A leptospirose é uma zoonose, amplamente difundida pelo mundo, causada por espiroquetas patogênicas do gênero Leptospira, que colonizam os túbulos renais de animais silvestres e domésticos. A transmissão ocorre, principalmente, pelo contato direto com água e solo contaminados com a urina de animais infectados que podem ser clinicamente assintomáticos. As leptospiras patogênicas invadem os tecidos do hospedeiro através da penetração da pele lesada ou mucosas da boca, narina e olhos. Logo após ultrapassar as superfícies de contato, as bactérias chegam rapidamente à corrente sanguínea e espalham-se para todos os órgãos causando lesões, principalmente, no fígado e rins onde produzem hemorragia e necrose tecidual. Após a entrada no hospedeiro, a progressão da infecção envolve a adesão das bactérias às células eucarióticas e às proteínas de matriz extracelular seguida pela invasão aos tecidos. Estudos recentes demonstraram que as leptospiras são capazes de se translocarem através das monocamadas celulares, o que poderia ser um mecanismo de evasão do sistema imune e também facilitaria a entrada e saída da corrente sanguínea para infectar órgãos-alvo. O mecanismo envolvido na invasão do patógeno através das barreiras extracelulares não está bem elucidado. Enzimas capazes de degradar proteínas da matriz extracelular poderiam contribuir com a motilidade e quimiotaxia das bactérias durante a invasão. Bactérias patogênicas sintetizam e secretam diferentes tipos de proteases, que atuam degradando colágeno e glicoproteínas entre outras proteínas do hospedeiro. Recentemente, um estudo, utilizando gelatina e caseína como substratos e lisado bacteriano, mostrou haver uma variedade de proteases em Leptospira spp. Análises do genoma indicam a presença de vários genes que codificam prováveis proteases. A comprovação experimental da existência e a caracterização funcional destas proteínas poderão contribuir no entendimento da patogenia da leptospirose. Neste sentido, este trabalho teve como objetivos a clonagem, expressão e caracterização funcional de uma provável colagenase (ColA) de L.interrogans sorovar Copenhageni. As sequências codificantes do domínio de colagenase 1 (D1), do domínio de colagenase 2 (D2) e de ambos os domínios (Full) da ColA foram amplificadas por PCR a partir de DNA genômico de Leptospira e clonadas no vetor de expressão pAE. Os fragmentos D1, D2 e Full da ColA foram expressos em E. coli BL21 - SI e purificados a partir das frações insolúveis por cromatografia de afinidade a níquel. Os fragmentos recombinantes purificados foram utilizados na obtenção dos antissoros policlonais, e as atividades enzimáticas de cada um foram avaliadas. Os antissoros policlonais produzidos em coelho apresentaram elevados níveis de anticorpos detectados por ELISA. Experimentos de Western - blotting demonstraram a presença de proteína ColA em diferentes sorovares patogênicos de Leptospira spp. As proteínas Full e D2 apresentaram atividade catalítica sobre o colágeno desnaturado e sobre peptídeo sintético e atividade hemorrágica em camundongos. Estes resultados indicam que ColA é provavelmente uma proteína de leptospira envolvidas na invasão de tecidos do hospedeiro. / Leptospirosis is a zoonosis widespread throughout the world, caused by pathogenic spirochetes of the genus Leptospira, which colonize the renal tubules of wild and domestic animals. Transmission occurs mainly through direct contact with water and soil contaminated with urine of infected animals that may be clinically asymptomatic. Pathogenic leptospires invade host tissues by penetrating damaged skin or the mucous membranes of the mouth, nostrils and eyes. Soon after passing the contact surfaces, leptospires come quickly into the bloodstream and spread to all organs causing damage mainly in the liver and kidneys where they produce hemorrhage and tissue necrosis after entering the host, the progression of the infection involves the adhesion of bacteria to eukaryotic