Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 192-203). Also available in print.

Thesis (Ph. D.)--Virginia Commonwealth University, 1997. / Prepared for: Dept. of Microbiology and Immunology. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 192-203). Also available online.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Medical Microbiology and Immunology. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 1997. / Prepared for: Dept. of Microbiology and Immunology. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Made available in DSpace on 2014-10-09T12:53:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:26Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:53:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:26Z (GMT). No. of bitstreams: 0 / As serino proteases participam de diversos processos fisiológicos (tal como o de coagulação) e patológicos. Essas enzimas estão amplamente distribuídas entre as espécies, são também toxinas dos venenos de serpentes, sendo denominadas SVSPs (snake venom serine proteases). Essas SVSPs são multifuncionais e contêm uma tríade catalítica formada pelos aminoácidos HDS. Algumas SVSPs são comercialmente disponíveis, sendo indicadas para o tratamento de infarto do miocárdio, tromboses e embolia pulmonar. No veneno de Crotalus durissus terrificus estão descritas até o momento, apenas duas SVSPs sendo que a mais estudada é a giroxina que representa cerca de 2,5% do veneno total. No presente estudo foi reportado a clonagem de sete serino proteases amplificadas a partir de uma biblioteca de cDNA de glândula de veneno de um único espécime adulto de Crotalus durissus terrificus. Estes clones foram analisados com relação à organização do cDNA, estrutura e prováveis funções. A construção do modelo tridimensional da giroxina permitiu verificar as similaridades com tripsina, trombina e outras SVSPs. A glicosilação e a presença de muitas pontes dissulfetos dificultam a obtenção das SVSP recombinantes na forma solúvel e com atividade, por expressão em E.coli. Assim, neste trabalho foi abordada a expressão em células de mamífero (que realiza as modificações pós-traducionais) com resultados promissores. Para tanto, o peptídeo sinal de Igk, a seqüência madura e a região 3 UTR da giroxina foram clonados no vetor pED, originando um novo vetor (pED-Giro). Este vetor carrega o peptídeo sinal de Igk, o que possibilitou a secreção da giroxina para o meio de cultura. O vetor pED-Giro foi transfectado em células CHO DXB11 dhfr e COS-7. A giroxina foi detectada no extrato total das células COS-7 por western blot e, em seguida, purificada do meio de cultura com coluna de afinidade (Benzamidina Sepharose) e demonstrado sua integridade pelo ensaio de atividade esterásica. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Plaquetas são fragmentos celulares anucleados, derivados de megacariócitos, que estão envolvidos em diversos processos fisiológicos e patológicos, como coagulação, inflamação, trombose, aterosclerose, e metástase e angiogênese tumorais. Para executar estas funções, plaquetas ativadas secretam uma fração solúvel de moléculas presentes em seus conteúdos granulares, que passam a interagir com outras moléculas e células adjacentes ao local da injúria, e com os próprios receptores plaquetários. No entanto, os mecanismos que regem a secreção em plaquetas ainda são pouco conhecidos. Neste sentido, o objetivo deste estudo foi analisar comparativamente a agregação de plaquetas ativadas por dois diferentes agonistas enzimáticos: a trombina, um dos mais importantes agonistas plaquetários, e a PA-BJ, uma serinoproteinase do veneno da Bothrops jararaca, que assim como a trombina, ativa plaquetas através dos receptores PAR-1 e PAR-4. Neste estudo foram utilizadas abordagens de espectrometria de massas e de bioinformática para caracterizar alterações nos proteomas do sedimento de plaquetas não ativadas e ativadas, e também, para em paralelo analisar as frações proteicas e peptídicas presentes no sobrenadante (secretoma). Nas análises do sedimento de plaquetas ativadas tanto por PA-BJ quanto por trombina, foi verificada a menor abundância das proteínas PBP, PF4, proteína S, fibronectina, fator V e alfa-1 antitripsina, entre outras, e que também foram identificadas no sobrenadante (secretadas), e o aumento de abundância das proteínas ADAM-10, tromboxano A2 sintase, integrina αlIb, miosina-9 e fosforilase b, que estão diretamente envolvidas na ativação/agregação. Por outro lado, verificamos que na secreção plaquetária induzida por trombina ocorreu o aumento de abundância de proteínas envolvidas na regulação da formação do coágulo, como a proteína S, PAI1 e antitrombina III, sugerindo que nos eventos disparados pela trombina, exista uma regulação rigorosa de sua ação no local da injúria vascular. Já na secreção induzida por PA-BJ, verificamos o aumento significativo