Orientador: Raghuvir Krishnaswamy Arni / Banca: Patrick Spencer / Banca: Jose Ramon Beltran Abrego / Resumo: Os venenos de serpentes são constituídos por uma complexa mistura de substâncias que apresentam características e proporções variáveis de acordo com as diferentes espécies, distribuição geográfica dos indivíduos, variação ontogenética e sexual e alimentação. Essa variabilidade na composição dos venenos é de grande interesse na pesquisa e desenvolvimento de novos fármacos, pois um elevado grau de variação aumenta o número de seus potenciais usos. De acordo com essas propriedades dos venenos de serpentes, no presente estudo procurou-se comparar algumas variações existentes nas frações do veneno de Bothrops leucurus, uma serpente da família Viperidae que se caracteriza por produzir um veneno com toxicidade variável devido a diferentes fatores. A toxicidade do veneno deve-se principalmente à sua fração proteica, constituída em sua maior parte por proteinases (metalo proteinases e serino proteinases), fosfolipases e L-aminoácido oxidases que apresentam efeitos potenciais no sistema homeostático, além de possuírem ação citotóxica, antimicrobiana e inflamatória. Neste estudo foram realizados ensaios cromatográficos para a purificação de algumas dessas enzimas e a caracterização das mesmas para a verificação de suas propriedades proteolíticas e fibrinogenolíticas, além do potencial antimicrobiano do veneno bruto. Foram purificadas duas metalo proteinases de 28 e 65 kDa, respectivamente, uma serino protease de 35 kDa, uma LAAO de 66 kDa e duas fosfolipases com 14 e 15kDa. Constatou-se que a metalo protease de 65 kDa apresentou a maior atividade fibrinogenolítica. Além disso, foi realizado o teste da mínima concentração inibitória com o veneno bruto para verificar seu efeito antimicrobiano contra uma bactéria gram-negativa e outra gram-positiva, sendo que nesta última, apresentou atividade inibitória / Abstract: The snake's venoms are constituted by a mixture of substances that exhibit character and variable proportion according to different species, geographic distribution of individuals, ontogenetic and sexual and feed variability. That variability on poisons compositions is of great interest on research and development of new drugs, because a high degree of variation increases the number of potential use. According to those properties of snake's poisons in the actual study it was sought to compare some variations existents on fractions of Bothrops leucurus venom, a snake of family Viperidae that is characterized by production of poison with variable toxicity due different factors. The poison toxicity should principally its protein fraction consisting mostly by proteinases (metalloproteinases and serine protease), phospholipases and L-aminoacid oxidases showing potential effects on homeostatic system, beyond having cytotoxic action, bactericidal and inflammatory. This study describe chromatographic separation of some enzymes and characterization of it and to verification of its proteolytic and fibrinogenolytic properties further the bactericidal potential of crude venom. Was purified two metalloproteinases of 28 and 65 kDa respectively, a serine protease of 35 kDa, a LAAO of 66 kDa and two phospholipases of 14 and 15kDa. It was found metalloproteinase of 65 kDa show a higher fibrinogenolytic activity. Futhermore crude venom was carried out the test of minimum inhibitory concentration with crude venom to check its antimicrobial effect against a gram-negative bacterium and other gram-positive, whereas in the latter showed inhibitory effect / Mestre