cells and extracellular matrix proteins followed by invasion of tissues. Recent studies have shown that leptospires are able to translocate across cell monolayers, which could be a mechanism for evasion of the immune system and also facilitate the entry and exit from the bloodstream to infect target organs. The mechanism involved in pathogen invasion through extracellular barriers is not well elucidated. Enzymes capable of degrading extracellular matrix proteins could contribute to motility and chemotaxis of bacteria during the invasion. Pathogenic bacteria synthesize and secrete different types of proteases that degrade collagen and glycoproteins among other host proteins. Recently, a study using gelatin and casein as substrates and bacterial lysate showed a variety of proteases in Leptospira spp. Analysis of the genome indicate the presence of several genes encoding probable protease. The experimental proof of the existence and functional characterization of these proteins may contribute to the understanding of the pathogenesis of leptospirosis. In this sense, this work aimed the cloning, expression and functional characterization of a probable collagenase (ColA) from L.interrogans serovar Copenhageni. Coding sequences of the collagenase domain 1 (D1), collagenase domain 2 (D2), and both domains (Full) of the ColA gene were amplified by PCR from genomic Leptospira DNA and cloned into the pAE expression vector. The D1, D2 and Full fragments of ColA protein were expressed in E. coli BL21-SI and purified from the insoluble fractions by nickel affinity chromatography. The purified fragments were used to obtain the polyclonal antiserum, and their enzymatic activities were evaluated by zymography. Rabbit polyclonal antiserum against the recombinant protein fragments were produced with a high antibody level detected by ELISA. Western-blotting experiments demonstrated the presence of ColA protein in different pathogenic serovars of Leptospira. The Full and D2 proteins showed catalytic activity on denatured collagen and synthetic peptide and hemorrhagic activity in mice. These results indicated that ColA is probably a leptospiral protein involved in invasion of host tissues.

No description available.

No description available.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include diverse groups of viruses that cause a wide variety of diseases in humans and animals. Most of these viruses encode proteases that cleave the viral polyprotein into intermediate or mature functional proteins during virus replication. As these proteases play a critical role in virus replication, they represent an attractive target for the development of antiviral drugs. In this study, the main goal was to establish assay systems and characterize the enzymatic activity of related proteases from Norwalk virus (NV), poliovirus, and transmissible gastroenteritis virus (TGEV). These proteases share several common characteristics including a typical chymotrypsin-like fold, a Cys residue as a nucleophile in the catalytic triad (or dyad) composed of Cys, His and Glu (or Asp) residues, and a preference for a Glu or Gln residue at the P1 position on the substrate. We cloned and expressed proteases from these viruses and characterized their enzymatic activities using a fluorescence resonance energy transfer (FRET) assay using a specific FRET substrate corresponding to each viral protease. First, assay conditions of the FRET assay was optimized for each virus protease. Second, inhibition profiles of each virus protein were investigated using five commercially available standard protease inhibitors (chymostatin, leupeptin, antipain, TPCK, and TLCK). The inhibition studies showed that TPCK inhibited NV, poliovirus, and TGEV proteases with varying strength, and chymostatin inhibited only NV protease. All other inhibitors had little effects on the virus proteases. The established FRET assays should facilitate screening potential antivirals.

