils induce NET release that amyloid fibrils
are digested by NET-associated elastase generating toxic peptides.
FAPERJ, CNPq
D19
EVALUATION OF ACUTE AND LONG-TERM EFFECTS OF
PSEUDOMONAS
AERUGINOSA
LUNG
INFECTION
IN
COGNITIVE
AND
NEUROINFLAMMATORY PARAMETERS
1- MAGNO, F.; NASCIMENTO, D. O.; ALEXANDRE, P.C.B.; CUNHA, M.G.A.T.; REIS, P.A.;
BOZZA, P. T.; CASTRO-FARIA-NETO, H. C.; 2- BOZZA, F.A.
1- Instituto Oswaldo Cruz, Fiocruz; 2- Instituto de Pesquisa Clínica Evandro Chagas,
Fiocruz
Sepsis is a severe medical condition characterized by systemic inflammatory response
secondary to infection, which progress to multiple organ dysfunction and death. It is the
leading cause of death in Intensive Care Units worldwide. Its most common source of
infection is the lung with high lethality rate. Cognitive impairment is a significant
consequence of sepsis among survivors. The encephalopathy associated with systemic
inflammation is not well understood so it is important the development of clinical
relevant models to help understand this sequelae. In this study we evaluated acute
inflammatory markers and established a long-term consequence in a murine model of
severe pneumonia. C57/BL6 mice were submitted to intratracheal instillation of 105
colony forming units of Pseudomonas aeruginosa (PA). 6 and 24 hours later the
bronchoalveolar fluid, the lungs and the brain were collected for cell migration, protein,
myeloperoxidase, cytokine, western-blotting, thiol and histological analysis. The survival
rate was observed during 7 days after the stimuli. These animals received one dose of
antibiotic, 6 hours after pneumonia induction. Cognitive damage was evaluated through
the freezing test. Our results showed expressive neutrophils recruitment and
myeloperoxidase levels in the lungs of PA infected mice. These animals showed a
significantly increase in IL-6, KC, proteins, IL1 , MCP-1 levels in the lung and brain.
Histological analysis showed an intense cell infiltrate in lung tissue and survival rate was
extensively lower in PA infected mice. Infected animals showed reduced expression of
PSD95 pos-synaptic protein and lower thiol levels in the brain. Infected mice had a loss
of aversive memory 13 days after stimuli and it remains 50 days later. We demonstrated
acute inflammatory response to Pseudomonas aeruginosa lung infection and indicated
that this pneumonia model can cause irreversible cognitive impairment. Our results
reveal an experimental model for the encephalopathy study associated to systemic
inflammation.
CNPq, Faperj, Fiocruz
D20
MODELOS
FARMACOFÓRICOS
TRIDIMENSIONAIS
DE
INIBIDORES DA PTERIDINA REDUTASE DE LEISHMANIA MAJOR
1,2-LEITE, F. H. A.; 2-CASTILHO, M. S.
1- Programa de pós-graduação em Biotecnologia, Universidade Estadual de Feira de
Santana; 2- Faculdade de Farmácia, Universidade Federal da Bahia.
Segundo a OMS, Leishmaniose é a segunda protozoose mais importante, em termos de
mortalidade e prevalência. O repertório de fármacos disponíveis para o tratamento
dessa parasitose é limitado e, geralmente, apresenta baixos índices de eficácia e
segurança. Embora os protozoários do gênero Leishmania sejam auxotróficos para
folatos, inibidores de diidrofolato redutase (DHFR) são pouco eficazes contra esse
parasito. A baixa suscetibilidade se explica pela presença da enzima pteridina redutase
(PTR1) que atua como via alternativa para a redução de ácido fólico. Diante desse
cenário, o objetivo deste estudo é a construção de modelos farmacofóricos que
auxiliem na identificação de inibidores que tenham afinidade por ambos os alvos
terapêuticos. Assim, 6 inibidores de DHFR e 9 inibidores da PTR1 de L. major,
previamente descritos na literatura, foram selecionados para a construção de modelos
farmacofóricos, os quais foram avaliados quanto a sua capacidade de diferenciar
inibidores conhecidos de moléculas com propriedades físico-químicas semelhantes, mas
que não tem atividade sobre as enzimas (falsos positivos). Adicionalmente, os modelos
foram avaliados quanto a sua habilidade de explicar a relação entre a estrutura química
e a atividade biológica de inibidores de DHFR (Ki: 0,13-363,1μM e de PTR1 Ki: 16,1436,0 μM n o utili ados ara constru o dos modelos farmacof ricos. 3 modelos
apresentaram valores de sensibilidade e especificidade adequados (AUC>0,7) para
discriminar moléculas ativas das inativas. Adicionalmente, a análise desses modelos
revela que moléculas com afinidade por PTR1 apresentam 3 pontos hidrofóbicos (2
deles no anel pteridina), dois grupos aceitadores de ligação de hidrogênio (grupos NH2
exo-cíclicos) e 4 grupos aceitadores de ligação de hidrogênio (3 deles no anel pteridina).
Moléculas com afinidade por DHFR também apresentam pontos hidrofóbicos e
aceitadores de ligação de hidrogênio no anel pteridina, contudo o número de disposição
do outros pontos farmacofóricos é diferente. A partir dessas informações é possível
planejar inibidores com afinidade frente a ambos os alvos terapêuticos.
FAPESB
Página 40
D21
TOLL-LIKE RECEPTOR 4 ACTIVATION PROMOTES CARDIAC
ARRHYTHMIAS BY DECREASING THE TRANSIENT OUTWARD POTASSIUM CURRENT
(ITO) THROUGH AN IRF3-DEPENDENT AND MYD88-INDEPENDENT PATHWAY.
1- MONNERAT-CAHLI, G.; 2- ALONSO, H.; 2- GALLEGO, M.; 1-ALARCON, M.L.; 3BASSANI, R.A.; 2-CASIS, O.; 1- MEDEI, E.
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Brasil. 2-Departamento de Fisiología, Facultad de Farmacia, Universidad del País Vasco,
Vitoria, Spain. 3-Departamento de Engenharia Biomédica – FEEC/Unicamp, Campinas,
Brasil
Cardiac arrhythmias are one of the main causes of death worldwide. Several studies
have shown that inflammation plays a key role in different cardiac diseases and Toll like
rece tors T R’s lay an im ortant role in cardiac com lications. In the resent study,
we investigated whether the activation of TLR4 induces cardiac electrical remodeling
and arrhythmias. Also the signaling pathway involved in these phenomena was studied.
Action potentials, the presence of cardiac arrhythmias and transient outward K+ current
Ito were recorded in Wistar rat’s hearts after 4h e osure to the T R4 agonist
ultrapure Lipopolysaccharide (LPS - 1µg/ml). TLR4 stimulation in vitro promotes a
cardiac electrical remodeling that leads to cardiac action potential prolongation which
evokes arrhythmic events such as delayed after depolarization (DAD's) and triggered
activity. The perfusion of LPS (1 μg/ml) during 30 minutes did not modify Ito.
Conversely, after 24h of LPS incubation Ito was reduced, with no changes in the
biophysical properties of the current. Major changes in Ca2+ cycling were not observed
in ventricular myocytes after 24 h exposure to LPS; however, extrasystolic activity was
present in a considerable number of cells (25%). Neither the blockade of Interleulink-1
receptor-associated kinase 4 nor nuclear factor kappa B (NF-kB) prevented the LPS
effect on Ito. However, interferon regulatory factor 3 (IRF3) inhibition prevented the
effect of TLR4 activation on Ito. Activation of TLR4 induced longer AP duration and
evoked DAD's and triggered activity because of a reduction in Ito. The mechanism
involved is MyD88-independent and IRF3-dependent.
INBEB, FAPERJ, CNPq, DECIT
D22
POTENCIAL DAS CÉLULAS TRONCO DA MEDULA ÓSSEA EM
LESÃO MIOCÁRDICA RADIOINDUZIDA
1-RAMOS,IP; 1-ANDRADE,CBV; 2-SUHETT,G; 3-SALATA,C; 4-CANARY PC; 1-BRASIL,GV;1GOLDENBERG,RCS
1-Laboratorio de Cardiologia Celular e Molecular do Instituto de Biofisica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro; 2-Hospital Albert Einstein;3-Laboratório
de Ciências Radiológicas, Universidade do Estado do Rio de Janeiro
"Introdução: No Brasil, as estimativas do INCA para o ano de 2013, apontam a
ocorrência de aproximadamente 518.000 casos novos de câncer, sendo o câncer de
mama (CM) feminino o mais incidente deles entre as mulheres, com uma estimativa de
aproximadamente 53 mil novos casos. Aproximadamente 50% das pacientes para CM
recebem radioterapia (RT) para tratamento de CM. Um paciente com neoplasia, que se
submete ao tratamento para câncer corre o risco substancial de deterioração da saúde
cardiovascular. A terapia celular parece promissora no tratamento de doenças crônicas
e degenerativas, dentre elas a cardiomiopatia radioinduzida, posto que os atuais
recursos terapêuticos são insuficientes.
Materiais e Métodos: Ratas Wistar, com 3 meses, foram divididas em 2 grupos,
irradiados (G0) e irradiados e tratados com células-tronco de medula óssea (G1) . Os
animais foram eutanasiados 3 meses após o término do tratamento. Antes da eutanásia
foi realizado ecocardiograma. Após a eutanásia foi retirado o ventriculo esquerdo para
as técnicas de RT-qPCR (VEGF e Pro Colágeno I) e histologia para picrosirius.
Resultados/Discussão: Após 45 dias da irradiação foi observado um aumento na fração
de ejeção entre os animais do grupo G1 em relação ao grupo G0. Houve um aumento na
expressão gênica de VEGF e diminuição de Pro Colágeno I nos animais do grupo G1 em
relação aos do grupo G0. Demonstrando que a deposição de colágeno no coração dos
animais tratados com células-tronco de medula óssea foi reduzida o que foi
corroborado com os resultados obtidos por picrosirius em comparação com o grupo só
irradiado. O aumento da expressão do VEGF no grupo G1 é importante, pois demonstra
que pode estar ocorrendo um aumento da neoangiogênese no coração irradiado e
tratado, o que não foi observado no grupo G0. Para confirmação desses dados será
realizada imunohistoquímica para colágeno tipo I e CD31."
CNPq, FAPERJ, CAPES, INBEB
D23
CARACTERIZAÇÃO MORFOLÓGICA E BIOQUÍMICA DA COSTA
DE TRICOMONAS
1,2 – DE ANDRADE ROSA, I; 3,4-DE SOUZA W; 1,5- BENCHIMOL B
1- Laboratório de Ultraestrutura Celular, Universidade Santa Úrsula, Rio de Janeiro,
Brasil 2- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Brasil 3- Laboratório Hertha Meyer, Universidade Federal do Rio de Janeiro,
Brazil 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de
Janeiro, Brasil 5-Instituto Nacional de Biologia Estrutural e Bioimagem, Universidade
Federal do Rio de Janeiro, Brasil
"Tritrichomonas foetus é um protista parasito causador da tricomonose bovina, uma
doença sexualmente transmissível que acarreta diversos problemas reprodutivos.
Embora seja considerado um organismo primitivo, este parasito exibe um citoesqueleto
incomum e complexo, formado por diversas estruturas ainda pouco estudadas. Entre
estas destacam-se a pelta e o axóstilo, formados por microtúbulos, os filamentos
parabasais e a costa que possuem composição proteica desconhecida. A costa trata-se
de uma estrutura peculiar por apresentar uma organização diferente de outros
constituintes de citoesqueleto. É considerada uma fibra estriada formada por bandas
claras e escuras disposta de forma alternada e bastante regular. Desta forma, esta
estrutura despertou interesse também na área de metrologia por poder ser usada como
uma ferramenta de medidas em escalas nanométricas. Assim, realizamos o
fracionamento celular para obter uma fração enriquecida de costas que foi
caracterizada morfologicamente por contrastação negativa. Três regiões distintas foram
classificadas como cabeça, pescoço e corpo, de acordo com o sua forma e padrão de
bandeamento. A rápida transformada de Fourier nos auxiliou a comprovar a
periodicidade da costa e a revelar a presença de sub-bandas também com padrões
regulares.
Com o intuito de caracterizar as proteínas que compõem a costa de T. foetus, a fração
de costas foi submetida a eletroforeses uni- e bidimensionais, onde as bandas e spots
foram identificados por espectrometria de massa. Ao todo, 50 proteínas hipotéticas de
T. foetus, sem domínios conservados descritos, foram identificadas.
Juntos, nossos resultados demostraram que a costa é uma estrutura periódica com
divisões e subdivisões regulares, o que a torna uma boa ferramenta para medidas em
escalas nanométricas. Além disso, verificamos também que sua composição proteica é
totalmente distinta, sendo necessários mais estudos que avaliem a localização e função
destas proteínas na estrutura da costa."
INBEB, AUSU, Inmetro, CNPq, FAPERJ, CAPES, PRONEX.
Página 41
D24
METABOLOMIC PROFILING OF ANIMAL SERUM SAMPLES
UNDER CORYNEBACTERIUM PSEUDOTUBERCULOSIS INFECTION
1- PONTES, J. G. M.; 1- BALLOTTIN, D. P. M.; 1- TASIC, L.*
1- Universidade Estadual de Campinas
"A Gram-positive and pathogenic bacterium, Corynebacterium pseudotuberculosis
affects mainly caprines and sheep causing caseous lymphadenitis, the disease
responsible for the enormous losses in agribusiness, since there is not effective
treatment for this disease and infected animals end up being euthanized. Our objective
was to evaluate and compare the metabolic changes caused in infected animals by
Corynebacterium pseudotuberculosis among seropositive, seronegative and
asym tomatic animal’ blood serum sam les, so that later we can better understand the
mechanism of infection and the disease through in silico studies. In this work we will be
presenting the first results of analyzes that have been made using 1H NMR recorded on
a Bruker Avance III 600 MHz spectrometer and SEM-EDS. Metabolomic profile changes
account to the chemical shift region from 0 to 2 ppm and change in signal intensities in
the region between 6 to 8 ppm referring to signals related to aminoacids and proteins.”
CNPq and INBEB
D25
KOJIC ACID INDUCES, IN VITRO, HUMAN MONOCYTES
DIFFERENTIATION INTO MACROPHAGES THROUGH AUTOPHAGY
COSTA, J.P. ¹,2*, SILVA, B. J. M 1,2*, FRADE, P.C.R1,2 , RODRIGUES, A. P. D. 1,2,3, FARIAS,
L.H.S. 1,2, NASCIMENTO, J.L.M. 4, SILVA, E.O. 1,2
1Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Belém, Brazil. 2Instituto Nacional de Ciência e
Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro,
Ilha do Fundão, Brazil 3Laboratório de Microscopia, Instituto Evandro Chagas, secretaria
de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil. 4Laboratório de
Neuroquímica, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém,
Pará, Brazil.
Monocytes are mononuclear phagocytes, present in the peripheral blood, that are able
to differentiate into macrophages and dendritic cells. Macrophages play a specific role
in the inflammatory process, being essential for the innate response. Given the
important role of monocytes/macrophages in the immune response, this study aimed to
investigate whether kojid acid, a natural product produced by some species of fungi, is
able to promote differentiation of human monocytes into macrophages in vitro through
differential expression of surface proteins and autophagic process. Monocytes were
isolated from peripheral blood collected in EDTA obtained from healthy human
volunteers (Blood collection center, Belém, Para, Brazil) and separated using
Histopaque®-1077. Cytotoxicity to the host cell was assessed by colorimetric MTT
assays. Autophagy was observed through the formation of autophagic vacuoles by
Transmission Electron Microscopy and expression of the lysosomal protein LC3b by
Immunofluorescence. The treatment of monocytes with 50 μg/mL KA for 48h induced
morphological alterations, such as an increase in cell area, numerous cellular
projections and increased organelle number. In addition, flow cytometry analysis
showed increased labeling LC3b and was observed the presence of autophagic vacuoles
in the cytoplasm indicating autophagic process in cells treated. Immunofluorescence of
F4/80 and flow cytometry analysis of F4/80, CD11b and CD11C protein revealed that KA
induces the differentiation of monocytes into macrophages in vitro. The cell viability of
the monocytes was also maintained following KA treatment. No significant production
of reactive oxygen species was detected by CellRox in treated cells. In conclusion, these
results support the hypothesis that kojic acid, a natural alternative molecule, may be
able to induce, in vitro, monocyte differentiation into macrophage.
INBEB
D26
THE ORIGIN OF LYSOSOMES RELATED ORGANELLES OF
TRYPANOSOMA CRUZI TRYPOMASTIGOTES
1,2- JULIANA CUNHA VIDAL, 1,2-CAROLINA DE LIMA ALCANTARA, 1,2,3-WANDERLEY DE
SOUZA, 1,2- NARCISA LEAL DA CUNHA-E-SILVA
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brazil; 2-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e
Bioimagens, Rio de Janeiro, Brazil. 3-Diretoria de Metrologia Aplicada às Ciências da
Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro), Rio de
Janeiro, Brazil.
"Trypanosoma cruzi is the causative agent of Chagas disease. The parasite presents
three life cycle stages: trypomastigotes, the infective forms, amastigotes, the
proliferative forms found in the cytoplasm of the vertebrate host cells and
epimastigotes that proliferate in the invertebrate host. In nature, triatomine bugs pick
up trypomastigotes together with the blood of infected hosts. Parasites travel along the
insect gut without leaving gut lumen and in their way undergo two differentiations:
from trypomastigotes to epimastigotes and, after proliferating, again to
trypomastigotes. The latter step, called metacyclogenesis is of particular interest, as the
parasites acquired the ability of infecting humans. Metacyclic trypomastigotes are layed
down together with triatomine feces at the local of the insect bite, penetrate human
cells, scape early from parasitophorous vacuole to the cytoplasm, where they
differentiate into amastigotes. Summarizing this complicate story, T. cruzi alternates
between vertebrate and invertebrate hosts by means of alternating between infective
and proliferative forms. Using previously established protocols, our laboratory is able to
reproduce in vitro the complete T. cruzi life cycle1.
Epimastigotes present a very efficient endocytic pathway that begins with a specialized
structure, the cytostome-cytopharinx complex, a deep invagination of the cell surface,
sustained by stable microtubules, that delivers cargo-containing vesicles to a tubulevesicular network and subsequently to reservosomes, the final endocytic
compartment2.
Reservosomes are able to store and digest macromolecules, present an acidic character,
maintained by an unusual P-type H+ATPase, concentrate cruzipain, the most important
T. cruzi protease, its natural inhibitor chagasin, and other hydrolases as well. Despite
these features, reservosomes were not considered genuine lysosomes because they
lack classic lysosome markers3.
