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Caractérisation fonctionnelle d'inhibiteurs de protéases lors de l'interaction Vigne/Botrytis cinerea / Characterization of protease inhibitors in the interaction between Vitis vinifera and Botrytis cinerea.Gerard, Clémentine 29 September 2014 (has links)
Caractérisation d'inhibiteurs de protéases lors de l'interaction entre la vigne et Botrytis cinerea. Il a été montré que lors de l'infection de la baie de raisin par B. cinerea, des protéases fongiques pourraient être à l'origine de la dégradation d'une des protéines PR majoritaires de la baie mûre, la chitinase VvChi4D (Thèse S. Colas, 2012 ; van Sluyter et al., 2013). L'hypothèse émise lors de notre étude est que des inhibiteurs de protéases de la vigne pourraient empêcher la dégradation de cette protéine de défense par les protéases de B. cinerea.L'expression de deux inhibiteurs de protéases, un Potato Inhibitor I (VvPin) et un Kunitz (VvKun), ainsi que celle de trois protéases fongiques, une protéase aspartique (BcAp8), une protéase glutamique (BcAcp) et une protéase à sérine (BcSer), ont été suivies lors de l'infection de la baie de Pinot noir et de la feuille de vitroplants. Les résultats obtenus montrent que l'expression des deux IP est induite en même temps que celle de la protéase à sérine mais après celle des deux protéases acides du champignon. La production en système hétérologue des deux IP ainsi que l'obtention de protéases acides et de protéases à sérine de B. cinerea a permis de montrer que la protéine VvKun est capable d'inhiber les protéases à sérine du champignon. En revanche, aucun des deux IP n'est capable d'inhiber les protéases acides du champignon, protéases responsables de la dégradation de la chitinase VvChi4D. / Characterization of protease inhibitors in the interaction between Vitis vinifera and Botrytis cinerea.It has been shown that upon infection of the grape berry by B. cinerea, fungal proteases may be responsible for the degradation of a PR protein of the mature berry VvChi4D chitinase (Thesis S. Colas, 2012; van Sluyter et al, 2013). The hypothesis of our study is that protease inhibitors could prevent the degradation of this defense protein by proteases of B. cinerea.The expression of two protease inhibitors, a Potato Inhibitor I (VvPin) and a Kunitz (VvKun), and that of three fungal proteases, an aspartic protease (BcAp8), a glutamic acid protease (BcAcp) and a serine protease (BcSer) were followed during infection of Pinot Noir berry and leaf plantlets. The results obtained show that the expression of IP is induced both in the same time as the serine protease, but after that the two fungal acid proteases. The heterologous production of both IPs and the production of acid and serine proteases from B. cinerea secretoms have shown that VvKun is capable to inhibit serine proteases of the fungus. However, neither IP is capable of inhibiting fungal acid proteases, responsible for the degradation of the chitinase VvChi4D.
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Avaliação do modelo de hamster para detecção das alterações lipídicas e cardiotoxicidade associadas à terapia contra o vírus da imunodeficiência humana / Evaluation of the hamster model for the detection of lipidic and cardiotoxicity alterations associated to therapy against human immunodeficiency virusSanchez, Eduardo Milton Ramos 04 February 2010 (has links)
Com a introdução de uma nova classe de antiretrovirais integrantes da terapia anti-retroviral altamente ativa (HAART) para o tratamento das infecções pelo vírus da imunodeficiência humana, começaram a ser descritos inúmeros efeitos secundários.Na tentativa de se estabelecer um modelo animal para o estudo destes efeitos buscou-se uma espécie com similaridade no perfil e metabolismo lipídico. Iniciou-se estudo em Mesocricetus auratus. Foram avaliados o perfil lipídico e glicêmico,função hepática e renal, níveis de auto-anticorpos anti ox-LDL, perfil eletrocardiográfico, alterações histopatológicas renais e cardíacas nos animais sob dieta hiperlipídica e normal,tratados com Indinavir, inibidor de protease utilizado na HAART. Observou-se uma diminuição da sobrevida nos animais tratados com indinavir, aumento do nível sérico de triglicérides e glicose, redução de auto-anticorpos anti ox-LDL,aumento do segmento QRS no eletrocardiograma, presença de fibrose renal e cardíaca, hipercelularidade glomerular nos animais tratados com a droga com ou sem dieta hiperlipídica quando comparados com os controles. Concluimos que Mesocricetus auratus se apresenta como um bom modelo para o desvendamento dos mecanismos patológicos observados na HAART. / With the introduction of a new antiretroviral class use, integrants of highly active anti-retroviral therapy (HAART) for the treatment of infections by human immunodeficiency virus, several side effects started to be described.To establish an animal model for the study of these side effects, was chosen specie that have similarities in the lipidic profile and metabolism. A study in Mesocricetus auratus was started. It was evaluated the lipidic and glicemic profile ,hepatic and renal function, the levels of auto-antibodies against ox-LDL, electrocardiographic profile and renal and cardiac histopathological alterations in these animals under hyperlipidic and normal diets,treated with Indinavir, a protease inhibitor used in HAART.It was observed a decrease in the survival rate in the animals treated with Indinavir; an increase of the triglycerides and glucose serum level; reduction of anti ox-LDL auto-antibodies; increased QRS segment in the electrocardiogram; presence of renal and cardiac fibrosis; glomerular hypercellularity in the animals treated with the drug, with or without hyperlipidic diet when compared with the controls. We conclude that the Mesocricetus auratus is a good model for disclosure of the pathological mechanisms generated by HAART.