das proteínas amiloide beta A4 e do fibrinogênio, envolvidas na ativação/agregação plaquetária, além da liberação e ativação de MMP1, indicando que esta metaloproteinase atue sinergicamente com a PA-BJ para a formação e estabilização do agregado plaquetário. Nas análises do secretoma de plaquetas não ativadas, identificamos pela primeira vez, a presença das proteínas catalase, anidrase carbônica, inibidor de elastase leucocitária e a glicoproteína rica em histidina, que estão envolvidas na inibição e regulação da ativação plaquetária. A análise da fração peptídica do sobrenadante plaquetário permitiu avaliar pela primeira vez o degradoma gerado no processo de agregação por PA-BJ e trombina. O conjunto de peptídeos resultante da ativação plaquetária pela PA-BJ é maior e mais complexo do que aquele gerado pela ação da trombina, sugerindo que as vias ativadas por ambas sejam diferenciais e sujeitas a diferentes controles de regulação da proteólise. Além disso, a degradação seletiva de algumas proteínas, e o conjunto de peptídeos gerados, poderiam ter um papel no controle da ativação e agregação plaquetárias. Em conjunto, nossos resultados demostram que, embora a PA-BJ e a trombina induzam a agregação plaquetária mediada pelos receptores PAR-1 e PAR-4, estas enzimas induzem vias diferentes, alterando a secreção plaquetária para levar à agregação. / Platelets are anucleated cell fragments derived from megakaryocytes which are involved in many physiological and pathological processes, such as coagulation, inflammation, thrombosis, atherosclerosis, and tumor angiogenesis and metastasis. To perform these functions, activated platelets secrete a soluble fraction of molecules present in their granules, which then interact with other molecules and cells adjacent to the site of injury, and with platelet receptors. However, the mechanisms governing secretion in platelets are still poorly understood. Therefore, the objective of this study was to comparatively analyze the aggregation of platelets activated by two different enzyme agonists: thrombin, one of the most important platelet agonists, and PA-BJ, a serine proteinase from Bothrops jararaca venom, which, like thrombin, causes platelet aggregation mediated by the receptors PAR-1 and PAR-4. For this purpose, approaches of mass spectrometry and bioinformatics were used to characterize changes in the proteome of non-activated and activated platelets, and also to analyze proteins and peptides present in the supernatant of aggregated platelets (secretome). In the analysis of the sediment of platelets activated by PA-BJ and thrombin, various proteins, such as PBP, PF4, protein S, fibronectin, factor V, and alpha-1 antitrypsin, were detected in lower abundance while they were also identified as secreted, in the supernatant; likewise, proteins that are directly involved in the activation/aggregation, such as ADAM-10, thromboxane A2 synthase, integrin αIIb, myosin-9 and phosphorylase b were identified in higher abundance in platelets activated by PA-BJ and thrombin. Moreover, we found that in the thrombin-induced platelet secretion there was increased abundance of proteins involved in the regulation of blood clot formation, such as protein S, and antithrombin III PAI1, suggesting that in the events triggered by thrombin, there is strict regulation of its action at the site of vascular injury. In the analysis of the secretion induced by PA-BJ, we found a significant increase in amyloid beta A4 protein and fibrinogen, which are involved in the platelet activation/aggregation, in addition to the release and activation of MMP-1, indicating that this metalloproteinase acts synergistically with PA-BJ in the formation and stabilization of the platelet thrombus. In the analysis of the non-activated platelet secretome, we identified for the first time the presence of catalase, carbonic anhydrase, leukocyte elastase inhibitor and histidine-rich glycoprotein, which are involved in the inhibition and regulation of platelet activation. The analysis of the peptide fraction of the supernatant of activated platelets enabled the characterization, for the first time, of the degradome generated in the process of aggregation by thrombin and PA-BJ. The resulting set of peptides generated upon platelet activation by PA-BJ is larger and more complex than that generated by the action of thrombin, suggesting that the pathways activated by both are differential and are subject to different controls of proteolysis. Furthermore, the selective degradation of some proteins, and the set of generated peptides could play a role in the control of platelet activation and aggregation. Taken together, our findings demonstrate that although both PA-BJ and thrombin induce platelet aggregation via PAR-1 and PAR-4, these enzymes activate different pathways to cause platelet secretion and aggregation.