Differently from epimastigotes, the endocytic pathway of amastigotes and
trypomastigotes was not described yet. In fact, after many unsuccessful experiments,
data from our group and from others indicate that trypomastigotes are unable to
uptake macromolecules from medium. Nevertheless, trypomastigotes and amastigotes
also present cruzipain, chagasin, and P-type H+ATPase concentrated in acidic organelles
placed between kinetoplast and nucleus. Few years ago, we proposed to classify these
compartments as lysosome related organelles (LROs)4.
The present study aimed to investigate the origin of metacyclic trypomastigotes LROs.
We have preloaded epimastigote reservosomes with uncoated fluorescent beads before
inducing metacyclogenesis. The rate of metacyclogenesis did not change compared to
control (not loaded) parasites: after 48h of differentiation we have obtained 53% of
trypomastigotes. Surprisingly, 25% of them contained beads between kinetoplast and
nucleus, consistent with the localization of LROs. These parasites were the first register
of trypomastigotes containing endocytic cargo. In a similar experiment, using 10 nm
gold-labeled transferrin (Tf-Au) as tracer for electron microscopy, we observed spherical
organelles containing Tf-Au in parasites at the end of metacyclogenesis. We are now
Página 42
characterizing these compartments by immunocytochemistry. Our observations are a
strong indication that LROs from metacyclic trypomastigotes originate directly from
reservosomes of epimastigotes."
CAPES, CNPq, INBEB
D27
ANG-(3-4) INHIBITS RENAL NA+-ATPASE IN HYPERTENSIVE
RATS THROUGH A MECHANISM THAT INVOLVES DISSOCIATION OF ANG II RECEPTORS
HETERODIMERS AND PKA
1,2- DIAS, J.; 1,2,3- FERRÃO, F.M.; 1,2- AXELBAND, F.; 4- CARMONA, A.K.; 2,3- LARA, L.S.;
1,2- VIEYRA, A.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem; 3Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 4Departamento de Biofísica, Universidade Federal de São Paulo
The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain
largely unknown. The present study: (i) investigates whether ANG-(3-4) modulates
ouabain-resistant Na+-ATPase resident in proximal tubule cells, and (ii) verifies whether
its possible action on pumping activity -considered the fine tuner of Na+ reabsorption in
this nephron segment- depends on blood pressure. ANG-(3-4) inhibited Na+-ATPase
activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar
concentrations, with no effect in Wistar-Kyoto rats (WKY) or on (Na++K+)-ATPase.
PD123319 (10-7 M) and PKA(5-24) (10-6 M), an AT2R antagonist and a specific PKA
inhibitor respectively, abrogated this inhibition, indicating that AT2R and PKA are
central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in
WKY rats, the peptide (10-8 M) completely blocked stimulation of Na+-ATPase induced
by 10-10 M ANG II in normotensive rats through a mechanism that also involves AT2R
and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R
heterodimers, which remain associated in 10-10 M ANG II and dissociate to a very low
dimerization state upon addition of 10-8 M ANG-(3-4). This lower level of heterodimers
was that found in SHR, and heterodimers did not dissociate when the same
concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg
body mass) increased [Na+]ur and UNaV with a simultaneous decrease in systolic
arterial pressure in SHR, but not in WKY rats. Thus, the influence of ANG-(3-4) on Na+
transport and its hypotensive action depend on receptor association and on blood
pressure.
CNPq, CAPES, FAPERJ, FAPESP, INBEB
D28
INTERAÇÃO PARÁCRINA ENTRE CÉLULAS RENAIS E CÉLULAS
ESTROMAIS MESENQUIMAIS: ESTUDO DOS MEDIADORES LIPÍDICOS
1,2- Santanna, J.F; 1,2- Andrade, A.S ; 1,2- Lindoso, R.S ; 1- Luna Gomes,T ; 1,2- Vieyra,
A.R ; 1- Bandeira-Melo, C. ; 1,2- Einicker-Lamas, M
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural de Bioimagem (INBEB)
Células Mononucleares de Medúla Óssea (CDMO), em especial Células Mesenquimais
(CM) possuem importante papel no reparo do tecido renal. Sabe-se que CDMO são
mobilizadas a locais de lesão e podem atuar através da secreção parácrina. Assim,
acredita-se que eicosanóides possam estar associados a repostas renoprotetoras
observadas durante interação parácrina entre células renais e CM pós lesão. CDMO/CM
foram co-cultivadas por 3 h com células HK-2 através de poços Millicell, possibilitando a
troca de moléculas bioativas por ambos os tipos celulares. A lesão foi induzida em
células HK-2 por depleção de soro 24 h e de ATP por Antimicina A (10 µM) 30 min. A
biogênese de corpúsculos lipídicos, um dos principais sítios de síntese dos eicosanóides,
foi avaliada por microscopia ótica após coloração com Tetróxido de Ósmio 1,5 %.
Eicosanóides foram dosados por Ensaio Imuno- Enzimático de competição do
sobrenadante das co-culturas e das culturas individuais de Hk-2 ou CDMO/CM. Como
resultados, eicosanóides são modulados nas diferentes condições experimentais. Níveis
de prostanóides totais decaem em cultura de células HK-submetidas a lesão e são cerca
de 5 x maiores em condições de interação parácrina com CDMO. Níveis de
prostaglandina E2 acompanham o mesmo padrão obsevado nos prostanóides totais.
Um aumento marcante de prostaglandina D2 é observado em condições de co-cultura
de HK-2 com CM. Corpúsculos lipídicos sofrem alterações nas HK-2 tanto em número,
aumentado pós lesão e pós co-cultura com CM; quanto em diâmetro, aumentado pós
lesão e reduzido em co-cultura com CM. O número de corpúsculos lipídicos em CDMO
também é aumentado pós co-cultura com HK-2. Concluindo que níveis de diferentes
prostanóides são regulados durante condições de lesão química e interação parácrina,
assim como seus sítios de síntese. Sendo possível que respostas de pró-sobrevivência
desencadeadas nas células renais em períodos de co-cultura possam estar relacionadas
a ambas alterações.
FAPERJ, CNPQ, INBEB, PROBITEC, CAPES.
D29
BRADYKININ
AND
C5A
ANAPHALATOXIN
FUEL
INTRACELLULAR GROWTH OF TRYPANOSOMA CRUZI IN MACROPHAGES
1-ALMEIDA, L.N.; CORDOVIL, T.; OLIVEIRA, A.C.; SCHARFSTEIN, J.
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
Involving two zymogens (FXII and PK) and the co-factor high molecular weight kininogen
(HK), the contact pathway of coagulation is a surface-assembled complex that promotes
pericellular proteolysis in neutrophils and macrophages. We have previously linked
bradykinin release in T. cruzi-infected tissues to development of Th1-protective
immunity. Conversely, we have recently demonstrated that T. cruzi trypomastigotes
may take advantage of the transient formation of inflammatory edema (fueled by
kinins, endothelin and C5a) to invade heart cells that naturally overexpress their
cognate GPCRs (BKRs, ETRs and C5aRs). Consistent with this, we have recently found
that mice deficient in BK1R display reduced intracardiac parasitism and are refractory to
chronic myocarditis/fibrosis (Andrade et al., abstract enclosed). Prompted by these
findings, here we investigated the impact of GPCR signaling (BK2R, BK1R, C5aR) on
intracellular outgrowth of T. cruzi amastigotes in macrophages. To this end, we added
specific antagonists of C5aR (A8B), BK2R (HOE-140) or BK1R (DAL8-BK) to monolayers of
WT or BK1R-/- bone-marrow derived macrophages (BMDM), pre-treated with IFN-γ and
infected 20 hours earlier with Dm28c T. cruzi. Measurements of the number of
intracellular amastigotes/100 cells at 72 h p.i. revealed that parasite load was markedly
reduced by each of these GPCR blockers (44%, 47% and 46%, respectively; P<0.001).
Pharmacological specificity of the host protective effect was confirmed in BK1R-/macrophages, which showed reduced amastigote outgrowth upon treatment with HOE140 or A8B, but not with the BK1R antagonist. Intriguingly, the increased resistance
conveyed by these GPCR blockers was not due to increased production of NO or ROS.
These in vitro observations are consistent with the hypothesis that generation of
infection-promoting GPCR ligands (BK, C5a, Des-Arg-BK) in inflammatory exudates might
favor parasite persistence in chronic inflamed tissues.
INBEB, FAPERJ, CNPq
D30
WHAT ARE THE GENERAL EVOLUTIONARY TRENDS OF AN OLD
PROTEIN RECRUITED FOR A NEW FUNCTION?
1,2 - FARIA, L.M.; 1 - PASCUTTI, P.G.; 2 - QUATTROCCHIO, F.M.
Página 43
1 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 Department of Genetics, Vrije Universiteit Amsterdam.
"One new member of the P-type-ATPase superfamily of transporters (PH5) is part of a
complex required for the building up of the proton gradient across the tonoplast of the
central vacuole, in petal cells. PH5 is strongly related to plasma membrane ATPases of
plants and yeast (Verweij et al., 2008). In this work we intend to investigate, by
colocalization experiments and analyses of different protein fusion constructs, some
evolutionary steps involved in the evolution of these P-H+-ATPases from plasma
membrane to the tonoplast. PH5 belongs to the plant-specific P3A subfamily of H+ATPases and has H+ pumping activity when expressed in yeast. However, PH5 is
different from the other P3A-ATPases characterized until now (e.g. Arabidopsis AHA2),
which are plasma-membrane H+ pumps, because it resides in the tonoplast (Verweij et
al., 2008). Because PH5 differs in only a few amino acids from other P3A-ATPases, those
few changes must have been sufficient to alter its location and to assume a new
function during evolution.
A set of constructs was generated in which different domains of PhAHA2 and PH5 were
exchanged and the resulting chimeras were fused to GFP. These protein chimeras were
then expressed in petunia petal cells together with the marker for the plasma
membrane RFP-SYP122. The study of the localization of these fusion proteins gives a
first indication of which domains contributed, during evolution, to the acquisition of
new characteristics by the ancestor of PH5 and to its recruitment for a new function.
Furthermore, normal modes analysis of PH5, PhAHA2 and the chimeras was carried out
as an attempt to address the evolutionary directions of their structural changes. One of
the points that has to be clarified now is whether or not amino acid changes have been
selected in order to allow proteins to follow a particular single normal mode direction.
CNPq, CAPES
D31
ISQUEMIA E REPERFUSÃO MIOCÁRDICA: ESTUDO DOS
FATORES CARDIOPROTETORES DO PRECONDICIONAMENTO ISQUÊMICO: AVALIAÇÃO
FUNCIONAL E PROTEÔMICA DE FATORES HUMORAIS COM ATIVIDADE
CARDIOPROTETORA
1-MACIEL ,L; 1,2-VERISSIMO-DA-COSTA, G. C; 1-OLIVEIRA, D.F; 1-BAPTISTA G; 1,2BISCH, P. M; 1-NASCIMENTO J.H.
1-Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho / 2- Laboratório
Multidisciplinar de Proteomica - Instituto de Biofísica Carlos Chagas Filho - Centro de
Ciências da Saúde - Universidade Federal do Rio de Janeiro
O precondicionamento isquêmico (PCI) é um mecanismo endógeno de cardioproteção
contra as in urias do infarto. PCI ode ser indu ido “distância”, em outros tecidos,
sugerindo a liberação de ativadores humorais. Objetivos: Identificar e caracterizar
fatores cardioprotetores liberados no PCI e elucidar as vias envolvidas nesta
cardioproteção. Métodos: Corações de ratos Wistar machos foram perfundidos em
aparato de Langendorf. Grupos experimentais, Controle: Submetidos a 30 min. de
isquemia e 60 min. de reperfusão (I/R); PCI: Submetidos a 3 ciclos de 5 min. de isquemia
e reperfusão, antes do I/R; Efl_pci: Perfusão do Efl-pci antes da I/R; Efl_fracionado:
Perfusão das frações do Efl_pci (cutoff <3kDa; 3-5 kDa; 5-10 kDa; 10-30 kDa; 30-50 kDa;
>50 kDa), antes da I/R. Perfusão das frações cardioprotetoras com bloqueadores: Canais
de K + sens eis a TP, glyburide 10 μM ou 5
100 μM J K- T T G490 10 μM
PKC ueleritrina 10 μM Rece tores ara adenosina - A1 (DPCPX 20 nM) e opióides
(Naloxone 10 nM). A determinação da área de infarto foi por coloração com TTC (1%). O
Efl_pci foi analisado por SDS-Page, e em LC-MS/MS, ESI-Q-Tof para identificação dos
fatores humorais. Resultados: As frações <3 kDa e entre 5 e 10 kDa, apresentaram
efetiva recuperação pós I/R. A cardioproteção pelas frações <3 kDa e entre 5 e 10 kDa
foi inibida pela glibenclamida, 5HD, queleritrina, AG490, DPCPX e naloxone. A análise
em SDS-PAGE mostrou proteínas em diversas faixas de peso molecular. A analise em LCMS/MS, identificou 987 proteínas. Onde 60 proteínas são descritas em vias de
cardioproteção. Das quais, 8 possuem tamanho menor que 10 kDa. Conclusão: Fatores
humorais, com atividade cardioprotetora, possuem peso molecular <3 kDa e entre 5 e
10 kDa. Esta cardioproteção foi antagonizada por bloqueadores para vias pré-descritas
como cardioprotetoras. Análise proteômica do Efl_pci mostrou a existência de proteínas
atuantes na cardioproteção.
CNPq, FAPERJ, CAPES
D32
CARACTERIZAÇÃO ESTRUTURAL DE CADEIAS POLIPEPTÍDICAS
NASCENTES USANDO RESSONÂNCIA MAGNÉTICA NUCLEAR EM SOLUÇÃO
1 - VAZQUEZ, L; 2 - ALMEIDA, M. S.; 3 - WÜTHRICH, K
1,2 - Instituto de Bioquímica Média de Meis/ INBEB; 3 - The Scripps Research Institute
"Proteínas incorretamente dobradas não tem um tempo de meia vida suficiente dentro
de um organismo, o que de outra forma que pode levar a doenças de proteínas mal
enoveladas, como a doença de príon, malde Alzheimer, câncer ou doença de Parkinson.
Os ribossomos sintetizam cadeias de polipeptídeo, e durante a sua síntese, as proteínas
nascentes têm a capacidade de se auto estruturar.
A exigência de ribossomo para a realização da conformação específica do peptídeo
nascente ainda é controversa. Com o melhor do conhecimento, alguns estudos já
indicaram que polipeptídeos nascentes, ainda ligados ao ribossomo, podem dobrar na
conformação nativa, após a sua cadeia completa ser quase alcançada. No entanto,
nenhum destes estudos mostra o calculo da estrutura 3D e do vínculo deste
polipeptídeo nascente com o complexo ribossomal, por que os obstáculos das técnicas
de se trabalhar com um grande complexo, intrinsecamente reduz a quantidade de sinais
de RMN, e limita a proposta nete sentido. RMN em solução, é uma técnica de escolha
para caracterização este processo uma vez que muiot provavelmente a cadeia nascente
polipéptido é muito dinamica, até atingir certo ponto de crescimento. Em face das
dificuldades da configuração experimental dos estudos anteriores e o desafio que pode
surgir a partir da solubilidade dos intermediários dobráveis, nós concebemos um
protocolo em que a proteína alvo pode ser produzida de forma recombinante, em
diferentes fases de alongamento, na proção c-terminal fundidos para um aumento da
solubilidade e de expressão (SET). Essas construções serão pequenas o suficiente (c.
9kDa para GB1 SET e 0,5-10 kDa para os polipeptídeos alvo) para permitir que as suas
estruturas 3D determinadas para frente sejam bem estabelecidos através de tripla
ressonância pelo métodos de espectroscopia de RMN."
INBEB, FAPERJ, CNPq
D33
ATIVIDADE PROMISSORA DA ESTEROL HIDRAZONA H3
CONTRA SPOROTHRIX SCHENCKII E SPOROTHRIX BRASILIENSIS.
1-BORBA-SANTOS, L.P.; 2- ISHIDA, K.; 3,4- VISBAL, G.; 5- RODRIGUES, A.M.; 5- DE
CAMARGO, Z.P.; 6- LOPES-BEZERRA, L.M.; 1- ROZENTAL, S.
1-IBCCF, UFRJ, Rio de Janeiro, Brasil; 2-ICB, USP, São Paulo, Brasil; 3-IVIC, Caracas,
Venezuela; 4-INMETRO, Rio de Janeiro, Brasil; 5-UNIFESP, São Paulo, Brasil; 6-UERJ, Rio
de Janeiro, Brasil.
"Sporothrix schenckii sensu stricto e Sporothrix brasiliensis são descritas como as
espécies mais virulentas do complexo Sporothrix schenckii, que agrupa fungos termodimórficos causadores da esporotricose. O objetivo deste trabalho foi avaliar a atividade
antifúngica da esterol hidrazona 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3),
um inibidor da enzima esterol metiltransferase da via de síntese do ergosterol, contra
isolados de S. schenckii e S. brasiliensis.
Página 44
A concentração inibitória mínima (CIM) para anfotericina B, itraconazol e H3 foi
determinada para 32 isolados (16 de cada espécie) na forma filamentosa e de leveduras
com base nos protocolos adaptados M38-A2 e M27-A3 do CLSI. A susceptibilidade
também foi avaliada segundo as concentrações fungicidas mínimas (CFM), ensaios de
“Checkerboard” e “Time kill”. s efeitos nas le eduras decorrentes dos tratamentos
foram avaliados por microscopia eletrônica de varredura e transmissão; e o acúmulo de
lipídios neutros e alterações na atividade mitocondrial foram avaliados através da
marcação com BODIPY 493/503 e MitoTracker Red CMRos e análise por citometria de
fluxo. Adicionalmente, foram determinadas a citotoxicidade em células de mamífero e a
atividade hemolítica de H3.
H3 apresentou maior atividade antifúngica do que anfotericina B e itraconazol frente ás
duas espécies na forma filamentosa e de leveduras, com CIMs variando de 0.015 a 0.5
μg/m e 0.03 a 0. 5 μg/m , res ecti amente. nsaios de “Time kill” demonstraram ue
H3 possui atividade fungistática. Combinações de H3 com itraconazol apresentaram
efeito sinérgico contra S. brasiliensis.
Leveduras de S. schenckii tratadas por 96 horas com concentrações sub-inibitórias de
H3 apresentaram acúmulo de lipídios neutros, aumento da espessura da parede celular
e interferência na conversão da levedura para a forma filamentosa. Por outro lado,
leveduras de S. brasiliensis tratadas apresentaram atividade mitocondrial reduzida,
inchaço mitocondrial e diminuição da camada microfibrilar.
H3 apresentou baixa citotoxicidade e efeito hemolítico em células de linhagem contínua
LLC-MK2 e hemácias humanas, respectivamente; e grande seletividade fúngica.
Juntos estes resultados refletem a grande atividade antifúngica in vitro de H3. Mais
esforços serão empregados para determinar seus mecanismos de ação e sua atividade
in vivo em modelo experimental de esporotricose."