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Papel dos proteassomas na interação e desenvolvimento de Leishmania chagasi em macrófagos murinos. / Role of parasite proteasomes in the infectivity and intracellular development of Leishmania chagasi in murine macrophages.Jardim, Izaltina Silva 30 March 2001 (has links)
Nas células eucariotas a maioria das proteínas citoplasmáticas não são degradadas nos lisossomas, mas em organelas altamente conservadas encontradas em humanos, arquibactérias, plantas e leveduras, os proteassomas. Esta estrutura multicatalítica é constituída por componentes menores, cujo núcleo funcional é o componente 20S, que contém várias atividades proteolíticas (tríptica, quimotríptica, de peptidilglutamil peptidase, BrAAP e SNAAP). Esse componente 20S, associado ao complexo regulatório 19S, que é composto de múltiplas ATPases, forma o complexo 26S, responsável pela degradação de proteínas conjugadas com a ubiquitina. Estas estruturas citosólicas certamente desempenham papel importante no desenvolvimento de protozoários parasitas e na sua interação com células dos hospedeiros permissivos. Nesta dissertação, apresentamos um estudo sobre o papel do proteassoma na interação e desenvolvimento de promastigotas de Leishmania chagasi em macrófagos murinos. Inicialmente, purificamos e caracterizamos parcialmente o proteassoma de promastigotas de L. chagasi. Observamos que o complexo presente na L. chagasi possui atividades proteolíticas frente a pelo menos dois substratos sintéticos, LLVY-AMC e LRR-AMC, que avaliam, respectivamente, as atividades quimiotripsina-símile e tripsina-símile. A atividade tripsina-simile é maior que a atividade quimiotripsina-simile; e além disso, esta última é totalmente inibida pela lactacistina, um inibidor específico do proteassoma, enquanto a atividade tripsina-simile é apenas parcialmente inibida. Utilizando a lactacistina foi possível analisar o papel desse complexo proteolítico durante a infecção e desenvolvimento intracelular da L. chagasi. Promastigotas mantidas em cultura na presença de 50μM de lactacistina tiveram seu crescimento bloqueado. Essas promastigotas eram capazes de infectar macrófagos peritoneais de camundongos BALB/c, mas não conseguiam sobreviver dentro desses macrófagos. Esta incapacidade de sobrevivência foi específica para os parasitas tratados com a lactacistina, não sendo observado nos parasitas tratados com outros inibidores de proteases. Estes resultados sugerem que o proteassoma pode ter um papel importante no desenvolvimento intracelular e na replicação das promastigotas de L. chagasi no hospedeiro vertebrado. / Proteasomes are multicatalitic and multisubunit endopeptidase complexes widely distributed in eukaryotic cells. These enzymes are central proteases in the cytosol and nucleus and are involved in removal of abnormal, misfolded or incorrectly assembled proteins, in processing and degradation of transcriptional regulators in stress response and in the processing of protein antigens. This multicatalytic proteinase complex is composed of a catalytic core, 20S proteasome, which have multiple proteolytic activities (trypsin-like, chymotrypsin-like, peptidylglutamtyl-peptide hydrolyzing, BrAAP and SNAAP). The 20S proteasome associates with the multisubunit complex 19S to produce the 26S proteasome. The 26S proteasome has specificity for ubiquitinylated protein substrates and hydrolyses ATP during proteolysis of ubiquitinylated proteins. In the present work we have purified a 20S form of proteasome from Leishmania chagasi and partially characterized it. The purified 20S proteasome has activity towards fluorogenic substrates that are cleaved by trypsin or chymotrypsin, and is sensitive to lactacystin, a specific inhibitor of the proteasome. We show that the L.chagasi proteasome the trypsin-like activity is higher than the chymotrypsin-like. Therefore the chymotrypsin-like activity is inhibited by lactacystin and the trypsin-like it is only partially inhibited. We show here that lactacystin blocks in vitro L chagasi promastigote replication at a final concentration of 50 µM. To evaluate the effect of proteasome inhibition on the infectivity and intracellular development of L. chagasi, murine macropages were challenged with promastigotes from early stationary phase treated with lactacystin. Infectivity of macrophages was the same in lactacystin-treated parasites as in the untreated ones. Contrarywise, the intracellular development of the parasite is impaired by pretreating promastigotes with lactacystin. These promastigotes were able to infect BALB/c peritoneal macrophages but they did not survive inside macrophages. These data indicate the important role of the proteasomes of L. chagasi promastigotes on the intracellular development and replication in host cells in vitro.