Foi desenvolvido um conjunto de peças plásticas que permitem a montagem e representação dos aminoácidos mais comuns, bem como a construção de estruturas protéicas. Durante e após o desenvolvimento o material foi submetido a várias etapas de avaliação por professores (do ensino básico e universitário), alunos de pós-graduação e de graduação. A primeira etapa foi o desenvolvimento dos modelos em ambiente computacional, seguida da prototipagem das peças. Após discussão com a comunidade científica (apresentados na XXXVI Reunião Anual da SBBq em 2007) as sugestões foram implementadas nos modelos computacionais. Quatro moldes para injeção termoplástica foram projetados, detalhados e construídos, sob nossa orientação. As peças representando as estruturas que compõe os aminoácidos e ligações foram produzidas em grande escala e iniciou-se o processo final de avaliação. As peças apresentaram boas relações geométricas com as fórmulas estruturais dos aminoácidos obtidas de bancos de dados e livros didáticos. As conexões C&alpha;-amina e C&alpha;-carboxila permitem verificar a liberdade de rotação característica das cadeias polipeptídicas e as possibilidades dos ângulos de torção &Psi; e &Phi;, visualizando a restrição de rotação da ligação peptídica. Montando um conjunto de aminoácidos é possível construir uma cadeia polipeptídica e, através das ligações de hidrogênio, montar as estruturas secundárias principais (hélice-&alpha; e estruturas &beta;). Duas avaliações preliminares foram realizadas e a avaliação final ocorreu em uma oficina de atividades com 256 professores das áreas de ciências da natureza da rede publica do Estado de SP. Os resultados da avaliação foram extremamente positivos, sendo importante destacar a quantidade e o teor dos comentários elogiosos ao potencial de utilização do material, notadamente, dos professores de biologia e química. O material poderá inclusive auxiliar no preenchimento de lacunas conceituais que existem na formação dos professores e que foram observadas durante as atividades de avaliação. Este conjunto de peças, organizado na forma de um kit: Construindo Estruturas de Aminoácidos e Proteínas, foi submetido à avaliação do MEC e certificado por este órgão, passando a integrar o Guia de Tecnologias Educacionais 2008.<b/> / It was developed a set of plastic pieces that allow the assembly and representation of the most common amino acids, as well as the construction of protein structures. During and after development the material was submitted to several stages of evaluation by teachers (primary and university), graduate and undergraduate students. The first step was the development of models in the computing environment, followed by prototyping of parts. After discussion with the scientific community (presented at the XXXVI Annual Meeting of SBBq in 2007) suggestions were implemented in the computational models. Four thermoplastic injection molds were designed, detailed and constructed under our supervision. Parts representing the structures of amino acids and bonds were produced in large scale and it was started the final process of evaluation. The pieces had good geometric relationships with the structural formulas of amino acids obtained from databases and textbooks. The connections C&alpha;-amine and C&alpha;-arboxyl permit to check the freedom of rotation of the polypeptide chains and the possibility of torsion angles &Phi; and &Psi;, visualizing the restriction of rotation of the peptide bond. Assembling a set of amino acids is possible to build a polypeptide chain and, through hydrogen bonding, to assemble the main secondary structures (&alpha;-helix and &beta;-structures). Two preliminary evaluations were conducted and the final evaluation took place in a workshop with 256 teachers of the fields of natural sciences from public schools of the São Paulo State. The results of the evaluation were extremely positive and it is important to highlight the amount and content of approving comments for the potential of use of the material, especially from biology and chemistry teachers. The material may even assist in filling in conceptual gaps that exist in teacher instruction and that were observed during the evaluation activities. This set of pieces, arranged in the form of a kit: Building Structures of Amino Acids and Proteins, was submitted to MEC and certified by this organization, starting to integrate the Guide of Educational Technology 2008<b/>.

Abstract not available

Graduation date: 2005

In this thesis, we have advanced a set of distinct bioinformatic and computational tools to address the structure and function of proteins. Using data mining of the protein data bank (PDB), we have collected statistics connecting the propensity between the protein sequence and the secondary structure. This new tool has enabled us to evaluate new structures as well as a family of structures. A comparison of the wild type staphylococcal nuclease to various mutants using the proposed tool has indicated long-range conformational deviations spatially distant from the mutation point. The energetics of protein unfolding has been studied in terms of the forces observed in molecular dynamics simulations. An adaptive integration of the steered molecular dynamics is proposed to reduce ground state dominance by the rare low energy trajectories on the estimated free energy profile. The proposed adaptive algorithm is utilized to reproduce the potential of mean force of the stretching of decaalanine in vacuum at lower computational cost. It is then used to construct the potential of mean force of this transition in solvent for the first time as to observe the hydration effect on the helix-coil transformation. Adaptive steered molecular dynamics is also implemented to obtain the free energy change during the unfolding of neuropeptide Y and to confirm that the monomeric form of neuropeptide Y adopts halical-hairpin like pancreatic-polypeptide fold.

There is no abstract available for this thesis. / Department of Chemistry

Recent studies of the relationship between protein sequence and protein structure are reviewed. A detailed discussion of past attempts to predict the structure of a protein from its amino acid sequence, the protein folding problem, is presented and the strengths and weaknesses of these methods are examined. The root-mean-square deviation is studied and a benchmark for structural comparisons is established. A combinatorial approach to the protein folding problem is outlined and its advantages over existing methods is discussed. Specific algorithms based on the combinatorial approach are developed and applied to a variety of proteins. The success of this approach in terms of the root-mean-square deviation benchmark as well as the drawbacks of this method are presented.