INBEB, FAPERJ, FAPESP, CAPES, CNPq
D34
ANTILEISHMANIAL ACTIVITY OF THE CROTOXIN DERIVED
FROM CROTALUS DURISSUS TERRIFICUS SNAKE VENOM.
1,2 - Farias, L.H.S.; 2,3 - Rodrigues, A. P. D.; 4 -Sampaio, S. C.; 1,2 -Silva, E.O.
1-Laboratório de Parasitologia/ Laboratório de Biologia Estrutural, UFPa; 2- Instituto
Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade
Federal do Rio de Janeiro, Ilha do Fundão, Brazil; 3-Laboratório de Microscopia
Eletrônica, Instituto Evandro Chagas, Pa. 4-Laboratório de Fisiopatologia, Instituto
Butantan, SP.
American Tegumentary Leishmaniasis (ATL) is a parasitic disease, widely spread in most
countries of Latin America, and caused by different species of the genus Leishmania.
This protozoan is an obligate intracellular parasite that developed mechanisms to
subvert the microbicidal activity of macrophages, such as inhibition of superoxide and
nitric oxide (NO) production. The chemotherapy is one of the most effective treatments
for this disease. The antileishmanial drugs available are in general toxic, expensive and
require long-term treatment. Thus, the development of new natural products to treat
leishmaniasis has become a priority. Ophidian toxins are natural sources of bioactive
products with therapeutic properties already described. Therefore, we consider analyze
the activity of crotoxin (CTX), a dimeric protein and the main neurotoxic component of
Crotalus durissus terrificus snake venom, against promastigotes of Leishmania (L.)
amazonensis and macrophages. The toxin significantly decreasing of 32,5% on the
growth of promastigotes at 1,2µg/mL and 24,9% at 4,8µg/mL after 96 hours of
treatment (IC50= 22,86µg/mL). The colorimetric assay (MTT), that measures metabolic
viability of mitochondria, showed that this compound presented no cytotoxic effects
against macrophages. Interestingly, CTX treated macrophages presented a significant
higher capacity to metabolize the MTT substrate (mean= 59,78% ±3,31, higher) when
compared with untreated control, indicating intense mitochondrial activity.
Furthermore, we analyze the capacity of host cell to produce reactive oxygen species
(ROS) when treated with CTX, using CellRox green® (Molecular Probes) labeling. It was
observed that treated macrophages presented intense production of ROS (mean=
35,95% ±2,76, higher) when compared with untreated cells, indicating the capacity of
CTX to stimulate the microbicide response of host cell. These results demonstrated that
CTX inhibits the growth of parasites and does not have cytotoxic effects on the host cell.
Taken together, our results demonstrated that this compound could be a promising
antileishmanial agent.
CAPES, UFPA, CNPq, INBEB, FAPERJ
D35
PKC EPSILON STIMULATES CU(I)-ATPASE ACTIVITY OF ATP7B
FROM PORCINE LIVER
1,2- CARDOSO, L.H.D.; 1,2- VIEYRA, A.; 1,2- LOWE,J.
1 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem
"Copper is essential to several organisms, although in high concentrations it is extremely
toxic. Therefore, copper levels in the organism must be finely regulated. ATP7B is one of
the two mammalian copper-transporting ATPases, and it is essential to hepatic copper
excretion into the bile. Previous studies from our laboratory have demonstrated that
PKA inhibits ATP7B activity. The aim of this work is to determine the involvement of PKC
on the modulation of ATP7B activity.
Golgi-enriched membrane preparations were obtained from pig liver, and different PKC
isoforms (alfa, epsilon, zeta) were detected by western blotting. Cu(I)-ATPase activity
was measured using a colorimetric assay to detect inorganic phosphate. The PKC
activator PMA (10-8 M) stimulated Cu(I)-ATPase activity by 55%, while PKC inhibitor,
calphostin C (10-8 M) decreased activity by 40%. Addition of lambda phosphatase (80
U/mL) after pre-incubation with PMA decreased ATP7B activity to the same levels of
lambda phosphatase alone, showing that a kinase-mediated phosphorylation step
occurs in the modulation of ATP7B activity, which was later confirmed by a kinasemediated phosphorylation assay. The inhibition of phospholipase C by U73122 (10-10
M) decreased ATP7B activity. Using PMA combined with the Ca2+ chelator EGTA, ATP7B
activity increased, indicating the involvement of a novel PKC, not stimulated by Ca2+.
ATP7B activity decreased in the presence of PKC epsilon specific inhibitor, showing that
this novel PKC isoform is responsible for the stimulation of ATP7B activity. Enzyme
kinetics assays demonstrated that PKC signaling pathway changed Vmax, but not the
affinity for ATP and copper. Catalytic phosphorylation assays did not present any
changes in ATP7B phosphorylation or dephosphorylation steps, indicating that the
change occurs in a step after ATP7B dephosphorylation.
In conclusion, signaling pathways that activate phospholipase C and PKC epsilon may
stimulate ATP7B activity, consequently increasing active copper transport which could
change the hepatic copper metabolism."
FAPERJ, CNPq, INBEB
D36
EFEITO
CARDIOPROTETOR
DO
PROPRANOLOL
NA
INSUFICIÊNCIA CARDÍACA ESTABELECIDA EM RATOS WISTAR ADULTOS DESNUTRIDOS
CRONICAMENTE.
Mendes, L.V.P.1; Oliveira-Pinto, L.M.2; Nascimento, J.H.M.2; Vieyra, A.2,3; Cunha,
V.M.N.1; Lara, L.S.1,3 Mendes, L.V.P.1; Oliveira-Pinto, L.M.2; Nascimento, J.H.M.2;
Vieyra, A.2,
1-Instituto de Ciências Biomédicas - UFRJ; 2-Instituto de Biofísica Carlos Chagas Filho –
UFRJ; 3-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem.
Página 45
A desnutrição crônica promovida em ratos wistar (DBR-CR) modifica a hemodinâmica
cardíaca estabelecendo um quadro de insuficiência cardíaca (IC). O objetivo deste
trabalho foi determinar se o tratamento com propranolol (30 mg/Kg) previne o
desenvolvimento da IC, avaliando a sinali a o β-adrenérgica. Após o desmame, ratos
Wistar foram divididos em: Controle e Controlep (alimentados com dieta convencional
sem e com tratamento com propranolol, respectivamente); DBR-CR e DBR-CRp
(desnutridos sem ou com tratamento, respectivamente). Após o sacrifício (13 semanas),
os corações foram utilizados para ensaios de Langendorff e bioquímicos (n=5, por
grupo; procedimentos aprovados pelo CEUA da UFRJ; DFCICB030). Foi observada a
diminuição da pressão ventricular esquerda máxima (PDmáx) do grupo DBR-CR
comparado ao controle (45 %); bem como a do índice de contratilidade (+dP/dTmax) (40
%) e a do índice de relaxamento (-dP/dTmáx) (30 %). Houve redução do Emax do
isoproterenol (143±15 vs 184±11 %). Foi observado deslocamento para a esquerda da
curva de Frank-Starling, e a redução da capacitância venosa em 27%. O tratamento com
propranolol promoveu: (1) a manutenção da PDmáx; (2) o aumento da +dP/dTMax e
da -dP/dTMax e (3) redução em 60% do Emáx do isoproterenol. Em relação as proteínas
cinases, o tratamento reverteu a diminuição da razão entre atividade e expressão da
proteína cinase A (PKA) observado no grupo DBR-CR, identificando-se aumento do
conte do roteico de PKCα isoforma rotetora da IC e diminui o de PKCε en ol ida
no desenvolvimento da IC). Estes dados indicam que a redução da contratilidade e da
complacência, estabelecida no grupo DBR-CR está associado a dessensibilização da via
β-adrenérgica. O tratamento com propranol preveniu a redução da função
hemodinâmica cardíaca, re-estabelecendo a ia de sinali a o β-adrenérgica.
CNPq
D37
THE ENDOCANNABINOID SYSTEM AS A POTENTIAL
REGULATOR FOR Na+ HOMEOSTASIS IN KIDNEY CELLS
1, 2- SAMPAIO, L. S., 1, 2- TAVEIRA-SILVA, R., 1, 2- VIEYRA, A., 3- DI MARZO, V.,2- REIS, R.
A. M., 1, 2- EINICKER-LAMAS, M.
1- Instututo Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem. 2Instituto de Biofísica Carlos Chagas Filho – UFRJ, 3- Endocanabinnoid Research Group,
Istituto di Chimica Biomolecolare, CNR, Pozzuoli
"The endocannabinoid system (ECS) is classically formed by cannabinoid receptors,
endogenous lipids, enzymes for the synthesis and degradation. ECS is primarily involved
in the modulation of rapid responses in various cellular models, including regulating the
intracellular concentration of ions.
The aim of this study was to identify the ECS in renal proximal tubule and evaluate the
role of cannabinoids on the sodium active primary transport.
For this purpose, LLC-PK1 cell line was grown at 90% confluence on DMEM+FBS 10%. To
evaluate the presence of ECS, immunofluorescence and PCR were performed. The
measure of activity of the Na+-K+ATPase was by colorimetric method. cAMP was
assayed by the [3H] cAMP incorporation.
Our data show that main enzymes and receptors of the ECS are expressed in LLC-PK1.
Treatment with Win-55-212-2 (WIN), CB1/2 agonist, increases Na+-K+ATPase activity at
1 or 30min of incubation. Hemopressin (HP), CB1 inverse agonist, increased the activity
with 1min, but decreased in 30min of treatment. Addition of AM251, CB1 antagonist,
reverted the effect of both in 30min, but had no effect upon 1min. Pretreatment with
Capsazepine, TRPV1 antagonist, abolished the effect of WIN or HP in 1min. Pretreated
with PTX not showed difference on WIN or HP effect. The cAMP level increased 30min
after addition of HP and was inhibited by AM251. The H-89, PKA inhibitor, returned the
HP effect and Calphostin C, PKC inhibitor, blocked the WIN action. Our data suggests
that in LLC-PK1 there are different pathways of ECS regulated the Na+-K+ATPase
between CB1/TRPV1, cAMP/PKA and PKC"
INBEB, CAPES, CNPQ, FAPERJ
D38
ESTUDO DOS EFEITOS DO METILMERCÚRIO SOBRE OS CANAIS
DE POTÁSSIO Kv4.3, hERG e KCNQ1/KCNE1 EM CÉLULAS HEK-293
1-SANTOS, M.C.P.; 2-GALLEGO, M.; 1-MEDEI, E.; 2-CASIS, O.; 1-NASCIMENTO, J.H.
1 – Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho, IBCCF, UFRJ – RJ Brasil 2 – Departamento de Fisiologia, Escola de Farmácia, UPV/EHU - Pais Vasco Espanha
"INTRODUÇÃO: O metilmercúrio (MeHg) é um organomercurial encontrado em peixes e
outros animais aquáticos da Região Amazônica em áreas expostas à contaminação
mercurial. Estudos epidemiológicos têm mostrado que a exposição humana ao MeHg
pode contribuir para o desenvolvimento e progressão de desordens cardiovasculares,
tais como aumento do risco de arritmias cardíacas e morte súbita. Atualmente é bem
aceito que mudanças (preponderantemente diminuição) nas correntes repolarizantes
de potássio são uma das principais causas destes eventos arrítmicos. OBJETIVO:
Investigar os efeitos do MeHg sobre as correntes iônicas repolarizantes de potássio:
Kv4.3; hERG e KCNQ1/KCNE1, envolvidas diretamente na repolarização celular, em
células HEK-293. MATERIAIS E MÉTODOS: Para o estudo eletrofisiológico, foi utilizado o
método de Patch-clamp na configuração Whole-cell. Assim, protocolos de voltagem
específicos foram utilizados para registro das correntes de potássio permeadas pelas
seguintes subunidades: Kv4.3, hERG e KCNQ1/KCNE1. Células HEK-293, foram
transfectadas com as subunidades acima descritas. Uma curva dose-resposta com as
seguintes concentrações de MeHg foi realizada: 0 nM; 0,01 nM; 0,1 nM; 1 nM visando
analisar as mudanças nas correntes iônicas correspondentes. RESULTADOS: O MeHg
promoveu redução na amplitude de todas as correntes iônicas estudadas (p<0,05). A
cinética de ativação e inativação de Kv4.3 e de ativação e desativação de hERG não
sofreu alterações com a utilização do MeHg. Entretanto, a corrente permeada pelas
subunidades KCNQ1/KCNE1 apresentou mudança na dependência de voltagem da
ativação, tendo seu V1/2 deslocado para potenciais mais positivos. CONCLUSÃO: Os
resultados apresentados mostram que o MeHg é capaz de promover mudanças nas
correntes repolarizantes semelhantes àquelas classicamente encontradas em corações
com remodelamento elétrico e maior risco de arritmias cardíacas."
CNPq, Ciência sem Fronteiras
D39
SYNTHESIS, CHARACTERIZATION AND EVALUATION OF
XANTHENEDIONES AS TYROSINASE ACTIVATORS
1 FERREIRA, M.S.; 2 PIRES, D.A.T.;1 FIGUEROA-VILLAR, J.D.
1- Departamento de Química, Instituto Militar de Engenharia; 2- Instituto de Química,
Universidade de Brasília
Tyrosinase is the enzyme responsible for production of melanin. The appearance of
melanoma, the most serious form of skin cancer, can be related to disorders of this
enzyme. Therefore is important to develop tyrosinase inhibitors to avoid skin cancer.
One of the tested compounds as inhibitors of this enzyme were tetraketones. However,
these compounds only display relatively limited inhibition activity. We discovered using
NMR spectroscopy that some of these tetraketones suffer cyclization in solution at
room temperature, forming the respective xanthenedione. For this reason, we
considered the possibility of these xanthenediones displaying an opposite effect of
tetraketones with tyrosinase, acting as activators. Therefore, seven of these compounds
were synthesized in two steps: reaction of dimedone with benzaldehydes, forming
tetraketones, followed by cyclization using p-toluenesulfonic acid at 100 oC, leading to
Página 46
good yield (83 to 94%). The structure of these compounds were determined by NMR
and IR spectroscopy and tested as inhibitors or activators of tyrosinase using as
substrates L-tyrosine or L-Dopa by UV spectroscopy. The obtained results confirmed
that six xanthenediones are activators of this enzyme. The two more effective
tyrosinase activators were para-methoxyphenyl xanthenedione and phenyl
xanthenedione, which increased the activity of the enzyme 89.88 and 172.56%,
respectively. In function of these results we consider necessary the preparation of new
tetraketones or similar compounds which could not be cyclized, a condition that could
lead to better anticancer activity.
INBEB, CNPq e CAPES
D40
TLR2 KNOCKOUT (TLR2-/-) ATTENUATE STREPTOZOTOCININDUCED CARDIAC ELECTRICAL REMODELING IN GENDER NON-SPECIFIC MANNER
Micaela López Alarcón, Gustavo Monnerat-Cahli, Gabriel Manso, Guilherme Brasil,
Emiliano Medei.
IBCCF
Several studies have shown that inflammation plays a key role in diabetes.
Cardiomyopathy is one of the main causes of death in this disease. The present study
investigates whether Toll like receptors (TLR) 2 regulates cardiac electrical activity in
diabetic mice and whether its effects is genderspecific. Methods and Results: Diabetes
was induced by 5 consecutives low doses of streptozotocin (STZ) in C57BL/6 mice from
both sexes. The animals were divided in eight groups (female and male): WT; WT+STZ;
TLR2 -/- and TLR2-/-+STZ. Eight weeks after diabetes induction, metabolic and biometric
parameters were analyzed. The cardiac electrical function was analyzed by ECG and
cardiac AP. The TLR2 knockout did not prevent the hyperglycemia neither preserved
biometrics alterations in both sexes. Despite the chronic hyperglycemic state, the TLR2/-+STZ preserves in both sexes the ECG and action potential parameters analyzed. In
addition the TLR2-/-+STZ prevented the plasmatic increase of IL-1beta and TNF-alpha
induced by diabetes. The direct stimulation of either TLR2/1 or TLR2/6 by its specific
agonists promotes a cardiac electrical remodeling, prolonging the AP duration. Cardiac
function assess by MRI was similar among groups. Conclusion: The data obtained with
the agonist upon cardiac electrical activity and the plasmatic cytokines demonstrated
that, at least, two different mechanisms could be involved inTLR2-/- preserve cardiac
electrical remodeling in hyperglycemic condition.
FAPERJ, CNPq, INBEB
D41
ROLE OF CCR2 IN LEARNING PROCESS AND MEMORY
CONSOLIDATION
1-TEIXEIRA-DA-CUNHA, M.G.; 1-ALVES-JÚNIOR, 1-S.C; CHAVES, D.; 1-ALBUQUERQUE, D.;
2-RAJAO, M.; 3-GUERCIO, G.; 1- ’ VI , J.C.P. 3-PIAZINUTI, R.; 3-MENDEZ-OTERO, R.; 1REIS, P.A.; 1-BOZZA, P. T. ; 1-BOZZA, F.A.; 1-CASTRO-FARIA-NETO, H.C.; 1-GOMES, R.N.
1-Instituto Oswaldo Cruz, FIOCRUZ; 2- INCA; 3- UFRJ
The CCR2 is a chemokine receptor coupled to G protein. Its major ligand is the CCL2, in
wich is correlated with neuroinflammatory response in infection process, brain trauma,
Alzheimer, multiple sclerosis. But recent studies demonstrated that both CCR2 and CCL2
are expressed constitutively in important places of the central nervous system, such as
in hippocampus. The CCL2 increases the neuronal intracellular calcium and act as a
neuron-excitatory mediator in neurons of the hippocampus. Any studies showed the
role of CCR2 in memory consolidation under normal physiological conditions. To clarify
this point, we analyzed the role of CCR2 in learning process and in memory
consolidation of short time memory and longtime memory. For this we used CCR2
deficient mice, and submitted this animals to water maze test to evaluate the
contextual memory, to passive avoidance test to analyze the aversive memory, to open
field and PPI to examined both motor response and reflex response. We observed that
the absence of CCR2: impaired the memory consolidation of contextual memory in
comparison with wild type animals, increased the number of trials that the animals
require to learn in passive avoidance test, decreased the consolidation of short time
memory and longtime memory. The cognitive dysfunction is not associated to motor or
reflex impaired response. This decreased consolidation of memory was correlated with
lower expression of MAPK p-42-44 phosphorylated 1 hour after training in passive
avoidance test and decreased expression of mature BDNF in hippocampus 24h hours
after training in passive avoidance test. In CCR2 deficient mice we observed decreased
co-localization of PSD95 and Synaptophysin. These data suggest that the CCR2 is
important to function of synapsis in hippocampus, to neuronal function involved with
learning process and to sustentation of the memory of contextual and aversive memory.