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Atividade leishmanicida de constituintes químicos dos frutos de Garcinia brasiliensisPEREIRA, Ivan de Oliveira 02 February 2009 (has links)
As plantas são utilizadas no tratamento e na cura de enfermidades desde a
antiguidade, quando o homem começou a usar a natureza em seu favor. Estas
plantas, pela sua riqueza química e farmacológica, têm sido estudadas no intuito
de comprovar atividades atribuídas pela crença popular ou mesmo obter novos
compostos ativos. Neste contexto, a família Clusiaceae que compreende quarenta
e nove gêneros de plantas de ampla distribuição tem sido investigada. Entre eles
está o gênero Garcinia, também conhecido como Rheedia, que é rico em uma
considerável diversidade de compostos, tais como benzofenonas polipreniladas e
flavonóides. Neste trabalho foram obtidos os extratos hexânico, acetato-etílico e
etanólico do pericarpo dos frutos de Garcinia brasiliensis. Após avaliação da
atividade leishmanicida in vitro, o extrato hexânico se mostrou o mais ativo. Desta
forma, foi fracionado e dele obteve-se três benzofenonas, 7-epi-clusianona,
garciniafenona e guttiferona-a, que foram avaliadas quanto as suas atividades
leishmanicida e toxicidade. No intuito de determinar um possível mecanismo de
ação, os extratos foram avaliados quanto a sua capacidade de inibir proteases
isoladas de amastigotas, sendo o extrato acetato etílico o que apresentou melhor
resultado. Assim, este foi fracionado e dele se obteve o biflavonóide fukugetina,
que também foi avaliado quanto à capacidade de inibir proteases, apresentando
resultados muito satisfatórios. Todavia, quando o extrato acetato etílico e a
fukugetina foram avaliados quanto à atividade leishmanicida in vitro não foi
observado o mesmo efeito. Com estes resultados podemos observar que a
polaridade dos extratos e das moléculas testados neste estudo pode ser um fator
imprescindível na atividade leishmanicida, possivelmente por dificultar o transporte
destas substâncias através de membrana plasmática. / The plants have been used in the treatment and cure of illnesses since the
antique. When the men started to use the nature in your service. These plants,
due to their chemical and pharmaceutical characteristics, have been studied in
order to prove their theoretical medicinal capabilities or to obtain new active
components. In this context, the Clusiaceae family is made up of forty nine
species that are widely distributed, have been studied. Among these species is the
genus Rheedia, also known as Garcinia, which is rich in a wide range of
components, such as polyprenylated benzophenones and flavonoids. In this work
were obtained the hexanic, ethyl-acetate and ethanolic extracts from pericarp of
fruits from Garcinia brasiliensis. After the evaluation of leishmanicidal activity in
vitro, the hexanic extract shown the more active, thus, this was fractioned yielding
three benzophenones - 7-epi-clusianone, garciniaphenone and guttiferone-a – that
were evaluated how their leishmanicidal and toxicity activities. With the intention
of to determine a possible mechanism of action, these components were then
evaluated of their capacity of to inhibit proteases isolated from amastigote forms of
Leishmania, have been the ethyl-acetate extract the more effective. Thus this was
fractioned yielding the biflavonoid fukugetin, witch also was evaluated in relation
your proteases inhibition capacity, showing results very satisfactory. However,
when the ethyl-acetate extract and the fukugetin were evaluated in relation the
leishmanicidal activity in vitro, not was find the same effect. With these results, we
might realize that the hydrophilic of extracts and molecules evaluated in this study
can be an essential factor in the leishmanicidal activity, possible by to make
transport difficult of this compounds across the plasmatic membrane. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
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Atividade anticoagulante da catepsina L-like protease BmCL1 do carrapato Rhipicephalus (Boophilus) microplusXavier, Marina Amaral January 2016 (has links)