Na indústria coureira há uma grande preocupação com o meio ambiente, já que a maioria das etapas para o processamento do couro são realizadas através de processos químicos. Estes processos devem ser controlados para um baixo impacto ambiental e menor custo de tratamento de resíduos. Este trabalho visa desenvolver um extrato enzimático completamente biodegradável, constituído por lipases de microrganismos, para diminuir o uso de tensoativos nos curtumes. Para tal foi necessário o uso de microrganismos que produzem lipases de maneira eficiente para substratos específicos com a variação no espectro de temperatura e pH utilizados nos processos de processamento do couro. Portanto, foi realizado o isolamento de diversos microrganismos em um curtume e estes foram submetidos à análise da produção de lipases induzida por gordura animal. A partir destes isolados foram selecionados e identificados os melhores produtores de lipases: Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Pichia pastoris e Proteus sp. Dentre estes microrganismos, o mais promissor, foi a levedura P. pastoris. A produção de lipases é afetada por diferentes fatores ambientais, assim foram testados, em escala piloto, a influência do tempo de cultivo, pH, temperatura de cultivo e agitação. Além disso, também foram testados o uso de goma arábica e três diferentes surfactantes no cultivo para o possível aumento da produção da enzima. As melhores condições de crescimento para uma maior produção de lipase por P. pastoris foram otimizadas em 72 h, 28ºC, pH 8,0 e 200 rpm. A adição de Triton X-100 ao final do cultivo também aumentou a atividade de lipase. A partir destes resultados a levedura foi submetida ao cultivo em reator de 10 L e determinados o melhor tempo de cultivo, o consumo de glicose e a densidade óptica a 600 nm. O melhor tempo de cultivo manteve-se em 72 h. Ao final deste cultivo, o extrato bruto foi utilizado para determinar a estabilidade da enzima à formulação empregada nos processos de curtimento, já que as condições destes processos podem afetar a atividade de algumas enzimas. A enzima não se mostrou estável aos químicos utilizados no processamento de couros. Por isto, para testar a eficácia da enzima em relação aos produtos encontrados no mercado algumas etapas foram reformuladas para que a enzima pudesse manter a sua atividade original. Os resultados destes experimentos mostraram-se satisfatórios, provando que a produção e utilização desta enzima no mercado é promissora. / The tanning industry has a great concern with the environment, since the majority of stages for leather processing is carried out using chemicals. These processes must be controlled for a low ambient impact and lesser cost of residues treatment. This work aims to develop a completely biodegrading enzymatic extract consisting of microbial lipases, to diminish the use of surfactants in the tanneries. For this product production is necessary the use of efficient microorganisms producing lipases for specific substrates with the variation in the specter of temperature and pH used in the tannery. Therefore, the screening of diverse microorganisms in a tannery was carried out and these had been submitted to the analysis of the induced lipases production with animal fat. It had been selected and identified the best lipases producers: Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Pichia pastoris and Proteus sp. Among these microorganisms, the most promising was P. pastoris. The lipase production is affected by different ambiental factors, thus it was tested, in scale pilot, the influence of the growth time, pH, growth temperature and agitation. Moreover, also the use of arabic gum and three different surfactants in the culture for the possible increase of the enzyme production was tested. The best growth conditions for the best lipase production of P. pastoris are 72 h, 28ºC, pH 8.0 and 200 rpm. The addition of Triton X-100 in the cultivation end also increases the lipase activity. Through these results the yeast was grown in 10 L culture reactor. During this cultivation the best growth time, the glucose consumption and the optical density at 600 nm were determined. The best growth time remained in 72 h. This culture crude extract was used to determine the enzyme stability to the formulation used in the tanning processes, since the conditions of these processes may affect enzyme activity. The enzyme was not stable in the presence of these chemicals. To compare the enzyme effectiveness with the chemicals and commercial enzymes, some processing stages were improved, so that the enzyme could keep its original activity. These results were satisfactory, suggesting that this enzyme production and its commercial use are promising.