IOC - FIOCRUZ, FAPERJ
D42
STUDIES BY MOLECULAR DYNAMICS AND GENERALIZED
SIMULATED ANNEALING OF ANTIMICROBIAL PEPTIDE DERMADISTINCTIN K
Batista, Moema M..1 Fernandes,T cio V. .1, antoro, Marcelo M.3† Pascutti, Pedro
G.1,2
1Lab. de Modelagem e Dinâmica Molecular, IBCCF-UFRJ, RJ, 2INMETRO - Instituto
Nacional de Metrologia, Qualidade e Tecnologia, RJ, Brazil. Dep de Bioquímica-ICBUFMG, MG, 3 (deceased in march 10, 2014)
"INTRODUCTION: Dermadistinctin (DD K) is an antimicrobial peptide from Phylomedusa
distincta frog skin. It has an α-helix structure that appears only in biomembrane or in
presence of amphipathic cosolvent. The aim of this work was to study conformationaldynamics-structure of DD K peptide in a solvent used to stabilize peptides (2,2,2trifluorethanol-TFE/water mixture) by molecular dynamics (MD) simulations and
generalized simulated annealing (GSA). METHODOLOGY: The structural properties of
this peptide have been simulated in TFE/water mixture using GROMACS 4.5.5 and
Gromos53a6 force field. DD K peptide was fitted in a cubic box solvated with SPC (single
point charge) water model and TFE (50%/50%). 5 ions of chloride were added to this
system for balance of charge. The pressure used was 1 atm and the temperature was
293,15 K. The reference structure was taken from PDB website by code: 2JX6. The GSA
program was developed in the LMDM at UFRJ to predict peptides structures. It
randomly searches for the global minimum in energy hyper-surface. We could build a
hydrophobic environment to DD K by MD simulation and GSA algorithm where this
peptide could fold. RESULTS: We observed a coil structure in the first 10 residues that in
MD simulations presented a transition to β-sheet. This structure change suggests an
alternative mechanism for DD K insertion in membrane. GSA results did not show this
transition. We also studied the statistic of the solvent radial distribution around each
peptide residue. As result ALA, GLU, ILE, LEU, TRP were solvated in TFE and LYS, GLY
were solvated in water. When native contacts were calculated, 80% of conformations
were next the reference structure in GSA and MD simulations. MD simulations in
TFE/water mixtures were important to study DD K dynamics, showing standard of
conformation that could help to understand the biological activity of the peptide in
biomembranes."
INBEB, FAPEAM, INMETRO, FAPERJ
D43
IDENTIFICATION OF PROMISING ANTI-PRION
CANDIDATES THROUGH EX-VIVO, IN VITRO AND IN SILICO METHODOLOGIES
DRUG
Página 47
NATALIA C. FERREIRA1, WESLEY A. CONCEIÇÃO1, BRUNO MACEDO1, CLARICE S.
MACHADO1, ALESSANDRA MASCARELLO2, LOUISE D. CHIARADIA-DELATORRE2,
ROSENDO A. YUNES2, RICARDO J. NUNES2, ANDREW G. HUGHSON3, JASON
HOLLISTER3, GERALD S. BARON3, BYRON CAUGHEY3, YRAIMA CORDEIRO1
1- Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brasil. 2- Departamento de Química,
Universidade Federal de Santa Catarina, Santa Catarina, Brasil. 3- Laboratory of
Persistent Viral Diseases, Rocky Mountain Laboratories, National Institutes of Health,
Montana, United States of America.
Introduction: Transmissible spongiform encephalopathies (TSEs) are infectious
neurodegenerative disorders that do not have a symptomatic, curative or prophylactic
treatment. TSEs arise after the conversion of the cellular prion protein (PrPsen) which is
soluble, into the scrapie isoform (PrPres) that aggregates and accumulates in the brain.
Although many compounds have been effective in vitro none worked in vivo, mainly
because of their inability to cross the blood-brain-barrier (BBB). Screening in prioninfected cell culture (ScN2a) is commonly used to identify new anti-prion compounds.
Material and methods: In this work we tested 200 aromatic chemical compounds in
neuroblastoma cell infected with RML, 22L and CWD strains. Real-time quaking induced
conversion (RT-QuIC) assay tested the ability of these compounds to inhibit the
conversion in a cell-free system. Confocal microscopy verified if the active compounds
change PrP cellular location. In silico predictors were used to deduce about
pharmacokinetic and pharmacodynamic properties of these molecules. MTT reduction
assay allowed verifying their cytotoxicity in neuroblastoma cells. Molecular docking
provided structural models and binding affinities for the interaction between PrP and
the most promising compounds. Results: 47 compounds were able to inhibit in more
than 50% the amount of PrP in ScN2a cells infected with RML strain. Many compounds
were also effective against 22L and CWD strains. Three compounds were effective in the
RT-QuIC when brains from hamster and a patient infected with CJD were used as seed.
We found that some compounds induce the internalization of PrP in neuroblastoma
cells, decreasing the availability of PrP in the cell surface to be converted into PrPres.
These compounds were not cytotoxic and the 5 most promising ones were predicted as
non-mutagenic and able to cross the BBB. Conclusions: We identified new anti-prion
drug candidates which are effective in vitro and ex-vivo, and posses pharmacokinetic
properties that support their drugability.
CNPq, FAPERJ, INBEB
D44
A MEMBRANOTROPIC HEPATITIS C VIRUS PEPTIDE INTERACTS
WITH MODEL MEMBRANES AND HUMAN CELL SURFACE AND GAINS HELIX CONTENT
1-ALVES, N.S.; 1-MENDES, Y.S.; 2-SOUZA, T.L.F.; 1-BIANCONI, M.L.; 1-CARVALHO, C.A.M.;
3-ANOBOM, C.D.; 1-VALENTE, A.P.; 1-SILVA, J.L.; 1-GOMES, A.M.O; 1-OLIVEIRA, A.C.
1-Instituto de Bioquímica Médica Leopoldo de Meis, Univerisdade Federal do Rio de
Janeiro; 2-Faculdade de Farmácia, Univerisdade Federal do Rio de Janeiro; 3-Instituto de
Química, Univerisdade Federal do Rio de Janeiro
The Hepatitis C virus (HCV) is the major cause of viral hepatitis, becoming a worldwide
health problem. HCV is an enveloped RNA virus, and the glycoproteins, E1 and E2,
mediate cell entry and membrane fusion. Unfortunately, the localization of a fusion
peptide remains unknown. The structural characterization of HCV fusion mechanisms is
ery im ortant for the de elo ment of anti− CV strategies. ere we characteri e the
interaction of an E2 membranotropic peptide, HCV421-445, with different membrane
models. Peptide-membrane interactions and changes resulting from these interactions
were evaluated by circular dichroism (CD), isothermal titration calorimetry (ITC),
differential scanning calorimetry (DSC), dynamic light scattering (DLS), two-photon
excitation microscopy and nuclear magnetic resonance (NMR). ITC data show that
peptide-micelle interaction is endothermic and is favored by micelle charge. This
membrane-charge dependence is also observed by DSC, when changes in transition
temperature are only observed when phosphatidylglycerol is present. HCV421-445
induces vesicle aggregation in a pH-dependent manner, as observed by DLS. Twophoton microscopy reveals that FITC-labeled peptide binds to HepG2 membrane and
remains at cell surface for at least 30 minutes. CD and NMR analysis show that the
peptide is unstructured in solution and gains helix content when in the presence of
micelles. This helix could be stabilized by a helix stabilization motif, GXXXG. Our results
indicate that HCV421-445 is able to perturb lipid membranes, and becomes structured,
a common feature of fusion peptides. As this peptide may be important for cell fusion,
these studies are important to better understand the HCV fusion mechanisms, which
could help in the rational development of new antiviral therapies.
Capes, CNPq, FAPERJ, PRONEX, INBEB
D45
POTENCIAL
TERAPÊUTICO
DE
CÉLULAS-TRONCO
PLURIPOTENTES HUMANAS EM MODELO RENAL DE ISQUEMIA E REPERFUSÃO
1, 2- SIGNORETTI, P. V.P.; 2- FERNANDES, A. M.; 2- ASSIS, J. L.; 1- VIEYRA, A.; 1VALVERDE, R. R. F. H.; 2- LAMAS, M. E.
1- Laboratório de Físico-Química Biológica Aída Hassón Voloch, Instituto de Biofísica
Carlos Chagas Filho; 2- Laboratório de Biomembrana,Instituto de Biofísica Carlos Chagas
Filho
"INTRODUÇÃO: As doenças renais (DRs) representam um grave problema de saúde
pública onde cerca de 50% dos pacientes não sobrevivem. Tratamentos como a diálise,
aumentam a qualidade de vida dos pacientes mas tem efeito meramente paliativo e
grande impacto financeiro no SUS, principal financiador do tratamento de
aproximadamente 85% destes pacientes. Uma alternativa para o tratamento das DRs é
o transplantes, contudo, a disponibilidade do órgão e a compatibilidade
doador/receptor continuam sendo fatores determinantes e limitantes. Diante deste
cenário, a utilização de células-tronco visando o reparo de órgãos lesados é sugerida
como principal alternativa. Células-Tronco Embrionárias Humanas (hES) são relevantes
em estratégias regenerativas principalmente por oferecerem possibilidade de reposição
das células afetadas devido a sua pluripotencialidade. OBJETIVO: Diante do exposto
acima, o presente trabalho teve por objetivo analisar o potencial terapêutico de hES em
modelo animal de lesão renal por isquemia e reperfusão (I/R). METODOLOGIA: 1 x 106
células/100 μ foram injetadas na cápsula renal de ratos machos Wistar 1 hora antes da
lesão por isquemia bilateral (30 minutos) e reperfusão (24 h) (CEUA: IBCCF148). Os
animais foram acompanhados 4 e 24 horas após injeção por SPECT (CENABIO) via
marcação com DMSA-Tc99m e análises para marcadores de células humanas (anti-HNA)
e de morte celular além de swelling mitocondrial foram realizadas. RESULTADOS: Foi
observada a melhora no parênquima renal 24 h após administração de hES. Marcação
para HNA indicou a presença das hES no tecido renal isquêmico. Além disso, houve
aumento de 5x na expressão de Bcl-2 (sem alteração significativa de Bax) e redução do
swelling mitocondrial nos animais submetidos à injeção com as hES quando comparado
aos animais isquêmicos.
CONCLUSÃO: A partir desses dados preliminares, podemos sugerir que as hES parecem
recuperar a função e a integridade tecidual renal neste modelo de lesão por I/R."
CAPES/PROBITEC, CAPES/INL, FAPERJ
D46
EFEITO CRÔNICO DE ESTERÓIDES ANABÓLICOS SOBRE AS
CORRENTES ICA,L E ITO E A MOBILIZAÇÃO DE CÁLCIO EM MIÓCITOS VENTRICULARES.
1-ARANTES, P. C.; 2-MEDEI, E.; 3- NASCIMENTO, J. H
Página 48
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
Esteroides anabólicos (EAs) são derivados sintéticos da testosterona utilizados
ilicitamente por atletas e não atletas, em doses supra-fisiológicas, para promover ganho
de massa muscular, força e resistência ou para fins estéticos. Causam efeitos colaterais,
dentre eles alterações cardiovasculares, como hipertensão arterial, hipertrofia cardíaca,
arritmias e infarto do miocárdio. Este estudo objetivou investigar o remodelamento
elétrico cardíaco e sua repercussão no acoplamento excitação-contração, induzido pelo
tratamento crônico com EAs. Ratos sedentários, foram tratados por 8 semanas com 10
mg/kg/sem. (i.m.) de decanoato de nandrolona (Deca) ou veículo (Ctrl). Foram usadas
técnicas de ECG na derivação D2, microeletrodo intracelular e patch clamp na
configuração Whole cell, para análise da repolarização ventricular e a técnica de tensão
isométrica em fibra cardíaca desnuda avaliação do acoplamento excitação-contração. O
grupo Deca apresentou aumento do intervalo QT e QTc (p<0,001), sem alteração da
frequência cardíaca; aumento na duração do potencial de ação a 30% e a 90%
(p<0,001) da repolarização máxima; e aumento da triangulação do potencial de ação
(p<0,001). A amplitude de Ito nas camadas endocárdica e epicárdica do ventrículo
esquerdo foi menor (p<0,05 e p<0,001, respectivamente), enquanto a amplitude da
ICa,L foi maior no grupo Deca (p<0.01). Na avaliação do acoplamento excitaçãocontração, as medidas de tensão isométrica mostraram que o grupo Deca desenvolve
maior tensão nos pCa 6,0; 5,6; e 4,8 (p<0,001) e maior sensibilidade ao Ca2+ (p<0,05).
O grupo Deca apresentou menor resposta contrátil a diferentes concentrações de
cafeína após tempo fixo (3 min) de carregamento de Ca2+ (p<0,001) e a 20 mM de
cafeína após 1, 3 e 5 min de carregamento do reticulo sarcoplasmático (p<0,001).
Concluímos que o tratamento crônico com decanoato de nandrolona causou
remodelamento elétrico cardíaco nas alterações nas amplitudes de ICa,L e Ito refletiram
no acoplamento excitação-contração, causando menor mobilização e maior
sensibilidade ao Ca2+.
Capes,CNPq
D47
ANÁLISE ESTRUTURAL DO FRAGMENTO N-TERMINAL DA
ENDOSTATINA E SUA INTERAÇÃO PUTATIVA COM A INTEGRINA ΑVΒ3
1,2- TORRES, P.H.M.; 1- PASCUTTI, P.G.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto Nacional de Metrologia, Qualidade e Tecnologia
A angiogênese, ou neovascularização, é um processo fisiológico complexo que culmina
com a proliferação e migração de células endoteliais e a formação de novos vasos
sanguíneos. É um evento importante em processos de cicatrização e na formação do
endométrio uterino, mas também pode ser estimulado por determinadas patologias
como o câncer. Compostos antiangiogênicos, como a endostatina, a canstatina e o
arresteno podem ser, portanto, utilizados no tratamento de pacientes acometidos por
estas patologias. Muitos destes compostos, entre eles a endostatina, exercem parte de
suas funções através da interação com integrinas, que desempenham papel central na
migração das células endoteliais e no estabelecimento de novos vasos. Recentemente,
foi demonstrado que a atividade antiangiogênica da endostatina pode ser
desempenhada por um peptídeo de 27 resíduos pertencente à região N-terminal da
proteína. O presente estudo tem como objetivo contribuir para o entendimento do
mecanismo de ação deste peptídeo e a possível interação do mesmo com a integrina
alfa-v-beta-3, bem como elucidar mecanismos envolvidos na ativação desta integrina.
Para tanto, foram utilizadas técnicas de ressonância magnética nuclear, dicroísmo
circular, modelagem e dinâmica molecular. Os resultados sugerem que o peptídeo
assume uma conformação em grampo-beta, capaz de interagir com uma fenda
existente na interface entre as subunidades da integrina alfa-v-beta-3 que apresenta
complementariedade estérica e eletrostática. Identificamos também regiões
importantes para a manutenção da conformação inativa da integrina, como uma alça
pertencente à 5ª lâmina do domínio beta-propeller e um grupo hidrofóbico formado
por resíduos dos domínios beta-A e beta-tail. Sendo assim, propusemos que este
peptídeo, bem como outras moléculas antiangiogênicas, podem exercer suas funções
biológicas através de uma interação ainda não descrita na literatura e a exploração
destas regiões pode, portanto, mostrar-se relevante para o desenho de fármacos
inibidores de integrina.
INBEB, FAPERJ, CNPq
D48
NMR-SUPPORTED STRUCTURAL GENOMIC STUDIES OF
TRYPANOSOMES
1 - RACHEL SANTOS DE MENEZES; 1 - ARACELYS LÓPEZ CASTILLA; 1 - EVERTON DIAS
D'ANDREA; 1 - GABRIELA PINHEIRO HEREDIA; 1 - RAQUEL DA MOTTA; 2 - CHRISTOPH
RADEMACHER; 3 - ANNE DIEHL; 3 - HARTMUT OSCHKINAT; 1 - JOSÉ RICARDO PIRES
1 - 1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2 - 2Max
Planck Institute of Colloids and Interfaces , Berlin, Germany; 3 - 3Leibniz Institutute of
Molecular Pharmacology, Berlin, Germany
Chagas disease, Sleeping sickness and Leishmaniasis are between the so called
neglected diseases, endemic in poor countries of south-America and Africa. Sequencing
of the genome of the kinetoplastids Trypanosoma cruzi, Trypanosoma brucei and
Leishmania major (Tritryp databank) open up new perspectives for drug research
against these diseases. In each genome ca. 20,000 genes were identified ca. 50 % coding
for proteins of unknown function. Proteomic studies confirmed the expression of
several of these proteins in a life-cycle dependent manner. In the present work using
bioinformatics tools available we mined the tritryp databank to end up with a list of 409
proteins up to 30 kDa, conserved in kinetoplastids, without orthologues in mammals,
plant or fungi, without trans-membrane regions and without homologous sequences in
the PDB that should be suitable for structural studies by solution NMR. From these
proteins 20 were cloned in expression plasmids, and at least 6 were expressed as
soluble and folded proteins in E. coli, as evidenced by their 1H or 1H-15N HSQC nmr
spectra. The expression of the remaining proteins is still being optimized. The NMR
structures of four proteins were solved: Chagasin, a cysteine-protease inhibitor; FKBP12,
a peptidyl-prolyl cis-trans isomerase; a protein containing a single domain of unknown
function (DUF 1935), kinetoplastid specific related to the cysteine-protease Calpain; and
the hypothetical protein Q4D059. Interaction studies for some of these proteins with
potential ligands are also described in this work.
CAPES, CNPq, FAPERJ
D49
DESENVOLVIMENTO
DE
FÁRMACOS
ANTI-HIV:
PLANEJAMENTO DE INIBIDORES PARA O VÍRUS HIV-1 NÃO-B
1-REIS, R.R.; 1-PASCUTTI, P.G.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.
"O vírus da imunodeficiência humana (HIV) afeta o sistema imunológico caracterizando
a Síndrome da Imunodeficiência Adquirida (AIDS). Teve seus primeiros casos reportados
nos EUA, Haiti e África Central nos anos de 1977 e 1978. Segundo dados estatísticos,
esta doença matou 1,7 milhões de pessoas em 2011, tendo atualmente 34 milhões de
infectados no mundo. As proteínas do vírus são expressas como longas cadeias
polipeptídicas que devem ser processadas, via proteólise pela aspartil-protease do vírus,
para adquirirem atividade. Dessa forma, a protease é um eficiente alvo para terapia
anti-HIV, resultando em uma nova classe de fármacos, os inibidores de protease.
Existem dois tipos prevalentes do vírus: HIV-1 e HIV-2. O mais comum, HIV-1, pode ser
Página 49
dividido em grupos (M, N, O e P) e subtipos: A-K. A maior parte das infecções por HIV na
África são causadas pelos subtipos virais A e C. No Brasil, a lista de subtipos presentes
inclui os subtipos B (o principal em circulação), F (18% de prevalência), C, D, e A. O
subtipo F é distribuído em todo o país e corresponde aproximadamente a 10% das
amostras analisadas nas cidades do Rio de Janeiro e São Paulo.
