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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.

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.

No description available.

In Zellkulturexperimenten wurden die Effekte von Simvastatin (Prodrug), Atorvastatin, Pravastatin und dem Proteasomhemmer Lactacystin suf zwei Endothelzelllinien (CPAE und Ea.hy926) und primäre vaskuläre glatte Muskelzellen (VSMCs) bezüglich ihres Verhaltens auf die Morphologie, die Proliferation, die Viabilität und die Proteasomaktivität untersucht. Sowohl die Statine als auch Lactacystin induzierten vergleichbare morphologische Veränderungen und beeinflussten die Proliferation von CPAE-Zellen. In den eigenen Versuchen konnten durch Statine induzierte Effekte durch Mevalonat revertiert werden. Die durch Lactacystin verursachten Veränderungen wurden durch Mevalonsäure nicht beeinflusst. Wie erwartet hemmte Lactacystin in den untersuchten CPAE-Zellen signifikant die proteasomale Aktivität. Im Gegensatz dazu blieb die Proteasomaktivität nach einer Statinbehandlung unbeeinflusst. Ähnliche Ergebnisse wurden auch in den Ea.hy926 und den VSMCs deutlich. Weiterhin konnte gezeigt werden, dass sogar hohe Dosen der Statine die Aktivität der gereinigten 20S Proteasomen nicht modulieren. Aus diesen Ergebnissen lässt sich schlußfolgern, dass die ähnlichen biologischen Effekte der Statine und des Lactacystins nicht über einen gemeinsamen Mechanismus der Proteasominhibition funktionieren. / The effect of simvastatin, atrovastatin and pravastatin as well as of the proteasom inhibitor beta-clasto lactacystin was studied on morphology, proliferation viability and on proteasomaö activityin two mammalian endothelial cell lines (CPAE and Ey.hy926) and in primary vascular smooth muscle cells (VSMCs). Both statins and lactacystin induced comparable morphological changes and attenuated proliferation of CPAE. Whereas the statin-induced effects were reversed by mevalonic acid, however, the lactacystin-induced alterations were not influenced by mevalonic acid. As expected, lactacystin caused a significant loss of proteasomal activity measured in the extracts of trested cells. The extracts of statin-treated CPAEs exhibited unchanged activities. This result was also confirmed in Ea.hy926 cells and in primary rat VSMCs. It is shown, that even high dosis of statins do not modulate the activity of purified human 20S proteasomes. The conclusion was that similar biological effects of statins and the well known proteasome inhibitor lactacystin in vascular cells are not caused by a common inhibitory mechanism ofaction on the proteasome.

Ce travail de thèse s'intéresse à la cycloaddition [2+2] asymétrique de cétènes et à son application en synthèse totale. La meilleure réactivité des oléfines Z comparées aux oléfines E, une des caractéristiques de la cycloaddition [2+2], est étudiée à la fois d'un point vue synthétique et théorique via des calculs DFT. Une méthodologie de cycloaddition [2+2] asymétrique entre des éthers d'énol chiraux et divers cétènes générés in situ a ensuite été développée. Cette cycloaddition limitée jusqu'ici à l'utilisation du dichlorocétène a été étendue à une grande variété de cétènes, permettant l'obtention de cyclobutanones variées hautement fonctionnalisées. Les cyclobutanones chirales obtenues ont été engagées dans la synthèse totale de produits naturels : la lactacystine et la salinosporamide A, inhibiteurs du protéasome et potentiels anticancéreux. Ces synthèses présentent trois étapes clés communes : une cycloaddition [2+2] asymétrique, une expansion de cycle de type Beckmann et la fonctionnalisation des chaînes latérales. La synthèse formelle de la lactacystine est présentée dans ce manuscrit, ainsi que les travaux en cours vers la synthèse de la salinosporamide A. / This thesis work focuses on the [2+2] asymmetric cycloaddition of ketenes and its application in total synthesis. The best reactivity of Z olefins compared to E olefins, one of the features of the [2+2] cycloaddition, is studied both from a synthetic and theoretically point of view, via DFT calculations. A methodology of [2+2] asymmetric cycloaddition between chiral enol ethers and various in situ generated ketenes was then developed. Cycloadditions limited to the use of dichloroketene hitherto was extended to a wide range of ketenes, leading to various highly functionalized cyclobutanones. Those chiral cyclobutanones were engaged in the total synthesis of natural products: lactacystin and salinosporamide A, proteasome inhibitors and potent anticancer agent. These syntheses have three common key steps : a [2+2] asymmetric cycloaddition, a Beckmann type ring expansion and a side chains functionalization. The formal synthesis of lactacystin is presented in this manuscript, as well as the work in progress towards the synthesis of salinosporamide A.

Germ-line mutations in the breast cancer susceptibility gene BRCA1 strongly increase the risk of developing breast and ovarian cancer in women. Different hypothesis have been proposed to explain this tissue specificity. One of the most argued hypothesis is the one that proposes a link between BRCA1 and ovarian hormones' action. Much data have been published in the last years pointing to an important role of progesterone receptor (PR) in inducing normal mammary development and also breast cancer formation. This study aimed to search for a functional relationship between BRCA1 and PR in breast cancer cells. We have found that BRCA1 inhibits the transcriptional activity of PR. We have investigated in more detail the mechanism of this effect. BRCA1 and PR interact in vivo in a ligand-independent fashion. Most importantly, BRCA1 alters the ligand-independent and dependent degradation of PR protein through its ubiquitination and this might have a direct effect on the level of PR recruitment on regulated promoters. BRCA1 is recruited to the hormone-responsive regions of PR-target genes and affects the presence of histone deacetylase activity and the level of monoubiquitinated histone H2A, linking BRCA1 action with chromatin status. These findings support a connection between BRCA1, the principal tumour suppressor responsible for familial breast cancer, and the progesterone receptor transcriptional activity. This relationship can be hypothesized to be reflected in the BRCA1-related breast tumourigenesis. / Mutaciones germinales en el gen breast cancer susceptibility gene BRCA1 aumentan altamente el riesgo de padecer cáncer de mama y ovario en mujeres. Se han propuesto diferentes hipótesis para explicar esta especificidad de tejido. Una de las hipótesis más argumentadas es la que propone una relación entre BRCA1 y la acción de las hormonas ováricas. En los últimos años se han publicado numerosos datos señalando al papel esencial del receptor de progesterona (PR) en la inducción del desarrollo normal de la mama y en la formación del cáncer de mama. Este estudio pretendía buscar una relación funcional entre BRCA1 y PR en células de cáncer de mama. Hemos demostrado que BRCA1 inhibe la actividad transcripcional de PR. Hemos investigado en más detalle el mecanismo de este efecto. BRCA1 y PR interaccionan in vivo de una manera independiente de ligando. Y lo que es más, BRCA1 altera la degradación independiente y dependiente de ligando de PR a través de su ubiquitinización y esto podría tener un efecto directo en el nivel de reclutamiento de PR en promotores regulados. BRCA1 es reclutado a las regiones de respuesta a hormona de genes diana de PR y afecta la presencia de actividad histona desacetilasa y el nivel de histona H2A monoubiquitinada, estableciendo un enlace entre la acción de BRCA1 y el estado de la cromatina. Estos hallazgos apoyan una conexión entre BRCA1, el principal supresor de tumor responsable del cáncer de mama hereditario, y la actividad transcripcional del receptor de progesterona. Se puede hipotetizar que esta relación se ve reflejada en el proceso de tumorigénesis BRCA1-dependiente.