A família das serino proteases inclui diversas proteínas envolvidas em uma variedade de processos fisiológicos, como digestão protéica, regulação da pressão arterial e da coagulação sanguínea, entre outros. Quando disfuncionais, relacionam-se à condições patológicas graves como trombose vascular, embolismo pulmonar, infarto agudo do miocárdio, isquemia cerebral, bem como com o quadro da hemofilia B, ocasionada pela deficiência de fator IX. A investigação dos mecanismos de ação e a compreensão do funcionamento de serino proteases e de seus inibidores, as serpinas, é essencial para desvendar mecanismos de doenças e para a modelagem de fármacos mais específicos e eficientes. A pesquisa de fármacos anti-trombóticos busca desenvolver produtos capazes de interferir no processo hemostático que pode resultar no grave quadro trombo-embólico. A nitroforina-2 (NP-2), produzida na secreção salivar do inseto barbeiro Rhodnius prolixus, é uma proteína dotada de potente ação anti-hemostática, atuando como um inibidor específico do complexo tenase intrínseco (TF/FVIIa/FIX). Em trabalho anterior, nosso grupo identificou que o fragmento correspondente ao segmento 90-110 de NP-2 possui atividade anticoagulante. Um modelo do complexo fator IXa (fIXa)-NP-2 foi gerado a partir do “docking” do peptídeo de NP-2 na provável região de ligação com fIXa. Este complexo foi usado como ponto de partida para a simulação por dinâmica molecular e refinamento da estrutura. Novos peptídeos foram propostos como o objetivo de gerar estruturas com maior afinidade por fIXa. Identificamos o hexapeptídeo LKEADE como apresentando melhor afinidade por fIXa, o qual sugerimos como “template” para o planejamento racional de fármacos com ação anticoagulante e anti-trombótica. Numa outra vertente de nosso trabalho, estudamos a superfamília de proteínas denominada serpinas (serpins, ou “SERine Protease INhibitors”) que engloba um grande conjunto de proteínas dotadas de estruturas similares e provenientes de vários e distintos organismos. A função inicialmente identificada para as serpinas foi de inibição de serino proteases da coagulação sangüínea; entretanto, muitas serpinas perderam esta função.Nestes estudos foram usados modelos ocultos de Markov para criar modelos e descrições possibilitando classificar as serpinas em relação à sua função biológica. Foram criadas assinaturas para cada grupo: seqüências consenso e padrões de aminoácidos distintos para cada modelo/função correspondente. Um modelo específico para serpinas da coagulação sangüínea foi criado. Para este modelo, foi gerada a expressão regular [IVTLM]-[FLVA]- F-S-P-[VLWYF]-[SG]-[IV] que descreve tal função. Além disso, ela codifica a seqüência de uma importante região envolvida na mudança conformacional e no funcionamento da serpina. Tanto esta seqüência quanto a estrutura secundária correspondente podem ser melhor investigadas, por sugerirem um alvo para inibição ou ativação. / The protein family of serine proteases comprises molecules involved in a wide range of physiological processes, such as regulation of blood pressure and coagulation. Dysfunctional serine proteases are related to pathological conditions such as embolism, heart failure, cerebral ischemia and also hemophilia B (the latter being a consequence of factor IX deficiency). The understanding of serine proteases mechanism of action, as well as of their inhibitors is a key step to the discover and develop new drugs. In the study of homeostasis, the search for anti-thrombotic drugs seek to avoid thrombosis and related conditions. The protein nitrophorin-2 (NP-2), found in the salivary glands of the hematophagous insect Rodnius prolixus, is a specific inhibitor of the intrinsic tenase complex (TF/fVIIa/fIX). In previous studies, we identified the NP-2 fragment (amino acid sequence 90-110) showing anticoagulant activity. Models of the complex factor IXa (fIXa) – NP-2 were created. This complex was used as a starting point for the molecular dynamic simulations and structure refinement. New NP-2 peptides were suggested, in order to achieve higher affinity complexes. We identified the hexapeptide LKEADE as showing higher affinity for fIXa, which we suggest to be used on rational drug design studies for anticoagulant drugs. The superfamily of proteins known as serpins (“Serine Protease Inhibitors”) involve a number of similar structures, found in a wide variety of organisms. Its function was initially identified as an inhibitor of blood clotting serine proteases. However, many serpins have lost that function in the course of natural evolution. Hidden Markov models were then used to create profiles classifying those proteins due to their biological function. We created signatures for each group of functional serpins in the form of consensus sequences and distinct amino acid patterns. A blood clotting-specific model was generated, which has yielded the regular expression [IVTLM]-[FLVA]-F-S-P-[VLWYF]-[SG]-[IV]. Such pattern also codes for a region of the structure involved in an extensive conformational change. The change is related to the activation of the serpin, and, therefore, both pattern and sequence should be investigated. Combined, these information suggest a powerful target for blood clotting inhibition.

The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses. Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases. Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.