Neste trabalho, estamos estudando os subtipos A, B, C, e F da protease complexada
com novos inibidores (já utilizados na clínica) e com três inibidores clássicos (ritonavir,
indinavir e nelfinavir). Como resultados preliminares, temos a análise da protease por
meio de simulações de dinâmica molecular que sugerem que a proteina é bastante
estável. Simulações de docking molecular dos inibidores no sítio ativo
dos diferentes subtipos e simulações de dinâmica molecular do complexo estão em
curso visando a proposição de novos fármacos."
CNPq
D50
STRUCTURAL STABILITY IN AMYLOIDOGENIC AND NONAMYLOIDOGENIC VARIANTS OF THE TRANSTHYRETIN PROTEIN BY MD SIMULATIONS
UNDER HIGH PRESSURE
1-REINALDO S. DE OLIVEIRA JÚNIOR; 2-FERNANDO L. PALHANO; 2-DEBORA FOGUEL; 1PEDRO G. PASCUTTI
1-Instituto de Biofísica Carlos Chagas Filho - UFRJ; 2-Instituto de Bioquímica Medica UFRJ
INTRODUCTION: The formation of insoluble amyloid fibers is a characteristic of many
diseases known as amyloidosis. The transthyretin (TTR) is a homo-tetramer protein of
55 kDa and 127 residues per monomer. Over of one hundred mutations have been
described for the structure of transthyretin related to amyloid diseases such as Familial
Amyloid Polyneuropathy (FAP), characterized by the deposition of amyloid aggregates in
the peripheral nervous system. METHODOLOGY: The dissociation and denaturation of
TTR variants have been studied in different conditions of temperature, pH and pressure.
In the previous studies our group has described that the non-amyloidogenic variant as
T119M has great stability under 3.0 Kbar, pH 7.5, and 1 °C compared with the
amyloidogenic one. We used Gromos96_53a6 Force Field, Reaction Field (radius 1.4 nm)
in electrostatic treatment; SPC/E water model, cubical box, 5 ns simulation for
stabilization the hydrated layer of protein and ions. Consecutive simulations totalize 400
ns, 25 ns for each pressure step of 0,5 kbar, temperature of 1 °C, pH 3,0, and pressure
enhancement from 1,0 bar ≅ 1,0 atm to 3,5 kbar. RESULTS AND DISCUSSION: Multiple
and sequential Molecular Dynamics (MD) simulations of WT - and V30M-TTR dimers
were performed at high pressure (up to 3,5 kbar) and in explicit water to assess the
structural stability of transthyretin. The conformational space upon protein unfolding
was explored, and it was identified structural changes that may lead to the amyloid
assembly. The analysis of molecular properties such as secondary structure, hydrogen
bonds, and solvent accessible surface area along the MD unfolding trajectories clearly
demonstrate that V30M-TTR has a much higher tendency to unfold than WT-TTR. These
results are in agreement with previously published experimental data on the
conformational stability of dimers of several TTR variants.
CNPq, FAPERJ, INBEB
D51
INSIGHTS INTO IONIZATION STATES OF THE CATALYTIC
RESIDUES IN HIV-1 PROTEASE BY HYBRID QM/MM MOLECULAR DYNAMICS
SIMULATIONS
1-SOARES, R. O.; 2-GONÇALVES, A. S.; 1-PASCUTTI, P. G.
1-Instituto de Biofísica, UFRJ; 2-Instituto Federal de Educação, Ciência e Tecnologia do
Espírito Santo,Unidade Guarapari
"The HIV protease (HIV-PR) is an enzyme whose catalytic residue consists of two
aspartate side chains (Asp25/Asp25'). This protein is a relevant therapeutic target for
antiretroviral (ARV) treatment against AIDS. Several works suggest a monoprotonation
states for the aspartate residues in the active site of HIV-PR, with one of the residues
charged and the other neutral or both sharing a proton. However, there are
experimental evidences that HIV could be found in this protonation state, but also in
other two different states. In the present work we used the combined method of
quantum-mechanics and molecular-mechanics simulations (QM/MM) to investigate the
functional role of protonation in HIV-PR complexed with a substrate. We investigated
three different protonation states for the two aspartate catalytic residues:
monoprotonation, diprotonation and deprotonation and analyzed the binding of Gag
peptide substrate p2/NC (sequence of aminoacid residues SATIM/MQRGN) to the
enzyme in these three different states. Our results demonstrate that the non
protonation of both aspartic acids has a strong influence on the dynamic behavior of the
HIV-PR flaps, making ease its opening with the greater amplitude. Beside, this work
showed that the monoprotonation in one of the Asp25 residues results in the strongest
interaction between these catalytic residues by sharing the proton. This indicates that
the alterations in the flaps's dynamics are likely relationship as the different states of
protonation on residue catalytic and we expect these findings to contribute to a better
understanding of the protonation state's role in the catalytic action of HIV-PR."
CNPq, FAPERJ, INBEB
D52
O TRATAMENTO COM LPA (ÁCIDO LISOFOSFATÍDICO)
PREVINE OS DANOS RENAIS E A ETAPA INICIAL DA ATIVAÇÃO DO ESTRESSE DO
RETÍCULO ENDOPLASMÁTICO EM RATOS WISTAR SUBMETIDO AO PROCESSO DE
ISQUEMIA-REPERFUSÃO RENAL.
1-GONSALEZ, S.R.; 1-LEAL, A.C.; 2,3-EINICKER-LAMAS, M.; 1,3-LARA, L.S.
1-INSTITUTO DE CIÊNCIAS BIOMÉDICAS - UFRJ; 2 - INSTITUTO DE BIOFÍSICA CARLOS
CHAGAS FILHO - UFRJ; 3 - INSTITUTO NACIONAL DE CIÊNCIA E TECNOLOGIA DE
BIOLOGIA ESTRUTURAL E BIOMAGEM – UFRJ.
A clínica carece de tratamentos eficazes na prevenção do dano renal mediado pela
isquemia-reperfusão (I/R). O ácido lisofosfatídico (LPA) surge como um potencial agente
terapêutico. O objetivo foi caracterizar o efeito do tratamento com LPA no modelo de
I/R renal com ênfase ao transporte de Na+ e a proteção contra o estresse do retículo
endoplasmático. Foram utilizados 3 grupos experimentais: controle; I/R: sofreu
isquemia das artérias renais por 30 min e 24h de reperfusão; I/R+LPA: administração de
LPA (1 mg/Kg) durante a isquemia. Foram realizados ensaios funcionais renais e
morfológicos, Western blot, e atividade enzimática. O conteúdo de nitrogênio ureico no
sangue aumentou em 241% na I/R e o LPA preveniu esse aumento, sem alteração da
proteinúria. O ritmo de filtração glomerular diminuiu em 40% na I/R, retornando aos
valores controle com o tratamento. Já a excreção de Na+ diminui em 79%, e não foi
prevenida pelo LPA. Durante a I/R, a atividade e a expressão da NKA aumentam cerca de
50 % enquanto a atividade da NaA diminui em 46%; este efeito pode estar associado a
diminuição de 40% da atividade da proteína cinase C (PKC). O tratamento com LPA
previne o efeito da I/R sobre a NKA e a PKC, mas não sobre a NaA. O mecanismo de
ação do LPA envolve: a recuperação exclusiva da perda de sensibilidade da NKA pela via
PLC/PKC ocasionada pela I/R, a diminuição da expressão do receptor de LPA2 (50%) e a
manutenção da expressão de GRP78, reduzida em 40% na I/R. O LPA protege o rim do
dano glomerular, mas não do tubular, este está associada à insensibilidade da NaA ao
tratamento. A manutenção da via PLC/PKC é o mecanismo central sobre as atividades
ATPásicas e sobre na manutenção da homeostasia do retículo endoplasmático,
evidenciado pela prevenção do aumento de GRP78.
Página 50
FAPERJ, CNPq, INBEB
D53
IMPAIRMENT OF MEGAKARYOPOIESIS BY YELLOW FEVER
VIRUS AND DENGUE VIRUS
1- Campos, S.P.C.; 1- Ferreira, D.L.; 1- Castro, M. G.; 1- Sanches; D.; 2- Rodrigues, M. F.;
3- Paredes, B. D.; 1- Silva, J.L.; 1- Gomes, A.M.O. & 1- Oliveira, A.C.
1 Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber IBqM/UFRJ; 2 Laboratório de Biologia Molecular do Câncer - IBqM/UFRJ; 3 Laboratório
de Cardiologia Celular e Molecular – IBCCF/UFRJ
"Introduction: Dengue Virus (DENV) and Yellow Fever Virus (YFV) have great importance
in Africa, South America and Asia and cause acute hemorrhagic fevers that are related
to hemostasis dysfunction, with thrombocytopenia. The thrombocytopenia is related to
the diseases severity evolution. Platelets play a crucial role in hemostasis and are
cytoplasmic fragments of megakaryocytes. The megakaryocytes are derived from their
hematopoietic progenitors, megakaryoblast. Each of them gives rise to various
megakaryocytes and single megakaryocyte produces from 5.000 to 10.000 platelets. The
processes that lead to dengue and yellow fever diseases severity evolution and how
these viruses alters platelets production have not yet been elucidated.
Aim: To better clarify the processes in which viral infection leads to thrombocytopenia,
we aim to study the interaction between DENV and YFV with megakaryocyte
progenitors, analyzing possible events of megakaryopoiesis impairment.
Material and Methods: We infected MEG-01 cells (Human Megakaryoblastic cell line)
with DENV-2 and YFV 17 DD in a multiplicity of infection of 1.
Results and Discussion: We detected intracellular YFV proteins since 24h post infection
(p.i.) by confocal microscopy, confirming infection. We analyzed the production of
infectious particles by plaque assay and observed increasing production until 96h p.i.,
followed by decrease. We analyzed cell viability by extracellular activity of LDH and
trypan blue exclusion. We observed higher LDH activity from 96h p.i. with YFV but not
with DENV-2. We observed an increase in cell death from 120h p.i. for both viruses.
During YFV infection, we also observed reduction on infected 4N cell population from
144h p.i. compared to control.
Conclusion: Our data suggest that YFV infects and replicates in MEG-01 cells. Our data
also suggest that DENV-2 and YFV can induce cell death from 120h p.i. Furthermore, YFV
infections also changes differentiation profile by reducing the 4N cell population 144h
p.i."
CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX
D54
EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF
DIETHYLCARBAMAZINE (DEC) ON CARBON TETRACHLORIDE–INDUCED HEPATIC
DAMAGE IN MICE
1-ROCHA,SWS*; 1-FRANÇA, MER; 1-RODRIGUES, GB; 1-BARBOSA, KPS; 1-NUNES, AKS; 3PASTOR, AF; 2-OLIVEIRA, AGV; 1-OLIVEIRA, WH; 1-LUNA, RL; 1-PEIXOTO, AC.
1-Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães - FIOCRUZ. 2Laboratório Microscopia e Microanálise, Centro de Tecnologias Estratégicas do
Nordeste-MCT. 3-Laboratório de Virologia e Terapia Experimental, CPqAM- FIOCRUZ.
Pharmacological studies showed that DEC interferes in the arachidonic acid metabolism,
acting as an anti-inflammatory drug. Rocha et al (2012) demonstrated that DEC can be a
potential drug for the treatment of chronic inflammation induced by chronic alcoholism
and as a hepatoprotective drug in reducing lesions in mice malnourished. In the present
study we investigated the effect of DEC on chronic liver inflammation induced by carbon
tetrachloride (CCl4). Forty C57BL/6J male mice were separated in groups (n=10): control
group, DEC 50mg kg group, CCl4 group and CCl4 + DEC group. DEC (50mg/kg) was
administered in bottle for 12 days. CCl4 (0.5 ml/g) was administered for 6 weeks (2
injections/week). Liver fragments were processed for histological (HE and Sirius red),
immunohistochemical, immunofluorescence and molecular analysis. Histological
analyses of the CCl4 group showed large areas of extensive necrosis and fibrosis, lipid
accumulation and inflammatory cell infiltration. The CCl4+DEC group exhibited reduced
inflammatory process and prevented liver necrosis and fibrosis. CCl4 group increased
collagen deposition, assessed by Sirius red-positive areas, which were markedly reduced
in the CCl4+DEC group. Immunohistochemical and immunofluorescence analyses of the
CCl4 group showed substantial COX-2, IL1β, MDA, TGFβ and αSMA immunopositivity.
On the other hand, substantially reduced enzyme and cytokines expression were found
in the CCl4+DEC group. The CCl4 group exhibited increased IL1β, COX-2, NFκB, IFNγ and
TGFβ expressions in the western blot analysis, all of which were reduced in the
CCl4+DEC group. The CCl4 group exhibited low expression of IL-10 (anti-inflammatory
cytokine), whereas the CCl4+DEC group enhanced significantly the IL-10 expression. The
persistence of hepatic injury in the CCl4 group was confirmed by the COX-2 and iNOS
mRNA expression levels. In contrast, the CCl4+DEC group reduced significantly the
mRNA enzymes expression. According to the present results, DEC is a potential
alternative treatment for chronic liver inflammation induced by CCl4.
INBEB, PAPES, FACEPE, CNPq.
D55
PREDICTION OF PROTEIN FOLDED IN NATIVE STRUCTURES BY
GENERALIZED SIMULATED ANNEALING COUPLED TO GROMOS FORCE FIELD
1,2- FERNANDES, T.V.A.; 1,2- PASCUTTI, P.G.
1- Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro; 2National Institute of Metrology Quality and Technology, INMETRO
"The genome mapping and sequencing advances are producing an exponential number
of amino acid sequences of new proteins, and the comprehension of these protein
structures becomes a crucial extension to these progresses. Currently, the threedimensional structure of a protein is obtained by experimental techniques such as X-ray
Crystallography or NMR. However, due to the limitations and high costs of these
techniques, determination of three-dimensional structure of a protein is a problem that
still challenges the scientists.
In this sense, theoretical and computational studies have increased the understanding
of the factors that drive a polypeptide sequence to its native state. This improvement is
due mainly to advances in computing power in recent years. The purpose of the protein
structure predictions is to provide the conformation of the native state for a given
amino acids sequence. In general, it is assumed that the protein sequence is folded in a
native state, or a collection of native states, which are found at (or near) the global
minimum (free) energy.
The goal of this work is the development of methodologies and protocols, based on the
Tsallis thermostatistics, for ab initio protein structure prediction using atomistic
molecular models. Thus, we use a set of 66 protein models, with length from 9 up to 60
amino acids residues in order to calculate their native structures.
In this work we use an optimization method, the Generalized Simulated Annealing
(GSA), coupled to the GROMOS Force Field to investigate the protein folding problem.
We were able to find structures very near of the native states. In some structures such
as the 1LCX, 2BP4, 1PEF, 1L2Y (PDB ID) we found deviations smaller than 3.0Å, what is
considered a high resolution prediction. Furthermore, 60% of the tested models showed
deviations of less than 4.0Å, which are considered good results."
Página 51
D56
EVALUATION OF HOST CELL MODIFICATIONS UNDER CYST
FORMATION
Tatiana C. Paredes-Santos1, 2, Marcia Attias1, 2and Rossiane C. Vommaro1,2*
Universidade Federal do Rio de Janeiro (UFRJ), 2 Instituto Nacional de Ciência e
Tecnologia -Biologia Estrutural e Bioimagem(INBEB)
The conversion of Toxoplasma gondii from the tachyzoite to the bradyzoite form leads
to the persistence of infection in intermediate hosts, including humans. In the process,
the reminiscent parasitophorous vacuole (PV) converts to an intracellular cyst
presenting a cyst wall; this stage can persist for long periods in the host. The
modifications of host cell organelles are poorly understood until now. Previous works
have already shown that T. gondii can recruit host cell organelles and cytoskeleton
filaments to the vicinity of the PV along the infection. Our aim was to investigate the
reorientation of host cell organelles during spontaneous in vitrocystogenesis. The
epithelial cell line LLC-MK2 infected with the cystogenic strain EGS was used as
experimental model. The distribution of host cytoskeleton was evaluated by electron
and fluorescence microscopy and compared to uninfected cells. Intermediate filaments
of cytokeratin were observed concentrated around the cysts by electron microscopy,
however, using the anti pan cytokeratin staining, it was not possible to observe this
phenomenon in the fluorescence mycroscopy. Fluorescence analysis showed that the
distribution of actin filaments was unaltered upon cyst formation. Furthermore,
microtubules were seen surrounding the cysts, forming a cage around it both by TEM
and IFA. The presence of microtubules around the cysts may be indicative that
endosomal compartments are able to reach the cyst wall vicinity and in an IFA assay it
was possible to observe lysosomes recruited to the border of the cysts. Besides, several
profiles of endoplasmic reticulum were seen closely related to the cyst wall membrane,
although mitochondria were seen not associated as shown previously for PV. These
preliminary results indicate that formation of T. gondii cysts in EGS strain modifies the
inner organization of host cells likely to obtain nutrients or to control host cell responses
not yet determined till now.
Faperj, CNPq and CAPES.
D57
CARACTERIZAÇÃO
DO
TRANSPORTE
DE
FOSFATO
INORGÂNICO EM TRYPANOSOMA BRUCEI
1,3- RUSSO-ABRAHÃO, T., 2,3- SILVA-RITO, S., 2,3- MARINS-LUCENA, T., 2- ALVESBEZERRA, 4- M., KOELLER, C.M., 2- DE PAULA, I. F., 4- HEISE, N., 2- GONDIM, K.C. E 1,2,3MEYER-FERNANDES, J.R.
1- Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, 2Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de
Janeiro, 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e
Bioimagens, 4-Instituto de Biofísica Carlos Chagas Filho, Universidade Federa do Rio de
Janeiro
INTRODUCTION. Trypanosoma brucei is an extracellular protozoan parasite that causes
human African trypanosomiasis or "sleeping sickness ". During his supply of blood from
the mammalian host an infected tsé-tsé fly (genus Glossina ) injects metacyclic
trypomastigotes into the skin . The parasites enter the lymphatic system and pass into
the bloodstream, where they differentiate into blood trypomastigotes, which are
transported to other locations throughout the body. The tsé-tsé fly becomes infected
with bloodstream trypomastigotes when it feeds on the blood of an infected
mammalian host. In the fly intestine, parasites differentiate into procyclic
trypomastigotes, leaving the intestine and differentiate into epimastigotes, affecting the
salivary glands of the fly. During the different phases of their life cycle, T. brucei
depends on exogenous inorganic phosphate (Pi), but little is known about the transport
of Pi across the plasma membrane in this organism. In addition, Pi transporters have
been described in Saccharomyces cerevisiae, Plasmodium, Trypanosoma rangeli,
Leishmania infantum , Trypanosoma cruzi and other microorganisms . OBJECTIVES.