Introdução: Rhipicephalus microplus é um parasito importante na bovinocultura. Novas estratégias de controle dependem de um maior entendimento da sua fisiologia e da relação parasito-hospedeiro, e moléculas envolvidas na aquisição e digestão de sangue são alvos interessantes. Objetivo: determinar o mecanismo de ação do inibidor de coagulação BmGTI (Boophilus microplus Midgut Thrombin Inhibitor). Metodologia: BmGTI foi obtido a partir do homogenato de intestino de fêmeas de R. microplus parcialmente ingurgitadas utilizando técnicas de cromatografia de troca iônica, gel filtração e afinidade por trombina. A atividade inibitória foi monitorada pelo ensaio de coagulação do fibrinogênio induzida por trombina. A preparação de BmGTI foi avaliada por SDS-PAGE e analisada por LC-MS/MS. A provável ORF de BmGTI foi clonada no plasmídeo pPIC9, expressa em Pichia pastoris e purificada. Diferentes razões molares de trombina:rBmGTI foram examinadas para atividade inibitória de trombina sobre a clivagem do fibrinogênio em pH 7,5. E64 foi usado para bloquear o sítio ativo de rBmGTI e o ensaio de inibição foi realizado. Diferentes razões molares de trombina:rBmGTI foram incubadas e analisadas por SDS-PAGE para verificar interações entre as proteases. A atividade de rBmGTI sobre o fibrinogênio foi analisada pela incubação de ambos. Resultados e Discussão: Baseado na m/z dos fragmentos trípticos de BmGTI sua sequência foi identificada como BmCL1, uma catepsina Llike protease ativa em pH ácido, mas sem capacidade de hidrolisar substrato sintético em pH ≥7,0. rBmCL1/BmGTI foi expressa em P. pastoris como próenzima e após ativação, a enzima foi purificada. Diferentes razões molares de trombina:rBmCL1/BmGTI foram ensaiadas; quando a concentração de rBmCL1/BmGTI estava 64 vezes maior do que a de trombina, esta apresentou atividade residual de 37%. Quando rBmCL1/BmGTI teve seu sítio ativo bloqueado por E64, não houve inibição da atividade de trombina sobre fibrinogênio. A análise do fibrinogênio por SDS-PAGE, após incubação com rBmCL1/BmGTI, mostrou que as cadeias α e β do fibrinogênio são hidrolisadas. Conclusão: A partir destes resultados, concluímos que BmGTI inibe a coagulação sanguínea pela hidrólise das cadeias α e β do fibrinogênio. / Introduction: Rhipicephalus microplus is an important parasite of cattle. New strategies for control depend on a better knowledge of its physiology and hostparasite relationship, and molecules involved in acquisition and digestion of blood meal are interesting targets. Objectives: to determine how BmGTI (Boophilus microplus Midgut Thrombin Inhibitor) inhibits coagulation. Materials and Methods: BmGTI was obtained from partially engorged females midguts homogenate through ion exchange, size exclusion and thrombin-affinity chromatographies. Thrombin inhibition activity was measured by thrombin-induced fibrinogen clotting assay. BmGTI preparation was checked by SDS-PAGE and analyzed by LCMS/ MS. BmGTI putative ORF was cloned in pPIC9 plasmid, expressed in Pichia pastoris and purified. Different molar ratios of thrombin:BmGTI were examined for inhibitory activity of thrombin upon fibrinogen cleavage at pH 7.5. E64 was used to block BmGTI active site and inhibitory activity was assayed. Different molar ratio of thrombin:BmGTI were incubated and analyzed by SDS-PAGE to verify proteaseinhibitor interactions. BmGTI direct activity upon fibrinogen was analyzed after incubation by SDS-PAGE. Results and Discussion: Based on m/z of the BmGTI tryptic fragments it was identified as BmCL1, a cathepsin L-like proteinase active at acidic pH, but unable to hydrolyze synthetic substrate at pH ≥7.0. BmCL1/BmGTI was expressed in P. pastoris as pro-enzyme and after activation the enzyme was purified. Different molar ratios of thrombin:rBmCL1/BmGTI were assayed, and when rBmCL1/BmGTI concentration was 64-fold higher than thrombin concentration, this enzyme residual activity was 37%. When rBmCL1/BmGTI has its active site blocked with E-64, it was unable to inhibit thrombin activity upon fibrinogen. Analysis by SDS-PAGE of fibrinogen after incubation with rBmCL1/BmGTI showed that fibrinogen chains α and β were hydrolyzed. Conclusion: Based on these results, we concluded that rBmGTI/BmCL1 inhibits blood coagulation through hydrolyzes of fibrinogen chain- α and β.