No description available.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2016. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2016-05-13T13:13:32Z No. of bitstreams: 1 2016_DiegoEliasHonda.pdf: 2169221 bytes, checksum: 3f749f9435181b7433ed52f969f2025d (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-05-20T21:34:43Z (GMT) No. of bitstreams: 1 2016_DiegoEliasHonda.pdf: 2169221 bytes, checksum: 3f749f9435181b7433ed52f969f2025d (MD5) / Made available in DSpace on 2016-05-20T21:34:43Z (GMT). No. of bitstreams: 1 2016_DiegoEliasHonda.pdf: 2169221 bytes, checksum: 3f749f9435181b7433ed52f969f2025d (MD5) / O inibidor de serinoproteases da família Bowman-Birk Black eyed-pea Trypsin/Chymotrypsin Inhibitor (BTCI), encontrado em grãos de feijão de corda Vigna unguiculata, é uma proteína com enorme potencial biotecnológico, sendo destacado seu aspecto farmacológico. Sua estrutura possuí sete ligações dissulfeto que estendem sua ação a condições mais extremas de temperatura e pH. Toda sua aplicabilidade decorre do fato de inibir as enzimas tripsina e quimotripsina. Neste trabalho, buscou-se encontrar metodologia semi-empírica que fosse capaz de extrair informações químicas sobre o processo inibitório que ocorre entre o BTCI e a tripsina, bem como a construção de um sistema que permita um olhar mais detalhado sobre os fenômenos que ocorrem na interface entre o inibidor e a enzima a ser inibida. Neste sentido, as propriedades avaliadas foram os orbitais de fronteira e seus quatro vizinhos imediatos. Não obstante, também foi mensurada a força de cada uma das sete ligações dissulfeto. Observou-se que, para o estudo do BTCI no vácuo, diferentes metodologias semi-empíricas forneceram resultados adversos. Conclusão semelhante foi observada para o caso do BTCI complexado com a tripsina. Todavia, quando se analisou a interface entre essas duas proteínas, os métodos em questão tiveram grande correspondência entre si, indicando que a Cys22 é um dos resíduos mais importantes na interface, provavelmente ajudando a manter a conformação durante o processo de ancoragem. Quanto às ligações dissulfeto, não foi possível afirmar sobre qual delas maior impacta a estrutura do BTCI. Contudo, verificou-se que as ligações entre os resíduos Cys34 e Cys19, e Cys61 e Cys46 foram as de menor contribuição energética. _______________________________________________________________________________________________ ABSTRACT / The Bowman-Birk Trypsin/Chymotrypsin inhibitor from Vigna unguiculata seeds (BTCI) is a protein with high biotechnological potential, especially due to its pharmacological aspects. Its structure has seven disulfide bonds which extends BTCI working range to some severe temperature and pH conditions. All BTCI applicability is due to its trypsin and chymotrypsin inhibition. This way, we tried to find some semi-empirical methodology capable to give chemical information about the inhibition process between BTCI and trypsin, as well as to construct a system that allows a closer look about what happens within those proteins interface. To accomplish this objective, we looked to the frontier orbitals and their four immediate neighbors. Likewise, each of its seven disulfide bonds had their energy determined. We conclude that the study of BTCI in vacuum with different methodologies give different results. Similar conclusion was seen for BTCI complexed with trypsin. However, when we analyzed the interface between those two proteins all methods are in agreement, pointing that Cys22 is responsible to maintain the interface conformation during the enzyme-inhibitor docking. About the disulfide bonds, it wasn’t possible to confirm which one has the greatest impact on BTCI structure. Nevertheless, we saw that bonds between Cys34 and Cys19, and Cys61 and Cys46 residues had the lowest energy contribution.