Investigate the kinetics of 32Pi transport, pH influence, H+ and K+ ionophores and
inhibitors influence, H+:Pi cotransporter gene expression and RNAi of PHO84 gene.
METHODOLOGY. RESULTS. The Pi transport is modulated by pH variation, with higher
activity at acidic pH. FCCP (H+-ionophore), nigericin (K+-ionophore), valinomycin (K+ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited the Pi transport, which
was not inhibited by bafilomicina A1 (vacuolar ATPase inhibitor) . In addition, the Pi
transport showed Michaelis- Menten kinetics. A sequence encoding a carrier of
phosphate was identified in the genome of T. brucei, and expression of the gene
TbPho84 was obtained. The RNAi methodology resulted in a decrease in cell growth and
gene expression. CONCLUSIONS. These results confirm the presence of a Pi carrier in T.
brucei, similar to Pho84 described in S. cerevisiae, which contributes to the acquisition
of inorganic phosphate and may be involved in the growth and survival of procyclic
forms of T. brucei. This work presents the first description of a Pi transporter - PHO84 in
T. brucei, parasite responsible for many infections worldwide, especially in sub -Saharan
Africa.
D58
DNA BINDING OF FEIIIZNII COMPLEXES PROBED BY DNASE I
FOOTPRINTING
1-BORTOLOTTO, T.; 2-CAMARGO, T.P.; 2-NEVES, A.; 1-TERENZI, H.
1-Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade
Federal de Santa Catarina, Santa Catarina, Brazil; 2-Departmento de Química,
Universidade Federal de Santa Catarina, Santa Catarina, Brazil.
In recent years, we have demonstrated the DNA cleavage ability of a FeIIIZnII complex
([FeIII-(μ-OH)ZnIIL-H]) using the unsymmetrical donor ligand 2-[N-bis-(2 pyridylmethyl)aminomethyl]-4-methyl-6-[ ’-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol
(H2L-H) (1). To improve this ability, the complex ligand was specifically altered by the
addition of one or two pyrene motifs that tightly bind to the DNA structure, facilitating
the phosphodiester bond hydrolysis. Herein, we report an analysis of the complex-DNA
binding features by 1 and by its parent complexes [FeIII-(μ-OH)ZnIIL-Pyrene] (2)
containing one pyrene motif and [FeIII-(μ-OH)ZnIIL-Pyrene2] (3), using DNAse I
footprinting. A 49-mer 5’-FAM-labeled (FAM is 6-fluorescein) self-complementary
oligonucleotide was used as DNA probe of DNAse I footprinting assays. Footprinting
assays revealed that 1 binds preferentially at an AT-rich region covering four nucleotides
from T9 to A12 (A, TTAA). Two another weak binding sites are located at C16-C17 (B)
and C23-T24 (C). The complex 2, showed further binding sites including an expanded
binding site A including T13 (TTAAT) and a fourth binding site (D, T41-A42 and A44) that
opposes to the binding site A forming a nearly complementary A/D binding site. Finally,
the complex 3 showed similar results to 2, with addition of an expanded binding site B
including a G15 and G18-C20 (E, GCCGGC). The presence of pyrene motifs in 2 and 3
showed enhance the binding affinity of these compounds to DNA, evidenced by the
increase of apparent binding strength among binding sites A/D and B, but also expanded
the range and the number of the binding sites. The addition of pyrene increases the
structural size of the complexes, affecting the number of nucleotides that interact with
2 and 3 inside each binding site. Since pyrene is a general intercalator, it can acts as a
“chemical anchor” allowing that and 3 interact to additional icinal nucleotides.
CNPq, CAPES, MCT, FINEP, FAPESC, INBEB
Página 52
Pós-doutorandos
PD1
REVEALING THE CAMPTOTHECIN MECHANISM OF ACTION IN
THE TRYPANOSOMA CRUZI EPIMASTIGOTE FORM
1-Zuma, A.A; 2- Mendes, I.C.; 1-de Souza, L.C.R; 3-Elias, M.C.; 1,4,5 -de Souza, W.; 2Machado, C.R.; 1-Motta, M.C.M
1 - Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos
Chagas Filho - UFRJ, RJ, Brasil.; 2 - Laboratório de Genética Bioquímica, Departamento
de Bioquímica e Imunologia - ICB, UFMG, MG, Brasil; 3 - Laboratório Especial de Ciclo
Celular - Instituto Butantan, SP, Brasil; 4 - Instituto Nacional de Metrologia, Qualidade e
Tecnologia (Inmetro); 5 - Instituto Nacional de Ciência e Tecnologia de Biologia
Estrutural e Bioimagem
Chagas’ disease, one of the most neglected tropical diseases in the world, affects
approximately 8 million people in Latin America. This illness is caused by Trypanosoma
cruzi, a parasite that has a single nucleus and a unique mitochondrion with an enlarged
portion (named kinetoplast) that harbors the mitochondrial DNA (kDNA). The DNA
topology is modulated by topoisomerases that act during replication, transcription and
repair reverting positive and negative supercoilings of the double-strand.
Trypanosomatids topoisomerases are distinct from human enzymes, what encourages
their use as targets in chemotherapeutic studies. In the present work, we evaluated the
effects of camptothecin, a topoisomerase I inhibitor, considering T. cruzi epimastigote
proliferation, ultrastructure, cell cycle, DNA lesions, mitochondrial activity and
apoptosis. Our data showed that camptothecin caused a strong proliferation inhibition
and only the lowest drug concentration used (1µM) led to a reversible effect on cell
growth, ultrastructure and phosphatidylserine exposure. At ultrastructural level, treated
parasites presented unpacking of the nuclear heterochromatin and the swelling of the
mitochondrion. Camptothecin also promoted cell cycle arrest at G2/M and caused
nuclear DNA lesions. On the other hand, some protozoa entered in early apoptosis, but
they do not progress to late apoptosis, which may indicate that the parasites stay alive
in a ˝senescence-like˝ state. We also obser ed that treated cells resented higher le els
of reactive oxygen species and loss of mitochondrial membrane potential, what may be
associated with apoptosis. Thus, this work indicates that the camptothecin mechanism
of action comprises linked events that block the cell cycle, affect DNA organization and
mitochondrial activity, which may result in apoptosis.
CNPq, FAPERJ, INBEB
PD2
FALCIPAÍNA 2 COMO PROVÁVEL SÍTIO DE AÇÃO DA
CLOROQUINA?
MENEZES, C. M. S.1; FELICIANO, D. N.1,2; DAMETTO, M.1, GOMES, D. E. B.3, PASCUTTI,
P. G.1
1 Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2 Grupo Biología
Molecular, Centro Nacional de Sanidad Agropecuaria, La Habana, Cuba. 3 Laboratório de
Biologia Computacional, Instituto Nacional de Metrologia, Qualidade de Tecnologia,
Duque de Caxias.
"Desde a Segunda Guerra Mundial, a cloroquina (CQ) tem sido considerada como
antimalárico ideal. Contudo, o uso contínuo e indiscriminado levou ao surgimento de
cepas resistentes ou multi-resistentes, em especial, do Plasmodium falciparum e P.
vivax, espécies responsáveis, respectivamente, pelos maiores índices de mortalidade e
morbidade desta infecção. O mecanismo de ação da CQ é associado à interferência no
processo de digestão da hemoglobina humana, durante a fase intra-eritrocítica do ciclo
de vida do parasito. Este processo é classificado como cooperativo por envolver sistema
enzimático múltiplo, como as cisteinil (falcipaínas 2 e 3), aspartil (plasmepsinas) e
metalo proteases, e posteriormente, aminopeptidases, com o objetivo do parasito obter
aminoácidos essenciais para seu crescimento, sobrevivência e reprodução. O grupo
heme é liberado como sub-produto e, por sua natureza oxidativa, é subsequentemente
olimeri ado β-hematina ou hemozoína. A CQ atuaria por inibição desta última etapa,
via interação com o grupo heme ou com a hemozoína, impedindo a polimerização e
causando a morte do parasito por estresse oxidativo, Recentemente, foi sugerido por
Chugh et al. (1) mecanismo adicional em que a CQ também atuaria por inibição da
falcipaína 2. A fim de verificar esta possibilidade, realizamos estudo de ancoramento
molecular da CQ, do grupo heme e do inibidor clássico E-64 à estrutura da falcipaína 2
(código pdb ID 3BPF). Em auxílio à interpretação destes resultados, a superfície
topográfica da protease foi avaliada com o servidor CASTP. A CQ (assim como o grupo
heme, em corroboração a trabalho anterior do grupo (2)), ocupa sítios secundários aos
do E-64, comprovando a hipótese de interação da CQ com a falcipaína, o que
prejudicaria a atividade proteolítica desta última. Estudos de dinâmica molecular serão
realizados para refinar estes resultados e validar esta hipótese.
PD3
-
UNRAVELING THE INFECTION ROUTE OF MAYARO VIRUS
1- CARVALHO, C.A.M.; 1- SILVA, J.L.; 1- GOMES, A.M.O.
1- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de
Janeiro
Mayaro virus (MAYV) is an alphavirus related to several sporadic outbreaks of a highly
debilitating febrile illness in many regions of South America. Although highly neglected,
Mayaro fever in humans is often more incapacitating than dengue and its urbanization
from the Amazon region is on the verge. Alphavirus entry into target cells is supposed to
occur by receptor-mediated endocytosis followed by fusion between the viral envelope
and the endosomal membrane, although non-endocytic penetration of the viral genetic
material into the cytoplasm without membrane fusion has also been suggested. The aim
of this work was to determine the infection route of MAYV, pointing out the cell
structures explored by the virus to get access to the inner of the cell. Purified virus
particles were labeled with the lipophilic fluorescent probe DiD without impairment to
viral infectivity and the fluorescent signals were tracked in susceptible cells transfected
with endocytic markers by laser-scanning confocal fluorescence microscopy in real time.
Our results show that labeled virus particles were endocytosed a few seconds after
binding to receptors on the cell surface. Following DiD fluorescence dequenching at the
single particle level, we could capture the moment of the fusion between the viral
envelope and the endosomal membrane, that was shown to occur around 3 min postbinding, and found that it spatially correlated with both early endosome and caveosome
markers. Altogether, our data indicates that MAYV entry into cells occurs by a fast
endocytic mechanism that involves acidic and sterol-enriched vesicles. This work
unravels important details about the entry of MAYV particles in living cells and may
provide important insights to the development of antiviral strategies.
CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX.
PD4
MAPEAMENTO DA TOPOLOGIA DE INTERAÇÃO DO PEPTÍDEO
DA REGIÃO DO PLASMINOGÊNIO HUMANO QUE INTERAGE COM A PLASMINA DE
YERSINIA PESTIS
1-Sarzedas, C.G., 1-Seraphim, A.C.V, 1-Tinoco, L.W.
Página 53
1-Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
"A peste bubônica é uma doença que coloca em risco a segurança nacional e foi
classificada na categoria A pelo CDC (Center for Disease Control). Desde o seu
aparecimento até os dias de hoje, a peste já matou em torno de 200 milhões de
pessoas. Esta doença é causada pela bactéria Y. pestis que produz na sua membrana
externa a proteína plasmina (Pla), a principal responsável pela invasão da célula
hospedeira pela bactéria. Após a infecção a Pla promove a clivagem (ativação) do
plasminogênio humano (Plg) gerando plasmina e causando hemorragias em vários
órgãos.
No Plg foi identificado o peptídeo PK2: PKKCPGRVVGGCV, que representa a região onde
acontece a interação com a Pla. As análises por CD, RMN e fluorescência mostraram que
o PK2 livre possui uma estrutura estendida, esperada para um peptídeo cíclico devido à
ponte de dissulfeto entre as cisteínas 4 e 12. Na presença da Pla, o PK2 sofre alterações
estruturais com a mudança do deslocamento químico dos hidrogênios amídicos na
região RVV, indicada como sendo a região de clivagem do Plg pela plasmina. Contudo
essa interação não foi capaz de romper a ponte de dissulfeto, responsável por manter a
estabilidade estrutural do PK2. Embora a Pla seja um barrilhidrofóbicos não sejam indicados como participantes diretos da clivagem, a presença do
PK2 aumentou a intensidade de fluorescência da Pla, sugerindo que, tanto a Pla quanto
o PK2, estão sofrendo modificações estruturais na interação Pla-PK2. Esses resultados
serão usados para o desenvolvimento de um protocolo de análise que seja rápido e
eficiente para avaliar a interação Pla-PK2 e ativação da Pla. Desta forma, estes testes
poderão ser usados na busca de compostos capazes de inibir essa interação."
INBEB, Capes, CNPq, Faperj
PD5
AVALIAÇÃO DE DOSES PARA INDUÇÃO DE LESÃO HEPÁTICA
INDUZIDA POR RADIAÇÃO IONIZANTE.
1-ANDRADE,CBV;1-RAMOS,IP;1-FACCIOLI,LP;2-RESENDE,CMC;2- CANARY PC; 1GOLDENBERG,RCS
1-Laboratorio de Cardiologia Celular e Molecular do Instituto de Biofisica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro; 2-Hospital Clementino Fraga Filho
"Introdução: A despeito da grande capacidade regenerativa do fígado, o dano tardio
para o tecido, tal como fibrose hepática radioinduzida, é inevitável. Assim, os esforços
para investigar os mecanismos que levam aos efeitos deletérios tardios da radioterapia
têm aumentado. A regeneração hepática tem sido estudada por muitos anos, no
entanto estudos mais aprofundados sobre os mecanismos que governam os processos
regenerativos ainda são pouco conhecidos e podem expandir as opções de tratamento
para os pacientes com doença hepática. Desta forma, o objetivo desse trabalho é
estabelecer um modelo de lesão hepática radioinduzida para posterior tratamento com
células-tronco adultas.Materiais e Métodos: camundongos C57/BL-6, com peso entre
25-30 gr, foram divididos em 3 grupos: controle e irradiados com 15 Gy e 20 Gy. Os
animais foram eutanasiados 30 e 60 dias após a irradiação (dpi). Antes da eutanásia foi
realizada ultrassonografia hepática dos animais. Foi realizada a coleta de sangue para
dosagem de enzimas hepáticas (albumina, ALT e AST) e obtidos fragmentos dos fígados
para coloração de picrosirius. Resultados/Discussão: A curva de sobrevivência dos
animais demonstrou que somente 6% dos animais da dose de 20 Gy chegaram a 60dpi,
enquanto que o outro grupo experimental teve uma sobrevida de 98% no mesmo
período. No ultrassom observou-se um aumento da ecogeneicidade do fígado em
relação ao rim tanto nos animais do grupo irradiado com 15Gy quanto com 20Gy,
característica de fígados com esteatose, nesses grupos também foi observado a
presença de grades de fibrose, sempre quando comparados ao grupo controle. As
dosagens séricas demonstraram redução significativa nos níveis de albumina e ALT
indicando que a irradiação gerou alteração funcional do fígado. Na análise histológica
por picrosirius foi observado alterações na distribuição do colágeno pela matriz
hepática, encontrou-se colágeno distribuído entre os hepatócitos, porém o esperado
era encontra-lo somente ao redor dos vasos."
PD6
INFLAMMATORY EDEMA ELICITED BY TRYPANOSOMA CRUZI
FUELS HEART TISSUE PARASITISM THROUGH THE PROTEOLYTIC ACTIVATION OF THE
MAST CELL/KALLIKREIN-KININ PATHWAY
1- Andrade D; 1-Nascimento CR, 1-Brazil G ; 2-Eponina Carvalho-Pinto C; 1-Oliveira AC;
1-Schnaider Ramos-Junior E, 1-Serra R; 1-Almeida L; 1-Cordovil da Silva T; 1-Vellasco L;
1-Vairo L; 3- Fortes F; 4-Andrade M; 5- Lannes-Viera J; 6-Juliano L; 1-Goldenberg R; 7Sirois P; 8- Köhl J; 9-Alvarenga P; 9-Monteiro RQ; 1-Campos de Carvalho A; 1-Svensjö E
and 1-Scharfstein J * first authorship should be shared between Daniele Andrade and
Clarissa Nascimento
1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutual e BioImagem (INBEB), Brazil;2Departamento de Patologia, Universidade Federal Fluminense,
UFF, Rio de Janeiro, Brazil;3 Centro Universitário Estadual da Zona Oeste, UENZO, Rio de
Janeiro, Brazil; 4 Faculty of Medicine, UFMG 5Instituto Oswaldo Cruz, Fundação
Oswaldo Cruz, Rio de Janeiro, Brazil; 6 Departament of Biophysics, UNIFESP, 7
Sherbrooke University, Canada. 8 Lubeck University, Germany, 9 Instituto de Bioquímica
Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Chagas disease (CD) is a major cause of myocardiopathy in Latin America. We
hypothesized that vasoactive kinins released in inflammatory exudates might fuel heart
tissue parasitism via signaling of bradykinin GPCRs (BKRs) and endothelin receptors
(ETRs). Here we show evidence that this survival strategy depends on the pathogen
ability to evoke edema through the mast cell (MC)-driven activation of the kallikreinkinin system (KKS). Using intravital microscopy, we showed that histamine potentiates
parasite-induced plasma leakage, the response being conversely blunted by cromalyn- a
MC stabilizer- or by inhibitors of FXIIa and PKa (KKS proteases). Guided by
echocardiography, we then inoculated TCT in the left ventricle of mice and found
evidence of trans-endothelial leakage of dextran-TRITC (2 h p.i.) in wt heart, but not in
B2R-/- mice nor in wt mice pretreated with specific antagonists of BK2R, BK1R, or
ETaR/ETbR. Heart parasitism (30 d p.i.) is decreased in MC-deficient mouse strain or in
infected WT mice pretreated with cromalyn, FXIIa inhibitor or BKR or ETR antagonists,
thus linking MC-KKS-driven edema to parasite infectivity. Strikingly, the mice that
received a single-dose of GPCR blockers prior to intracardiac challenge were
subsequently protected from myocarditis/fibrosis (30 d p.i.). We then infected wt and
BK1R-/- mice via the i.p. route and found that they displayed markedly reduced
intracardiac parasite load (qPCR; 14 d p.i.). Moreover, BK1R-/- mice were protected
from chronic myocarditis and heart fibrosis (90 d p.i.). Extending these studies to
skeletal muscles, we found that interstitial edema and T. cruzi load (3 d p.i.) in
sternomastoid muscles were blunted in B6 mice pretreated with HOE-140 or R954,
respectively BK2R or B1R antagonist. Combined, these results suggest that drugs that
stabilize endothelial barrier through the targeting of the MC-KKS pathway might limit
myocardial edema and heart parasitism, consequently relieving adverse heart
remodelling in CD.