Previous research has underscored the importance of protein dynamics during light-induced electron transfer; however, specific interactions have not been well characterized. It is of particular importance to understand the role of protein dynamics and cofactor interactions in controlling electron transfer in oxygenic photosynthesis. These factors include hydrogen bonding, ð-stacking and electrostatic interactions. Reaction-induced FT-IR spectroscopy is sensitive to these interactions as well as isotopic incorporation, and is useful to probe protein dynamics associated with light-induced electron transfer in Photosystem I (PSI). Density functional theory (DFT) provides information concerning the vibrational frequencies of molecules as well as the amplitudes of the vibrations and sensitivity to isotope incorporation. Combining these approaches, protein dynamics associated with light-induced electron transfer in PSI were studied. The work presented here describes specific protein cofactor interactions and specific protein relaxation events associated with light-induced electron transfer. The results reported here are consistent with noncovalent protein cofactor interactions that modulate the redox potential of the secondary electron acceptor of PSI. Furthermore, the studies presented here describe novel protein dynamics associated with the oxidation of the terminal electron donor of PSI. These results characterize specific protein dynamics that may be associated with interactions of the soluble electron donors. These studies highlight the importance of protein dynamics in oxygenic photosynthesis.

Proteins play an important role in cells, as the morphology, function and activities of the cell depend on the proteins they express. The key to understanding how different proteins function lies in an understanding of the molecular structure. The overall aim of this thesis was the determination of the structure of DWNN domains. This thesis described the preparation of samples of human DWNN suitable for structural analysis by nuclear magnetic resonance spectroscopy (NMR), as well as NMR analysis.

Orientador: Cristiane Damas Gil / Coorientador: Sonia Maria Oliani / Banca: Eloísa Amália Vieira Ferro / Banca: Newton Antonio Bordin Jr / Resumo: A endometriose é uma doença inflamatória crônica, de etiologia multifatorial caracterizada pela implantação e crescimento do endométrio fora da cavidade uterina (endométrio ectópico). Nas lesões de endometriose, os mastócitos aparecem em elevado número, no entanto, poucos estudos investigaram seus mediadores inflamatórios na patogênese dessa doença. Entre os mediadores dos mastócitos ressaltamos a anexina A1 (ANXA1), proteína de 37 kDa com múltiplas ações biológicas como inibição da transmigração de leucócitos, proliferação celular e apoptose. Tais efeitos são mediados por receptores para peptídeos formilados (FPRs), especialmente FPR1. Assim, o objetivo desse trabalho foi investigar o papel das células inflamatórias, particularmente a ativação e heterogeneidade dos mastócitos, sua correlação com a expressão e o mecanismo de ação da ANXA1 em biópsias de endométrios eutópicos (controle, n=10) e ectópicos (endometriose de parede abdominal, n=18). As amostras de endométrio ectópico foram coletadas entre julho de 2003 e janeiro de 2012, incluídas em parafina e cedidas pelo Departamento de Patologia da FAMERP. As biópsias de endométrios eutópicos foram coletadas de pacientes sem quadro clínico de endometriose, entre junho de 2011 e junho de 2012, com a colaboração do Departamento de Ginecologia e Obstetrícia da FAMERP. Essas biópsias foram processadas para: (i) análises histopatológicas, (ii) quantificação de células inflamatórias (neutrófilos e mastócitos) e (iii) imuno-histoquímica utilizando anticorpos específicos para estudo da heterogeneidade dos mastócitos (triptase e quimase), da expressão da ANXA1 e do receptor FPR1 nos tecidos. A análise histopatológica das lesões de endometriose mostrou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Endometriosis is a chronic inflammatory disease of multifactorial etiology, characterized by implantation and growth of endometrium outside the uterine cavity (ectopic endometrium). Endometriotic lesions presented a high number of mast cells, however, few studies have investigated their inflammatory mediators in the pathogenesis of this disease. Among these mediators we emphasize annexin A1 (ANXA1), a 37 kDa protein with multiple biological actions like inhibition of leukocyte transmigration, cellular proliferation and apoptosis. Such effects are mediated by formyl peptide receptors (FPRs), especially FPR1. Thus, the purpose of this study was to investigate the role of inflammatory cells, particularly the activation and heterogeneity of mast cells, and its correlation with expression and mechanism of action of ANXA1 in biopsies of eutopic (control, n = 10) and ectopic endometrium (abdominal wall endometriosis; n = 18). The samples of ectopic endometrium were collected between July 2003 and January 2012, embedded in paraffin and provided by the Department of Pathology of FAMERP. Biopsies of eutopic endometrium were collected from patients without clinical features of endometriosis, between June 2011 and June 2012, with the contribution of the Department of Obstetrics and Gynecology of FAMERP. These biopsies were processed for: (i) histopathology, (ii) the quantification of inflammatory cells (neutrophils and mast cells) and (iii) immunohistochemistry using specific antibodies to study the heterogeneity of mast cells (chymase and tryptase), and expression of ANXA1 and FPR1 receptor in tissues. Histopathological analysis of endometriotic lesions showed two morphological patterns: glandular pattern of mixed differentiation (presence of differentiated glands with simple prismatic epithelium and... (Complete abstract click electronic access below) / Mestre