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Papel dos proteassomas na interação e desenvolvimento de Leishmania chagasi em macrófagos murinos. / Role of parasite proteasomes in the infectivity and intracellular development of Leishmania chagasi in murine macrophages.Izaltina Silva Jardim 30 March 2001 (has links)
Nas células eucariotas a maioria das proteínas citoplasmáticas não são degradadas nos lisossomas, mas em organelas altamente conservadas encontradas em humanos, arquibactérias, plantas e leveduras, os proteassomas. Esta estrutura multicatalítica é constituída por componentes menores, cujo núcleo funcional é o componente 20S, que contém várias atividades proteolíticas (tríptica, quimotríptica, de peptidilglutamil peptidase, BrAAP e SNAAP). Esse componente 20S, associado ao complexo regulatório 19S, que é composto de múltiplas ATPases, forma o complexo 26S, responsável pela degradação de proteínas conjugadas com a ubiquitina. Estas estruturas citosólicas certamente desempenham papel importante no desenvolvimento de protozoários parasitas e na sua interação com células dos hospedeiros permissivos. Nesta dissertação, apresentamos um estudo sobre o papel do proteassoma na interação e desenvolvimento de promastigotas de Leishmania chagasi em macrófagos murinos. Inicialmente, purificamos e caracterizamos parcialmente o proteassoma de promastigotas de L. chagasi. Observamos que o complexo presente na L. chagasi possui atividades proteolíticas frente a pelo menos dois substratos sintéticos, LLVY-AMC e LRR-AMC, que avaliam, respectivamente, as atividades quimiotripsina-símile e tripsina-símile. A atividade tripsina-simile é maior que a atividade quimiotripsina-simile; e além disso, esta última é totalmente inibida pela lactacistina, um inibidor específico do proteassoma, enquanto a atividade tripsina-simile é apenas parcialmente inibida. Utilizando a lactacistina foi possível analisar o papel desse complexo proteolítico durante a infecção e desenvolvimento intracelular da L. chagasi. Promastigotas mantidas em cultura na presença de 50μM de lactacistina tiveram seu crescimento bloqueado. Essas promastigotas eram capazes de infectar macrófagos peritoneais de camundongos BALB/c, mas não conseguiam sobreviver dentro desses macrófagos. Esta incapacidade de sobrevivência foi específica para os parasitas tratados com a lactacistina, não sendo observado nos parasitas tratados com outros inibidores de proteases. Estes resultados sugerem que o proteassoma pode ter um papel importante no desenvolvimento intracelular e na replicação das promastigotas de L. chagasi no hospedeiro vertebrado. / Proteasomes are multicatalitic and multisubunit endopeptidase complexes widely distributed in eukaryotic cells. These enzymes are central proteases in the cytosol and nucleus and are involved in removal of abnormal, misfolded or incorrectly assembled proteins, in processing and degradation of transcriptional regulators in stress response and in the processing of protein antigens. This multicatalytic proteinase complex is composed of a catalytic core, 20S proteasome, which have multiple proteolytic activities (trypsin-like, chymotrypsin-like, peptidylglutamtyl-peptide hydrolyzing, BrAAP and SNAAP). The 20S proteasome associates with the multisubunit complex 19S to produce the 26S proteasome. The 26S proteasome has specificity for ubiquitinylated protein substrates and hydrolyses ATP during proteolysis of ubiquitinylated proteins. In the present work we have purified a 20S form of proteasome from Leishmania chagasi and partially characterized it. The purified 20S proteasome has activity towards fluorogenic substrates that are cleaved by trypsin or chymotrypsin, and is sensitive to lactacystin, a specific inhibitor of the proteasome. We show that the L.chagasi proteasome the trypsin-like activity is higher than the chymotrypsin-like. Therefore the chymotrypsin-like activity is inhibited by lactacystin and the trypsin-like it is only partially inhibited. We show here that lactacystin blocks in vitro L chagasi promastigote replication at a final concentration of 50 µM. To evaluate the effect of proteasome inhibition on the infectivity and intracellular development of L. chagasi, murine macropages were challenged with promastigotes from early stationary phase treated with lactacystin. Infectivity of macrophages was the same in lactacystin-treated parasites as in the untreated ones. Contrarywise, the intracellular development of the parasite is impaired by pretreating promastigotes with lactacystin. These promastigotes were able to infect BALB/c peritoneal macrophages but they did not survive inside macrophages. These data indicate the important role of the proteasomes of L. chagasi promastigotes on the intracellular development and replication in host cells in vitro.