A família das serino proteases inclui diversas proteínas envolvidas em uma variedade de processos fisiológicos, como digestão protéica, regulação da pressão arterial e da coagulação sanguínea, entre outros. Quando disfuncionais, relacionam-se à condições patológicas graves como trombose vascular, embolismo pulmonar, infarto agudo do miocárdio, isquemia cerebral, bem como com o quadro da hemofilia B, ocasionada pela deficiência de fator IX. A investigação dos mecanismos de ação e a compreensão do funcionamento de serino proteases e de seus inibidores, as serpinas, é essencial para desvendar mecanismos de doenças e para a modelagem de fármacos mais específicos e eficientes. A pesquisa de fármacos anti-trombóticos busca desenvolver produtos capazes de interferir no processo hemostático que pode resultar no grave quadro trombo-embólico. A nitroforina-2 (NP-2), produzida na secreção salivar do inseto barbeiro Rhodnius prolixus, é uma proteína dotada de potente ação anti-hemostática, atuando como um inibidor específico do complexo tenase intrínseco (TF/FVIIa/FIX). Em trabalho anterior, nosso grupo identificou que o fragmento correspondente ao segmento 90-110 de NP-2 possui atividade anticoagulante. Um modelo do complexo fator IXa (fIXa)-NP-2 foi gerado a partir do “docking” do peptídeo de NP-2 na provável região de ligação com fIXa. Este complexo foi usado como ponto de partida para a simulação por dinâmica molecular e refinamento da estrutura. Novos peptídeos foram propostos como o objetivo de gerar estruturas com maior afinidade por fIXa. Identificamos o hexapeptídeo LKEADE como apresentando melhor afinidade por fIXa, o qual sugerimos como “template” para o planejamento racional de fármacos com ação anticoagulante e anti-trombótica. Numa outra vertente de nosso trabalho, estudamos a superfamília de proteínas denominada serpinas (serpins, ou “SERine Protease INhibitors”) que engloba um grande conjunto de proteínas dotadas de estruturas similares e provenientes de vários e distintos organismos. A função inicialmente identificada para as serpinas foi de inibição de serino proteases da coagulação sangüínea; entretanto, muitas serpinas perderam esta função.Nestes estudos foram usados modelos ocultos de Markov para criar modelos e descrições possibilitando classificar as serpinas em relação à sua função biológica. Foram criadas assinaturas para cada grupo: seqüências consenso e padrões de aminoácidos distintos para cada modelo/função correspondente. Um modelo específico para serpinas da coagulação sangüínea foi criado. Para este modelo, foi gerada a expressão regular [IVTLM]-[FLVA]- F-S-P-[VLWYF]-[SG]-[IV] que descreve tal função. Além disso, ela codifica a seqüência de uma importante região envolvida na mudança conformacional e no funcionamento da serpina. Tanto esta seqüência quanto a estrutura secundária correspondente podem ser melhor investigadas, por sugerirem um alvo para inibição ou ativação. / The protein family of serine proteases comprises molecules involved in a wide range of physiological processes, such as regulation of blood pressure and coagulation. Dysfunctional serine proteases are related to pathological conditions such as embolism, heart failure, cerebral ischemia and also hemophilia B (the latter being a consequence of factor IX deficiency). The understanding of serine proteases mechanism of action, as well as of their inhibitors is a key step to the discover and develop new drugs. In the study of homeostasis, the search for anti-thrombotic drugs seek to avoid thrombosis and related conditions. The protein nitrophorin-2 (NP-2), found in the salivary glands of the hematophagous insect Rodnius prolixus, is a specific inhibitor of the intrinsic tenase complex (TF/fVIIa/fIX). In previous studies, we identified the NP-2 fragment (amino acid sequence 90-110) showing anticoagulant activity. Models of the complex factor IXa (fIXa) – NP-2 were created. This complex was used as a starting point for the molecular dynamic simulations and structure refinement. New NP-2 peptides were suggested, in order to achieve higher affinity complexes. We identified the hexapeptide LKEADE as showing higher affinity for fIXa, which we suggest to be used on rational drug design studies for anticoagulant drugs. The superfamily of proteins known as serpins (“Serine Protease Inhibitors”) involve a number of similar structures, found in a wide variety of organisms. Its function was initially identified as an inhibitor of blood clotting serine proteases. However, many serpins have lost that function in the course of natural evolution. Hidden Markov models were then used to create profiles classifying those proteins due to their biological function. We created signatures for each group of functional serpins in the form of consensus sequences and distinct amino acid patterns. A blood clotting-specific model was generated, which has yielded the regular expression [IVTLM]-[FLVA]-F-S-P-[VLWYF]-[SG]-[IV]. Such pattern also codes for a region of the structure involved in an extensive conformational change. The change is related to the activation of the serpin, and, therefore, both pattern and sequence should be investigated. Combined, these information suggest a powerful target for blood clotting inhibition.