CNPq, FAPERJ, INBEB
PD7
STRUCTURAL AND THERMODYNAMIC STUDIES OF THE
HSP70/HSP90 ORGANIZING PROTEIN – HOP FROM LEISHMANIA BRAZILIENSIS
Página 54
1,2- SANTOS, C.A.; 1- GONZAGA, M.R.; SERAPHIM, T.V.; 2,3- RAMOS, C.H.I.; 1- BORGES,
J.C.
1- Grupo de Biologia Molecular e Bioquímica, Instituto de Química de São Carlos,
Uni ersidade de o Paulo U P , 13560‐970, o Carlos, P, Bra il - Instituto de
Química, Universidade Estadual de Campinas (UNICAMP), 13083-970, Campinas, SP,
Brazil; 3- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem,
Brazil.
"The Hsp70/90-organizing protein (Hop) is a multi-domain protein containing three
tetratricopeptide repeat (TPR) motifs that cooperates with Hsp70 and Hsp90 in the
protein folding in the cellular cytosol. Hop is a co-chaperone that can modulate the
ATPase activity of both Hsp90 and Hsp70 and is directly associated with the quality
protein control. Despite numerous studies focusing on the role of Hop is not yet fully
understood how it interacts with Hsp90. From structural and biochemical studies it is
known that Hop binds to the C-terminal ME(E/Q)VD motif of Hsp90. However, recent
studies have shown that the Hsp90 middle domain may also interact with Hop and both
C-terminal and middle domain Hsp90 interactions are via TPR2A and TPR2B present in
the Hop structure. In this work the full-length Hop (LbHop) and a deletion mutant
(LbHopTPR2AB) containing the TPR2A e TPR2B domains of Leishmania braziliensis were
clonned, overexpressed and purified. Structural properties of the recombinant proteins
were initially evaluated by circular dichroism and analytical size exclusion
chromatography. Finally, thermodynamic parameters of the molecular bindings events
between L. braziliensis Hsp90 (LbHsp90), previously characterized by our group, and the
full-length LbHop and the deletion mutant LbHopTPR2AB, target of this study, were
obtained with isothermal titration calorimetry (ITC) measurements. Our findings
showed that the functional state LbHop is a monomer in solution and the stoichiometry
of interaction between LbHop and LbHsp90 is one monomer of LbHop to a dimer of
LbHsp90. Similar results were also observed for the Hop deletion mutant, confirming
the role of TPR2A and TPR2B motifs for LbHsp90 interaction/modulation. In conclusion,
our study provides novel contribution to the mechanism of interaction between Hsp90
and Hop co-chaperone, and sheds light on how this molecular chaperone system works
in protozoa, especially as regards to L. braziliensis.
Keywords: Hop, TPR domain, Hsp90, Leishmania braziliensis, ITC."
FAPESP
PD8
THERMODYNAMIC AND CONFORMATIONAL ANALYSIS OF THE
BINDING OF FEIIIZNII – PYRENE COMPLEXES TO DNA
1- NORBERTO D.R.; 1- BORTOLOTTO, T.; 1- CAMARGO, T.P.; 2- NEVES, A.; 1- TERENZI, H.
1- Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade
Federal de Santa Catarina-UFSC; 2- 2Laboratório de Bioinorgânica e Cristalografia,
Departamento de Química, Universidade Federal de Santa Catarina-UFSC
Metallonucleases have a natural ability for interacting with DNA. Since binding and
cleavage of DNA are at the central of action of the cellular transcription and translation,
it represents an important target for therapeutic intervention and the development of
diagnostic structural probes. In general, metallonucleases bind DNA in a non-covalent
interaction fashion, including electrostatic or groove binding, intercalation between
base-pairs, and catalysis of DNA cleavage through hydrolytic or oxidative mechanisms.
Despite of the progress of recent years in this field, there are many aspects that remain
unexplored. This work reports an investigation into the thermodynamic behavior of
FeIIIZnII - pyrene complexes interacting with calf thymus DNA (CT-DNA) and a tentative
model that aim to explain the properties used in certain families of antitumor agents
was developed. The complexes FeZnLAld devoid of pyrene ligands, and FeZnLP1 and
FeZnLP2, which are functionalized with one or two pyrene moieties, respectively, were
examined by isothermal titration calorimetry (ITC), and compared to UV-Vis and
fluorescence s ectrosco ies. The arameters Kb, ∆ b and n were determined using a
single set of site fittings and indicate that the binding has a positive enthalpy change
associated to the substitutions of pyrene, with affinity values from 4.8 x104 M-1 to
1.8x106 M-1. The results show some aspects of intercalation of a planar ligand of the
complex in the DNA base pairs stack, with drastic increasing in entropic contribution.
The binding free energy ∆Gb was calculated according to different representations and
they are in agreement with studies of other heterovalent dinuclear complexes. Our data
demonstrate that FeIIIZnII complexes modified with pyrene strongly bind to DNA, and
would be important in the development of novel chemical hydrolases capable of DNA
targeting and cleavage.
CNPq, CAPES.
PD9
LUMINAL ANG II ON CA2+ MOBILIZATION OF PORCINE
PROXIMAL TUBULE KIDNEY CELLS (LLC-PK1): INCREASE OF SERCA ACTIVITY VIA AT1
AND AT2 HETERODIMERS STIMULATING PLC/PKC PATHWAY.
1,2,3- FERRÃO, F.M.; 2,3- LARA, L.S.; 1,2- DIAS J.; 4- DRUMMOND, H.A.; 5- REIS, R.I.; 5COSTA-NETO, C.M.; 6- ZHUO, J.L.; 7- CARMONA A,K.; 1,2- VIEYRA, A.; 1,2- LOWE, J.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de
Janeiro; 3- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 4Department of Physiology and Biophysics and Center for Excellence in CardiovascularRenal Research, University of Mississippi Medical Center, Jackson, Mississippi; 5Departmento de Bioquímica e Imunologia, Escola de Medicina de Ribeirão Preto,
Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil; 6- Laboratory of Receptor
and Signal Transduction, Department of Pharmacology and Toxicology, University of
Mississippi Medical Center, Jackson, Mississippi; 7- Departmento de Biofísica,
Universidade Federal de São Paulo, São Paulo.
Angiotensin II (Ang II) is found in renal interstitium and tubular lumen at higher
concentrations than systemic Ang II. We demonstrated that Ang II facing luminal
membranes of porcine proximal tubule cells (LLC-PK1) stimulate SERCA activity (10-14 M
- 10-10 M), while in high concentrations (10-8 M - 10-6 M) this effect is not observed.
The objective of this study was to investigate molecular mechanisms involved in Ang II –
stimulated SERCA activity, evaluating its physiological relevance. Both Ang II
concentrations (10-10 M and 10-6 M) stimulated AT1/AT2 heterodimers formation in
LLC-PK1 cells, and the use of losartan and PD123319 demonstrated that Ang II is
endocyted. This event is microtubule-dependent, but not mediated by β-arrestin and
clathrin, since colchicin, but not Pitstop 2 blocked endocytosis. Co-localization of Alexa
Fluor 488-conjugated Ang II with ER tracker suggests the translocation of Ang II to the
reticulum after endocytosis. Calphostin C (5 × 10-8 M; PKC inhibitor) and PMA (0,1 µM e
1 µM; PKC activator) blocked and mimicked Ang II stimulation of SERCA activity,
respectively. Calphostin C –sensitive PKC activity is 40 % increased at 10-10 M and 37 %
inhibited at 10-6 M Ang II. Moreover, pre incubation of 10-9 M Ang II for 30 min,
induced a 10 times higher and 3 times longer Ca2+ mobilization than in absence of
previous incubation with Ang II. In conclusion, after endocytosis of luminal Ang II with
AT1/AT2 heterodimers and translocation to the reticulum, SERCA is the target Ca2+ATPase to regulate intracellular Ca2+ homeostasis. The PLC/DAG/PKC pathway mediates
this phenomenon. The Ca2+ mobilization study indicates that increased Ca2+
intracellular store by SERCA activation, results in a more efficient response to agonist
future stimulus contributing to increase fluid and solute reabsorption in renal proximal
tubule.
CNPq, FAPERJ, CAPES
Página 55
PD10
UNDERNUTRITION
DURING
FETAL
LIFE
IMPAIRS
REPRODUCTIVE SUCCESS OF ADULT MALE RATS UNDERPINNED BY ABNORMAL Ca2+
HANDLING IN VAS DEFERENS
1,2,3- Muzi-Filho, H.; 1- Souza, A.M.; 1- Bezerra, C.G.P.; 1,4- Boldrini, L.C.; 2- Takiya,
C.M.; 1- Oliveira, F.L.; 1- Nesi, R.T.; 1- Valença, S.S.; 2,3- Einicker-Lamas, M.; 2,3- Vieyra,
A.; 1,3- Lara, L.S.; 1- Cunha, V.M.N.
1- Institute of Biomedical Sciences, Federal University of Rio de Janeiro; 2- Carlos Chagas
Filho Institute of Biophysics, Federal University of Rio de Janeiro; 3- National Institute of
Science and Technology for Structural Biology and Bioimaging, Federal University of Rio
de Janeiro; 4- Directorate of Metrology Applied Life Sciences, National Institute of
Metrology, Quality and Technology.
"Introduction. Epidemiological and animal model studies show that placental
undernutrition impairs reproduction performance in the adult offspring, but the
underlying molecular mechanisms within the male genital tract are mostly unknown.
Due to its special physiological characteristics in the transport of seminal fluid, we
hypothesized that the vas deferens should be a highly sensitive target. Regarding this
organ, prenatal undernutrition that induces molecular alterations can compromise
reproductive success in adulthood, even with normal nutrition post-partum onwards.
We have investigated whether in utero malnutrition provokes structural and molecular
abnormalities that ultimately lead to diminished fecundity and fertility. Methods and
Results. Male adult rats that were malnourished during their intrauterine life had a
uniquely increased vas deferens weight associated to a thickening of the muscular coat,
decrease of total and haploid germ cells, a marked increase in immature cells, and an
accentuated decline in fertility and fecundity. At the molecular level, the vas deferens
has a marked decrease in Ca2+ transport due to uncoupling of Ca2+-stimulated ATP
hydrolysis and ATP-driven Ca2+ flux, downregulation of SERCA2 and coupling factor
FKBP12. A vast increase in protein carbonylation (a marker of oxidative damage), and an
abnormal balance between protein kinase C and cyclic AMP-dependent protein kinase,
which are the main signaling components of pathways involved in the regulation of
Ca2+ handling, are seen as the legacy of undernutrition in early life. Conclusion. Overall
the results provide structural and molecular basis to explain, at least partly, why adult
individuals experiencing undernutrition in early life have a deficient reproductive
performance as adults."
Financial Support. The Brazilian National Research Council (CNPq), the Carlos Chagas
Filho Rio de Janeiro State Research Foundation (FAPERJ) and the National Institute of
Science and Technology for Structural Biology and Bioimaging (INBEB).
PD
11
P75NTR: STRUCTURAL ANALYSIS OF A SIGNALING
PATHWAY INVOLVED IN NEURODEGENERATION.
1-HEIMFARTH, L; 1-ALMEIDA, M.S.; 1-CABRAL, K.; 1,2,3-WUTHRICH, K.
]
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps
Research Institute, La Jolla, CA, 92037; Institute of Molecular Biology and Biophysics,
Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland
Neuronal injury or stress often results in activation of signaling pathways that are active
normally only during embryonic and neonatal development. One of these signaling
pathways is initiated by the p75 neurotrophin receptor (p75NTR). Studies have
demonstrated that p75NTR signaling contributes to neuronal degeneration during injury
and cellular stress, including the exposure to -amyloid protein. p75NTR is a Type I
transmembrane receptor with an extracellular domain that contains four cysteine-rich
domains (CRDs), the intracellular domain and a type II death domain, a potential G
protein activating domain. The aim of this work was the determination of the 3D
structure of human p75NTR death domain ([u-13C,15N]-hp75DD) by NMR and the
determination of termal denaturation curve by Dichroism Circular. The results show that
the NMR structure of [u-13C,15N]-hp75DD is a globular protein with six alpha hélices.
The N-terminal polypeptide segment of the hp75DD is located inside the structure and
C-terminal polypeptide segment is flexible region. Furthermore, we determined the CD
thermal denaturation curve for hp75DD. The CD data show a decrease in the intensity of
λ max at 208 and 222 nm with increasing temperature. The hp75DD denaturation
melting temperature (Tm) is 67 +/- 2,0 ºC and renaturation Tm is 72 +-1 ºC. The
structural characterization of hp75DD is very important to understand the involvement
of this receptor signaling pathway in cell degeneration. To solve the structure of a
protein can facilitate the prediction of drug and contribute to cure of a disease.
CNPq, FAPERJ, INBEB
PD12
TOXOPLASMA GONDII EGRESS: IS HOST CELL CYTOSKELETON
A DEAD-LOCK?
1,2 - CALDAS, L.A.; 3 - SEABRA, S.H.; 1,2 - ATTIAS, M.; 1,2,4 - DE SOUZA, W.
1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil; 2- Instituto Nacional de
Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Inbeb, Brazil; 3- Universidade
Estadual da Zona Oeste, Rio de Janeiro, Brazil; 4- Instituto Nacional de Metrologia,
Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil
The obligate intracellular protozoan parasite Toxoplasma gondii, capable of infecting
almost every nucleated warm blooded cells, has raised medical and veterinary
importance. Nevertheless, its egress from host cell is still poorly understood. Herein, we
investigated this step of the protozoan cell cycle by colloidal gold and fluorescent
markers labeling of actin and tubulin in infected cells. 24 hours post infection with 5
parasites per cell, samples were treated for 5 minutes with 5µM calcium ionophore in
order to induce T. gondii egress. Fixation was then performed with 4% formaldehyde in
PBS, pH 7.2, for 20 min followed by permeabilization with 0.1% Triton X-100 in PBS for
10 min at room temperature. For electron microscopy, samples were probed with rabbit
anti-actin antibody at a 1:100 dilution, followed by a goat-anti-rabbit antibody
conjugated to 10 nm colloidal gold particles, both incubated for 1h. The monolayers
were washed and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2,
post-fixed for 1 h with 1% OsO4 in 0.1 M cacodylate buffer pH 7.2 and 0.8% potassium
ferrocyanide, dehydrated in ethanol, critical point dried in CO2, sputtered with carbon
and observed in a Jeol 6340 field emission scanning electron microscope. For confocal
microscopy, the monolayers were pre-incubated with 50 mM ammonium chloride and
3% BSA in PBS pH 8.0 for 45 min. The samples were then incubated with primary
antibodies at a 1:100 dilution for 1 h, rinsed, and incubated with 1:400 secondary
antibodies at room temperature for 1 h. After rinsing in PBS and mounting with a
prolong antifade, samples were examined in a Zeiss 510 LSM 510 NLO. We observed a
dual possible role for actin: as a substrate for intracellular gliding of the parasite,
contributing to its scape, and also as a barrier for T. gondii during egress since escape
from host cells occurred preferentially at actin free zones. As long as dynamin and actin
were shown to interact directly with each other, infected cells were treated with
dynasore, a potent dynamin inhibitor, and were labeled for actin and tubulin in order to
observe the cytoskeleton arrangement. Dynamin seemed not to be necessary for T.
gondii egress.
CNPq, FAPERJ e CAPES
Página 56
PD13
KING
PEN SS
Rĺ E
E
EP RIN
N
HEPARINASE PARA CATÁLISE DIRIGIDA
1,2- DAMETTO, M.; 1,2- PASCUTTI, P.G.
1- Instituto de Biofisica Carlos Chagas Filho, UFRJ; 2- INMETRO, Xerem.
A heparina é usualmente utilizada como fármaco anticoagulante na prática médica,
como em cirurgias cardíacas e hemodiálise. A capacidade anticoagulante da heparina
está relacionada à sua ligação com antitrombina. Após ligação com a heparina, a
antitrombina é ativada e interage com a trombina, inibindo-a e promovendo o processo
de anticoagulação. A ligação antitrombina-heparina ocorre especificamente e com alta
afinidade devido a um pentassacarídeo presente na molécula de heparina. Entretanto,
menos de 1/3 das sequências de heparina obtidas nas preparações comerciais são
ativas como anticoagulante. Além disso, a distribuição mundial de formulações de
heparinas contaminadas em 2007 causou preocupação quanto à confiabilidade e
segurança da heparina proveniente de animais. Desta forma, aumentou-se a busca por
um método de baixo custo para a preparação de heparina sintética. Contudo, a síntese
química heparina sintética comercial tem mais de 50 passos e um rendimento de ~
0.1%, portanto, é um fármaco muito caro. Neste trabalho, estamos desenvolvendo uma
heparinase que cliva a molécula de heparina num tamanho suficiente para que esta
tenha atividade anticoagulante. A enzima deve lisar a cadeia de heparina no terminal
redutor de seu pentasacarídeo reconhecido pela antitrombina, possibilitando a
obtenção de formulações farmacológicas mais puras e homogêneas, e evitando o risco
da presença de contaminantes. Nesta etapa inicial, simulações de docking indicaram
que o pentassacarideo de heparina interage de fato com o sítio ativo da heparinase e
sua posição no sitio ativo da enzima favorece a clivagem no anel de açúcar específico.
CAPES, FAPERJ, CNPq
PD14
CHRONIC UNDERNUTRITION AFFECTS Na+-ATPase KINETICS
AND REGULATION, SIGNALING PATHWAYS INVOLVED ANG II, KINASES AND
PHOSPHATASES AND HISTONE DEACTYLASE ACTIVITY IN PROXIMAL RENAL TUBULES
1,2- SILVA, P.A.; 1,2- MUZI-FILHO, H.; 1,2- PEREIRA-ACÁCIO, A.; 1,2- MARTINS, J.F.; 1,2LANDIM-VIEIRA, M.; 1,2- VIEYRA, A.
1- Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro; 2National Institute of Science and Technology for Structural Biology and Bioimaging,
Federal University of Rio de Janeiro.
"Introduction. Malnutrition is an important problem of health in developing countries.
Multideficient diets affect the renal tissue, leading to the appearance of renal diseases.
Objectives. We aimed to investigate: (i) alterations in the Na+-ATPase affinity for Na+
and kinetics; (ii) alterations in the signaling components (kinase-mediated pathways and
abundance of protein phosphatase 2A – PP2A) linked to the renin/angiotensin system;
(iii) impact on histone deacetylases (HDAC). Methods. Wistar rats aged 90 days were fed
from weaning with either a control (CTR) or a deficient diet (RBD). Homogenates and
membranes from proximal tubules were used (i) to study the kinetics of the Na+ATPase, (ii) to evaluate the abundance of PKA, PKC (alpha, epsilon, dzeta and lambda
isoforms), PP2A, AT1R and AT2R, and (iii) to quantify the total activity of the HDACs.