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Les protéases et leurs inhibiteurs sécrétés par la cellule épithéliale : acteurs de l'inflammation et de la douleur / Proteases and their inhibitors secreted by epithelial cell : actors of inflammation and painRolland-Fourcade, Claire 03 November 2017 (has links)
Les protéases sont impliquées dans de nombreux processus biologiques et ont des origines très variées (cellules immunitaires, cellules épithéliales...). Leur activité est régulée par des inhibiteurs de protéases. Cette étude s'intéresse a` la balance entre les protéases épithéliales et leurs inhibiteurs dans le contexte de pathologies affectant l'intégrité´ de l'épithélium intestinal. Les conséquences d'un déséquilibre de la balance protéolytique ont été étudiées dans deux maladies chroniques aux composantes différentes, les Maladies Inflammatoires Chroniques de l'Intestin (MICI) (cycles de poussées inflammatoires) et le Syndrome du Côlon Irritable (SCI) (cycles de douleur). La muqueuse colique de patients atteints de SCI sécrète une activité de type trypsine dont l'origine et les fonctions sont mal définies. Cette étude s'intéresse à la source de cette activité trypsique dans le côlon des patients atteints de SCI, sa nature et son rôle dans l'activation neuronale. L'activité trypsique chez les patients atteints de SCI est augmentée majoritairement dans les cellules épithéliales (CEI). La stimulation de monocouches de cellules épithéliales avec du LPS ou de l'épinéphrine induit une augmentation de la quantité de trypsine-3 et de sa sécrétion au pôle basal de la cellule épithéliale stimulée, en corrélation avec une augmentation de l'activité trypsique. L'hyperactivité de la trypsine-3 au pôle basal entraine une perte de la fonction barrière de l'épithélium, anomalie retrouvée aussi chez les patients atteints de SCI. De plus, nous avons mis en évidence que la trypsine-3 est capable d'activer les neurones humains myentériques et les neurones sensitifs murins. In vivo, son administration intra-rectale à des souris induit une hypersensibilité viscérale dépendante du récepteur PAR2 (Protease Activated Receptor-2). Ainsi, les cellules épithéliales intestinales des patients atteints de SCI produisent et sécrètent de la trypsine-3 spécifiquement au pôle basal. Cette activité trypsique active des neurones sensitifs, participant à l'hypersensibilité viscérale, symptôme majeur dont souffrent les patients atteints de SCI. Les pathologies inflammatoires peuvent aussi être source d'un dérèglement protéolytique. Les patients atteints de MICI présentent une dérégulation de la balance élastolytique colique. Notre équipe a montré que l'apport d'ELAFINE (un inhibiteur d'élastase) par la bactérie génétiquement modifiée L.lactis au contact de la muqueuse inflammatoire protège les souris traitées en réduisant l'inflammation intestinale. Cependant les mécanismes protecteurs médiés par L'Elafine restent encore à déterminer. L'ELAFINE est un inhibiteur d'élastases mais possède aussi des propriétés antimicrobiennes. Afin de mettre en lumière quelles fonctions de L'Elafine portent les propriétés anti-inflammatoires des mutants de L'Elafine ont été générés et insérés dans la bactérie L.lactis: une mutation pour annuler sa fonction inhibiteur de protéase, un second mutant de son domaine antimicrobien et un dernier mutant ayant perdu ces 2 fonctions. Dans des monocouches de cellules intestinales épithéliales, l'apport d'ELAFINE protège de l'inflammation médiée par un déséquilibre de l'activité élastolytique : on observe une restauration de la fonction barrière de l'épithélium et une diminution des cytokines pro-inflammatoires (CXCL8 et IP10). La mutation du domaine antimicrobien n'affecte pas ces propriétés. Cependant, l'absence de la boucle inhibitrice annihile les propriétés anti-inflammatoires de L'Elafine. Ces travaux ont pu mettre en évidence l'importance de l'équilibre protéolytique au sein de la cellule épithéliale dans les pathologies intestinales. La balance entre les protéases et leurs inhibiteurs joue donc un rôle dans l'homéostasie épithéliale et les pathologies inflammatoires. / Proteases are involved in some biologic processes and their origins are variable (immune cells, epithelial cells...). Their activity is regulated by antiproteases. This study investigates the balance between proteases and their inhibitors in pathologies which modify epithelium integrity. Consequences of an unbalance in proteolytic activity was studied in two chronic pathologies with different components: Inflammatory Bowel Disease (IBD) (cycles of inflammatory boost) and Irritable Bowel Syndrome (IBS) (cycles of pain symptoms). Colonic mucosa from IBS patients releases trypsin activity. The origin and the functions of this activity are not well defined. This study investigated the source of this trypsin activity in the côlon of IBS patients, its nature and its role in neuronal activation. Trypsin activity from IBS patients is increased mostly in epithelial cells. Stimulation of epithelial cell monolayers with LPS or