As serino proteases participam de diversos processos fisiológicos (tal como o de coagulação) e patológicos. Essas enzimas estão amplamente distribuídas entre as espécies, são também toxinas dos venenos de serpentes, sendo denominadas SVSPs (snake venom serine proteases). Essas SVSPs são multifuncionais e contêm uma tríade catalítica formada pelos aminoácidos HDS. Algumas SVSPs são comercialmente disponíveis, sendo indicadas para o tratamento de infarto do miocárdio, tromboses e embolia pulmonar. No veneno de Crotalus durissus terrificus estão descritas até o momento, apenas duas SVSPs sendo que a mais estudada é a giroxina que representa cerca de 2,5% do veneno total. No presente estudo foi reportado a clonagem de sete serino proteases amplificadas a partir de uma biblioteca de cDNA de glândula de veneno de um único espécime adulto de Crotalus durissus terrificus. Estes clones foram analisados com relação à organização do cDNA, estrutura e prováveis funções. A construção do modelo tridimensional da giroxina permitiu verificar as similaridades com tripsina, trombina e outras SVSPs. A glicosilação e a presença de muitas pontes dissulfetos dificultam a obtenção das SVSP recombinantes na forma solúvel e com atividade, por expressão em E.coli. Assim, neste trabalho foi abordada a expressão em células de mamífero (que realiza as modificações pós-traducionais) com resultados promissores. Para tanto, o peptídeo sinal de Igk, a seqüência madura e a região 3 UTR da giroxina foram clonados no vetor pED, originando um novo vetor (pED-Giro). Este vetor carrega o peptídeo sinal de Igk, o que possibilitou a secreção da giroxina para o meio de cultura. O vetor pED-Giro foi transfectado em células CHO DXB11 dhfr e COS-7. A giroxina foi detectada no extrato total das células COS-7 por western blot e, em seguida, purificada do meio de cultura com coluna de afinidade (Benzamidina Sepharose) e demonstrado sua integridade pelo ensaio de atividade esterásica. / The serine proteases affect several physiological processes (such as the coagulation cascade) and pathological ones. These enzymes are widely distributed beyond the species; they are also toxins from snake venoms and are called SVSPs (snake venom serine proteases). These SVSPs are multifunctional and have a catalytic triad formed by HDS amino acids. Some of them are commercially available for use in clinical treatment for heart attack, tromboses and pulmonary embolism. So far, in Crotalus durissus terrificus venom only two SVSPs are described and gyroxin is considered the most studied SVSP which represents about 2,5% of the total venom. In the present study was reported the cloning of seven serine proteases amplified from a cDNA library of a venomous gland of a single adult specimen from Crotalus durissus terrificus venom. These clones have been analyzed in relation to the cDNA organization, structure and probable functions. The three-dimensional model of the gyroxin made possible the analysis of similarities with trypsin, thrombin and other SVSPs. The glycosylation and many disulfide bonds of the SVSPs make difficult the expression in E.coli to obtain the soluble recombinant toxin with activity. The expression in mammalian cells is very promising, because it is possible to make pos translation modification and to obtain the recombinant toxin secreted to the culture medium. The IgK signal peptide, the mature sequence and 3\'UTR region of gyroxin were cloned in the pED expression vector resulting in a new vector (pED-Giro). This vector carries the Igk signal peptide, which allows the secretion of the protein to the culture medium. The pED-Giro vector was transfected in CHO DXB11 dhfr and COS-7 cells. The gyroxin was detected in COS-7 total extract by western blot and after, purified from the medium culture and its integrity was confirmed by esterase activity assay.