Results. RBD increased maximal velocity (118.0 ± 14.2 against 44.4 ± 3.9 nmol Pi.mg1.min-1), affinity for Na+ (Na+0.5 = 4.0 ± 1.2 mM in CTR group and 0.2 ± 0.02 mM in RBD
group) and phosphate liberation from the phosphorylated intermediate of the Na+ATPase in initial cycles (about 3 times). RBD group presents, when compared to the CTR
group, a reduced abundance of PKA (about 50%) and an increased abundance of 3
isoforms of PKC (50% for PKC-alpha, 90% for PKC-dzeta and 300% for PKC-lambda,
without alterations in the PKC-epsilon abundance). RBD rats presented with increased
abundance of AT1R (about 15%, without alterations in the AT2R abundance). RBD
reduced the activity of the HDACs in ~40%. Conclusion. These cellular and molecular
alterations, associated with increased Na+ reabsorption, culminate in the onset of
hypertension in the adulthood, and can be considered critical features in chronic
undernutrition."
The Brazilian National Research Council (CNPq), the Carlos Chagas Filho Rio de Janeiro
State Research Foundation (FAPERJ) and the National Institute of Science and
Technology for Structural Biology and Bioimaging (INBEB).
PD15
BILAYERS
-
NANOSCALE ELASTIC PROPERTIES OF SIMULATED LIPID
1- LAPIDO-LOUREIRO, P.A.; MASCARENHAS, W.F.; PASCUTTI, P.G.
1,3 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2
- Instituto de Matemática e Estatística - Universidade de São Paulo
"Cell membranes are not in a tensionless state, when they perform tasks such as
membrane fission, endocytosis, cell shape deformation or muscle contraction. Despite
the fact that the structural properties of lipid bilayers at equilibrium have been
extensively studied, much less is known about the molecular structure of lipid bilayers
at nonzero lateral tension. One of the approaches able to tackle these issues is the
molecular dynamics (MD) simulation technique. We carried out MD simulations of DPPC
bilayer patches composed of 128 lipid molecules stretched to different areas departing
from the equilibrium area, at 298, 310 and 323 K. Thousands of molecular
configurations derived from extensive simulations (more than 4 μsec simulation time)
were subjected to a computational geometry algorithm (2D Voronoi diagram - VD) that
partitions the plane in a natural way. Using atom-level VD, we measured the area
occupied by each constituent lipid atom. In this way, we were able to calculate more
precise values of the areas of individual molecules and a better picture of their
fluctuation properties. From the fluctuation-dissipation theorem, we calculated the area
elastic constant Ka of the simulated systems. We constructed a simplified Ka versus area
phase diagram that suggests that even at small area strains there are marked alterations
in the elastic behavior of bilayers. At large strains and according to temperature,
simulated bilayers undergo tension-induced phase transitions (either an interdigitated
gel or a liquid-expanded phase), that, in vivo, would preclude membrane protein
functioning or herald rupture, respectively. To our knowledge, this is the first work to
use atom-level tessellations to study fluctuation properties of molecular areas of lipid
bilayers. We were able to measure elastic constants of strained bilayers in a precise
way, stressing the important role of MD simulations in situations hardly amenable to
experimental studies."
INBEB, FAPERJ, CNPq
PD16
EXTRACELLULAR VESICLES RELEASED FROM MESENCHYMAL
STROMAL CELLS MODULATE MIRNA IN RENAL TUBULAR CELLS AND INHIBIT ATP
DEPLETION INJURY.
1,3,4- Lindoso, R.S.; 1- Collino, F.; 2- Bruno, S.; 3,4- Araujo, D.D.; 3,4- Sant'anna, J.; 5Tetta, C.; 2,6- Provero, P.; 7- Quesenberry, P.J.; 3,4- Vieyra, A.; 3,4- Einicker-Lamas,
M.;1- Camussi, G.
1- Department of Medical Sciences and Molecular Biotechnology Center, University of
Torino, Turin, Italy; 2- Department of Molecular Biotechnology and Health Science,
Turin; 3- Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro,
Rio de Janeiro, Brazil; 4- National Institute of Science and Technology for Structural
Biology and Bioimaging, Rio de Janeiro, Brazil; 5- EMEA LA Medical Board,Fresenius
Medical Care, Bad Homburg, Germany; 6- Center for Translational Genomics and
Página 57
Bioinformatics San Raffaele Scientific Institute, Milan, Italy;7- Department of Medicine,
the Warren Alpert Medical School of Brown University, Providence, RI, USA.
"Introduction: The extracellular vesicles (EVs) derived from human mesenchymal
stromal cells (hMSC) are possible mediators of the paracrine mechanism of these cells.
miRNAs are involved in the regulation of several mechanisms, including repair
processes. The aim of this study was to analyze the role of EVs in renal repair,
identifying the miRNAs involved in this process. Material & Methods: HK-2 cells (human
renal epithelial cells) were treated with antimycin A for 1 h, promoting ATP depletion.
EVs were obtained from hMSC cultured overnight in RPMI with 0.5% of BSA, submitted
to a 150,000 rcf for 1 h at 4°C. Incorporation of EVs double-stained with VybrantTM Dil
and YT ® R
elect™ was analy ed by confocal microsco y. Proliferation was
evaluated by Brdu incorporation). Cell death assay was performed by FACS analysis
(MuseTM Annexin V Dead cell kit). Profiling of 365 mature miRNAs was performed by
TaqMan® Arrays and qRT-PCR was used to confirm miRNAs and mRNA expression.
Predicted miRNA targets were obtained from Targetscan 6.1. Results: The hMSC-EVs
promote protection of renal cells after injury, leading to a significant cell death
reduction. The EVs incorporation was increased after damage (increase of 100%) and
the CD29, CD44 receptors were involved in this process. Several miRNAs were
modulated by injury and treatment with EVs. Some miRNAs were directly transferred
from EVs while others were induced or inhibited. Up-regulated miRNAs targets revealed
important genes related to recovery process as apoptosis, cytoskeleton reorganization
and hypoxia. The changes in expression of these genes during injury were reverted with
EVs incubation. Conclusions: The results show that the paracrine actions of hMSC are in
part mediated by EVs and the protective effects promoted by these vesicles were
mediated by the transfer or induction of miRNAs that regulate important targets related
to cell recovery."
CAPES, CNPq, FAPERJ, DECIT-MS, Fresenius Medical Care, Associazione Italiana per la
Ricerca sul Cancro (AIRC) and National Institutes of Health (NIH).
PD17
PRESSURE-INACTIVATED
AVIAN
INFLUENZA
VIRUS:
IMPLICATIONS FOR VACCINE DEVELOPMENT
Barroso, S.P.C.1, Nico D.2, Nascimento,D.3, Santos,A.C.V.1,Couceiro,J.N.S.S.2,
Bozza,F.A.3, Souza, T.M.L.3, Sacramento, C. Q.3,Ferreira,A.M.A.2, Palatinik,C.2,
Silva,J.L.1,Oliveira, A.C.1
1Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de
Janeiro, and Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e
Bioimagem, Brazil. 2Instituto de Microbiologia, Universidade Federal do Rio de Janeiro,
Brazil. 3Fundação Oswaldo Fiocruz, Rio de Janeiro, Brazil.
"Introduction: Influenza virus poses a serious global health threat. Vaccination remains
the primary method for preventing influenza or to avoid developing serious
complications related to the disease. To induce a greater mucosal immune response,
vaccines by intranasal administration are highly desired. An egg grown inactivated H3N8
whole virus vaccine was prepared using hydrostatic pressure as inactivating agent. The
hemagglutinin and neuraminidase activities were assayed by hemagglutination and
sialidase assay, respectively. Objective: Evaluate the immunogenic capacity of pressureinactivated vaccine. Material and methods: To evaluate the immunogenic capacity of
inactivated particles we used six-week-old female Balb/c mice. The animals were
immunized intranasally at weekly intervals, with 3 doses of 100 µg/mL pressurized virus
(1 TCID50, 265HAU/50 µL). On day 21, mice were challenged in intranasal route.After
vaccination and infection, IgG2a, IgG1 and IgA were measured in sera, faeces and nasal
wash using ELISA.To further investigate the ability of the pressure-inactivated virus in
stimulating the immune system we evaluated the production of cytokines(IL-2, IL-4, IL-6,
IL-10, IL-17, IFN-γand TNF-α) in the supernatants of splenocytes and broncheoalveolar
lavage (BAL). We also performed lung histopathology analysis by light microscope.
Results and Discussion: In this study we show that hydrostatic pressure is able to
inactivate the H3N8 avian influenza virus, while still maintaining hemaglutination and
neuramidase functionality. Challenged vaccinated-animals showed no disease signs. In
the same way, these animals showed less Evans blue leakage and cell counts in BAL
when compared to challenged non vaccinated group. We found that the whole
inactivated particles were capable to generate neutralizing antibody response in serum.
After vaccination and challenge we found Th1/Th2 cytokines secretion with prevalence
of IFN-γ. Vaccinated and further-challenged mice showed no lung damage. Conclusion:
Our results showed that the animals present a satisfactory immune response after
vaccination and are protected against challenge."
CAPES, INBEB, FAPERJ, CNPq.
PD18
LONGITUDINAL EVALUATION OF SALIVARY PROFILE FROM
CHILDREN WITH DENTAL CARIES BEFORE AND AFTER TREATMENT
1- FIDALGO, T.K.S.; 1- FREITAS-FERNANDES, L.B.; 2- ALMEIDA, F.C.L.; 2- VALENTE, A.P.; 1SOUZA, I.P.R
1- Faculdade de Odontologia, Universidade Federal do Rio de Janeiro; 2- Instituto de
Bioquímica Médica, Universidade Federal do Rio de Janeiro.
The saliva is a biofluid largely used in metabolomic for assessment of local and systemic
diseases. Our group was able to demonstrate salivary metabolomic signature of children
with dental caries (Fidalgo et al, 2013). Thus, the aim of the current study was to
investigate the changes observed for metabolites related caries caries-lesion before and
after dental treatment using NMR. Saliva samples from children without caries and with
dental caries before and after treatment. 1H-NMR spectra were submitted to Partial
Least Squared Discriminant Analysis (PLS-DA). Streptococcus mutans and Lactobacillus
sp and pH were also evaluated. As expected, caries-free children presented low levels of
microorganisms when comparing to children with dental caries (p < 0.05; MannWhitney test). Also, after dental treatment it was observed a reduction of
microorganisms (p < 0.05; Wilcoxon test) and the increase of saliva pH. PLS-DA showed
a clear separation of saliva from children with caries and caries-free. In addition, after
dental treatment it was observed a reduction in the levels of acetate, propionate, fatty
acid, butyrate, and saccharide region. PLS-DA applied on 1H-NMR saliva spectra
distinguished the metabolites related to dental caries before and after dental
treatment.
PD19
MITOSOME BEHAVIOR DURING THE LIFE CYCLE OF THE
PATHOGENIC PROTOZOAN GIARDIA INTESTINALIS
1,3- MIDLEJ, V.; 1- PENHA, L.; 1- SILVA, R.; 1,2,3- DE SOUZA, W.; 1,2,3- BENCHIMOL, M.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto Nacional de Metrologia e Qualidade Industrial – Inmetro; 3- Instituto Nacional
de Ciência e Tecnologia em Biologia Estrutural e Bioimagens-INBEB.
The mitosome is a double-membrane bounded organelle found in few unicellular
eukaryotes, one of which is the human intestinal parasitic protozoan Giardia intestinalis.
The discovery of mitosomes in Giardia was strongly supported by the identification of
the protein machinery components responsible for iron-sulfur (Fe-S) cluster assembly
(IscS and IscU proteins) in this organelle. G. intestinalis also lacks mitochondria and
peroxisomes and has been considered to be among the earliest-branching eukaryotes.
This flagellated protozoan grows in vitro as trophozoites and under some conditions
differentiates into cysts, characterized by the absence of flagella, a rounded shape, and
Página 58
the presence of a cyst wall. Using antibodies that recognize two proteins present in the
mitosome, heat-shock protein 70 (mit-HSP70) and giardial chaperonin 60 (GiaCpn60),
we used confocal laser scanning and electron tomography microscopy, western blots
and qRT-PCR to analyze the presence and distribution of the mitosomes during both the
cell cycle and the process of the trophozoite-to-cyst transformation. At early stages of
the differentiation process (~12 h), there was a significant decrease in the extent of
labeling in the cells and the numbers of mitosomes, which almost disappeared after 21
h, but were recovered during the cyst stage. This was confirmed by an mRNA expression
analysis, thus indicating a process that modulates the formation of mitosomes during
the G. intestinalis life cycle. Electron microscopy tomography, which allows for threedimensional reconstruction, revealed the presence of both rounded and elongated
mitosomes.
Página 59
Pesquisadores
P1
DANCE AND SCIENCE: STUDY UPON THE SCRIPTING AND THE
CREATIVE CHOREOGRAPHIC COMPOSITION PROCESS BY THE HELENITA SÁ EARP
DANCE FUNDAMENTALS BASED UPON BIOLOGICAL ORGANIZATION PATTERNS AND
FORMS.
1- MEYER, A; 2- VIEYRA, A.
1- Escola de Educação Física e Desportos, Universidade Federal do Rio de Janeiro; 2Instituto de Biofísica Carlos Chagas Filho
"This research demonstrates the scripting and the creative choreographic composition
process by the Helenita Sá Earp Dance Fundamentals involved in the making of a
multimedia dance spectacle which itself is based upon biological organization patterns
and forms. The interactions between science and dance that occurred from the
twentieth century up until the current days are then analyzed in both theoretical
aspects and the utilization of bioscience themes for choreographic spectacles creation
purposes. The work establishes that the Helenita Sá Earp Dance Fundamentals
possesses an ensemble of epistemologically and methodically principles which are
ca able of instituting body language di ersifying agents. ar ’s conce tion osits dance
as wide-scoped knowledge, including a “science of dance” where its e istemology is
made of an o en ty e of knowledge named “ ance Parameters”, namely Motion,
Space, Shape, Dynamics and Time. In this manner, they are shown in systematic
relations as references capable to generate a kind of access to body actions stimulating
interdisciplinarity, benefitting the interaction between dance language and other
knowledge areas. The work attests how the Helenita Sá Earp Dance Fundamentals were
applied in the choreographic aestheticization of sub-molecular, molecular and cellular
conformational and structural changes that are present in the copper homeostasis
model which was utili ed in the creation of the “Transi es” dance show. This artistic
work was resented in the “II Feira F P RJ Ci ncia, Tecnologia e Ino a o”, in the
“Fe B 011”, in the Teatro Munici al de o a Friburgo and in the o ening of the “VIII
Congresso Brasileiro de Farm cia omeo tica”.
FAPERJ
P2
INFLUÊNCIA DO ESTADO DE PROTONAÇÃO NA PLASTICIDADE
ESTRUTURAL DA CELULASE CEL9A-68 DE Thermobifida fusca
1,2- FERNANDES T.A.V.; 3- COSTA M.G. ; 3- BATISTA P.R. ; 2- PASCUTTI P.G. ; 3CAFFARENA E.R. ; 1- GOMES D.EB.
1- Instituto Nacional de Metrologia, Qualidade e Tecnologia; 2- Universidade Federal do
Rio de Janeiro; 3- Fundação Oswaldo Cruz
As celulases catalisam a hidrolise de celulose em açúcares simples, que podem ser
fermentados em etanol ou outros produtos químicos. Portanto, a enorme quantidade
de biomassa disponível é uma fonte em potencial para produção de biocombustíveis.
Entretanto, para a produção de etanol para o uso de energia em larga escala, é
necessário aperfeiçoar a conversão desta biomassa. Neste cenário, as celulases
termofílicas ganham destaque por sua capacidade de clivar cadeias de açúcares em
altas temperaturas. A hidrólise lignocelulósica utilizando celulases em altas
temperaturas oferece várias vantagens, incluindo aumento da carga sólida devido à
viscosidade reduzida, menor risco de contaminação microbiana, maior compatibilidade
com os pré-tratamentos de alta temperatura, aumento da transferência massa e taxas
mais rápidas de hidrólise. Uma das celulases termofílicas mais bem estudadas é a endoexo-celulase Cel9A-68 da bactéria Thermobifida fusca, que possui um domínio catalítico
(CD) e um módulo de ligação a celulose (CBM), conectados por um linker rico em
resíduos de Pro-Ser-Thr. Acredita-se que a ação conjunta dessas estruturas promove
uma hidrolise eficiente das fibras de celulose. A fim de tornar este processo mais
eficiente através de bioengenharia, precisamos de uma compreensão detalhada, em
nível molecular, da flexibilidade intrínseca da celulase. Visando descrever esta
plasticidade, em nível molecular, realizamos simulações computacionais em diversos
estados de protonação (pH de 2.5 à 12), na temperatura ótima da enzima (325K).
Observamos uma alta variação do potencial eletrostático (de 57.99 até -122.19 KT/e)
indicando possíveis instabilidades estruturais dependente do pH. Essas oscilações na
mobilidade estrutural foram observadas nos sistemas simulados com estado de
protonação dos pHs abaixo do ótimo (pH = 5.5), sugerindo propostas para sítios de
mutação, a fim de manter as características da flexibilidade das subunidades em
temperaturas maiores e/ou em pH mais ácidos.
CNPq, INBEB
P3
STRUCTURAL ANNOTATION OF SEQUENCES FROM THE
ENDOPHYTIC BACTERIUM GLUCONACETOBACTER DIAZOTROPHICUS USING ASAPROT
1,2- DA SILVA, M.L. ; 2- BISCH, P. M.
1 - Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de
Metrologia, Qualidade e Tecnologia; 2 - Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro
"The gram-negative endophytic bacterium Gluconacetobacter diazotrophicus is found in
plants such as sugarcane, pineapple, coffee and sweet potato. This bacterium fixes
atmospheric nitrogen, produces plant growth-promoting hormones, bacteriocins and
helps solubilization of zinc compounds. 3778 protein sequences were predicted in the G.
diazotrophicus PAL5 genome [Refseq: NC_010125]. We investigated these 3778
proteins in order to compare the predictions made by conventional annotation and
structural annotation. Conventional annotation is based on information from the
primary sequences of proteins and structural annotation is based on information from
the three-dimensional structure of these proteins.
Structural properties of these proteins were analyzed by ASAProt workflow. ASAProt is a
computational workflow for structural annotation projects.
The 3D models were built and evaluated with Ramachandran plot, RMSD and BATS
classification by MHOLline. Furthermore, structural domains and superfamilies of the
proteins were analyzed by fastSCOP and 3DBlast. Simultaneously, the primary
sequences of proteins were analyzed by conventional approaches like COG, SMART and
Uniprot.
We have successfully constructed 3D models with excellent stereochemistry quality for
1390 proteins. So it was possible to compare the conventional annotation and the
structural annotation for 1225 sequences. Similar functions have been predicted by
both approaches for 58% of these sequences. We note the importance of structural
annotation in structural genomics projects, since the structural annotation might
complement the conventional annotation. Moreover, we can infer function at 53
proteins before predicted as hypothetical proteins by conventional annotation."
CNPq.
Página 60

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