epinephrine induces an increase of trypsin-3 quantity and its secretion specifically in the basal side of epithelial cells. This is in correlation with the increase of trypsin activity. Trypsin-3 hyperactivity at the basal side provokes a loss of epithelium barrier function, which is also found in colons of IBS patients. Then, we have highlighted that trypsin-3 is able to activate human myenteric neurons and murine sensitive neurons. In vivo, its intra-rectal administration to mice induces a visceral hypersensitivity dependent of PAR2 (Protease Activated Receptors 2). Thus, intestinal epithelial cells from IBS patients produce and release trypsin-3 specifically on their basal side. This trypsin activity activates sensitive neurons which participate to visceral hypersensitivity, a major symptom of IBS patients. Inflammatory pathologies could be a source of proteolytic malfunction. IBD patients have a dysregulation of elastolytic balance in the colon. Our team has shown that ELAFIN (an elastase inhibitor) delivered by the bacteria genetically modified L.lactis near the inflamed mucosa, protects mice from intestinal inflammation. However, the protective mechanisms induced by ELAFIN need to be investigate. ELAFIN is an elastase inhibitor but have also antimicrobial properties. With the aim to highlight what function of ELAFIN owns anti-inflammatory properties, mutants of ELAFIN have been generated and were insered into L.lactis: a first mutant lacked its antiprotease function, a second lacked antimicrobial properties and a last mutant lacked both properties. In intestinal epithelial monolayers, ELAFIN delivered by L.lactis protects against inflammation: a restauration of epithelial barrier function and a decrease of pro-inflammatory cytokines (CXCL8 and IP10) are observed. Mutation of antimicrobial domain doesn't affected these properties. Nevertheless, the absence of inhibitory loop annihilates anti-inflammatory functions of ELAFIN. This work highlights the importance of proteolytic balance inside the epithelial cell in intestinal pathologies. The balance between proteases and antiproteases plays an important role in epithelial homeostasis.
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Characterization Of Rv2745c In The Pathogenesis Of Mycobacterium TuberculosisJanuary 2014 (has links)
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic Mtb:ΔRv2745c mutant is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb, implicating a role for ClgR in the management of intraphagosomal redox stress. Our data indicates that ClgR plays a role in multiple regulatory networks in response to different stress conditions. Thus, redox stress leads to dysregulation of the σH/σE regulon in Mtb:ΔRv2745c. Induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions. Disruption of genes involved in sulfate assimilation also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. On the other hand, the expression of clgR during hypoxia is known to result in Clp protease induction. As such, the isogenic mutant has a significantly different growth profile upon hypoxia and reaeration. Transcriptomics reveal disruption of the dosR regulon, σH/σE regulon, and mycolic acid synthesis genes. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in vivo. Our in vivo findings in a low dose aerosolized model reveal deficiencies of the isogenic mutant when establishing an infection, leading to skewed immune responses throughout the course of infection. Thus, clgR plays a critical role in both establishing an infection that influence the immunogenic outcome. Additional studies investigating the role of clgR in a nonhuman primate model will further elucidate the contributions of clgR to the pathogenesis of Mtb in an animal model that is more representative of human TB disease. / acase@tulane.edu
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Expression of stage-specific Fasciola proteases and their evaluation in vaccination trialsJayaraj, Ramamoorthi, Jayaraj@menzies.edu.au January 2008 (has links)
The liver flukes Fasciola hepatica and F. gigantica cause infectious disease in ruminants and humans. The geographical range of these two parasite species (temperate and tropical respectively) ensures that infection can occur worldwide. Although anthelmintic treatment is effective against disease, emerging drug resistant strains leads to the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available. The main challenge at present is to produce recombinant proteins in an immunologically active form using recombinant DNA technology. This is an essential step in Fasciola vaccine production. Cysteine proteases are probably the most important facilitators of virulence in flukes and are produced by all stages of the fluke life-cycle. Two classes of cysteine protease are found in the excretory and secretory material of liver flukes- these are cathepsin L and cathepsin B. As such, the major aims of this thesis were to investigate the expression and purification of Fasciola recombinant cysteine proteins, and characterisation by SDS-PAGE and immunoblotting using monoclonal and polyclonal antibodies. These studies demonstrate the production of functionally active cathepsin proteins in S. cerevisiae BJ3505 cells which will lead to vaccine candidate analysis. The second aim of this thesis was to determine the protective efficacy of stage specific target antigens against experimental infection. In addressing this issue, the protective efficacy of single and multivalent recombinant protein vaccinations of adult stage F. hepatica cathepsin L5, immature F. gigantica cathepsin L1g and juvenile F. hepatica cathepsin B were analysed in Sprague Dawley rats against F. hepatica infection. This study demonstrates that juvenile fluke target antigen-cathepsin B induces better immune protection than adult fluke antigen-cathepsin L5. Cocktails of juvenile and adult stage fluke recombinant proteins (cathepsin B and L5) elicited the highest protective immunity against experimental infection and this combination showed not only reduction in fluke recovery and size of flukes, but also marked diminution in the intensity of liver lesions in vaccinated rats. In order to assess the immunogenic property of an early infective stage fluke secreting cysteine protease as a vaccine candidate, DNA vaccination vectors encoding cathepsin B were analysed in BALB/c mice. In this study, the ability of four DNA vaccination strategies such as secretory, chemokine-activating, lymph node targeting vectors encoding cathepsin B were assessed by antibody titre, antibody avidity, western blotting and ELIPSOT assay. The results have further validated the immunoprophylactic potential of a cathepsin B vaccine against F. hepatica. In this study, we have expressed and attained high yields of F. gigantica cathepsin L1g from E. coli BL21, and compared this to a yeast-expressed system. This protease was over-expressed and formed insoluble inclusion bodies that were subsequently solubilised with urea or guanidine hydrochloride. In order to purify the urea-solubilised protein, step-wise urea gradient chromatography was used. For refolding of solubilised protein, a dilution and dialysis procedure was utilised. Proteolytic activity was confirmed by gelatin SDS-PAGE analysis. In conclusion, the determination of the immune potential of recombinant stage specific antigens allows the development of effective vaccines against Fasciola infection.
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Measurement, inhibition, and killing mechanisms of cytotoxic granule serine proteasesEwen, Catherine L 06 1900 (has links)
Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are critical for the protection of organisms against pathogens and cancer. The process by which these cells eliminate infected or transformed cells are through two basic mechanisms, receptor-mediated interactions, or delivery of contents from intracellular cytotoxic granules. Granules are comprised of perforin and a family of serine proteases, called granzymes. Upon entry into target cells, these proteins work together to initiate cellular death pathways. Previous and extensive biochemical studies had already established that granzyme B (GrB) was a powerful inducer of apoptosis, but sensitive assays to confirm its release from cytotoxic cells were lacking. We hypothesized that GrB release, measured by ELISPOT, directly assessed the lytic potential of antigen-specific cytotoxic cells. Indeed, data provided in this thesis established a strong correlation between GrB release and target cell lysis. Our results imply that GrB could be a promising tool to assess cell-mediated immunity during vaccine development.
However, several other independent studies in grB-/- mice demonstrated that additional granzymes were capable of clearing viruses and tumorigenic cells. Granzyme H (GrH) is highly and constitutively expressed in human NK cells, and therefore, we hypothesized that it was also an effective cytotoxic molecule. Our experiments established that GrH-induced cell death by a mechanism distinct from those of GrB and Fas. We identified a GrH substrate, DFF45/ICAD, and showed that GrH induced mitochondrial damage through a Bid-independent mechanism. Furthermore, cell death was dependent on Bax and/or Bak, but independent of caspase activation. Hence, we have elucidated an alternative cytotoxic pathway that could be employed to eliminate target cells with immune evasion strategies targeted to GrB or Fas.
Finally, control of serine proteases by endogenous inhibitors is important to numerous biological processes, including apoptosis. We hypothesized that as GrH displayed chymase activity, the serine protease inhibitor anti-chymotrypsin (ACT) would impair GrH function. Our data established that ACT effectively attenuated GrH cytotoxicity and prevented proteolysis of a GrH substrate. Collectively, this thesis describes a novel GrH inhibitor, provides a new tool to evaluate cell-mediated immunity, and provides evidence of an alternative mechanism of cytotoxicity.
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