Le VIH-1 a développé plusieurs mécanismes menant à la dégradation de son récepteur cellulaire, la molécule CD4, dans le but d’augmenter la relâche de particules virales infectieuses et d’éviter que la cellule soit surinfectée. L’un de ces mécanismes est la dégradation, induite par la protéine virale Vpu, du CD4 nouvellement synthétisé au niveau du réticulum endoplasmique (RE). Vpu doit lier CD4 et recruter l’ubiquitine ligase cellulaire SCFβ-TrCP, via sa liaison à β-TrCP, afin de dégrader CD4. Puisque CD4 doit être retenu au RE pour permettre à Vpu d’induire sa dégradation via le système ubiquitine-protéasome, il a été suggéré que ce processus implique un mécanisme semblable à une voie cellulaire de dégradation des protéines mal-repliées appelée ERAD (« endoplasmic reticulum-associated degradation »). La dégradation par ERAD implique généralement la dislocation des protéines du RE vers le cytoplasme afin de permettre leur poly-ubiquitination et leur dégradation par le protéasome. Nous avons démontré que Vpu induit la poly-ubiquitination de CD4 dans des cellules humaines. Nos résultats suggèrent aussi que CD4 doit subir une dislocation afin d’être dégradé par le protéasome en présence de Vpu. De plus, un mutant transdominant négatif de l’ATPase p97, qui est impliquée dans la dislocation des substrats ERAD, inhibe complètement la dégradation de CD4 par Vpu. Enfin, nos résultats ont montré que l’ubiquitination sur des résidus accepteurs de l’ubiquitine (lysines) de la queue cytoplasmique de CD4 n’était pas essentielle, mais que la mutation des lysines ralentit le processus de dégradation de CD4. Ce résultat suggère que l’ubiquitination de la queue cytosolique de CD4 pourrait représenter un événement important dans le processus de dégradation induit par Vpu. L’attachement de l’ubiquitine a généralement lieu sur les lysines de la protéine ciblée. Toutefois, l’ubiquitination sur des résidus non-lysine (sérine, thréonine et cystéine) a aussi été démontrée. Nous avons démontré que la mutation de tous les sites potentiels d’ubiquitination cytoplasmiques de CD4 (K, C, S et T) inhibe la dégradation par Vpu. De plus, la présence de cystéines dans la queue cytoplasmique apparaît suffisante pour rendre CD4 sensible à Vpu en absence de lysine, sérine et thréonine. Afin d’expliquer ces résultats, nous proposons un modèle dans lequel l’ubiquitination de la queue cytosolique de CD4 serait nécessaire à sa dégradation et où les sites d’ubiquitination de CD4 seraient sélectionnés de façon non spécifique par l’ubiquitine ligase recrutée par Vpu. Enfin, nous avons observé que la co-expression d’une protéine Vpu incapable de recruter β-TrCP (Vpu S52,56/D) semble stabiliser le CD4 qui est retenu au RE. De plus, d’autres mutants de Vpu qui semblent capables de recruter β-TrCP et CD4 sont toutefois incapables d’induire sa dégradation. Ces résultats suggèrent que l’association de Vpu à CD4 et β-TrCP est essentielle mais pas suffisante pour induire la dégradation de CD4. Par conséquent, ces résultats soulèvent la possibilité que Vpu puisse recruter d’autres facteurs cellulaires pour induire la dégradation de CD4. Les résultats présentés ont permis de mieux définir le mécanisme de dégradation de CD4 par Vpu dans des cellules humaines. De plus, ces résultats nous ont permis d’élaborer un modèle dans lequel l’ubiquitine ligase cellulaire SCFβ-TrCP démontre de la flexibilité dans le choix des résidus à ubiquitiner afin d’induire la dégradation de CD4. Enfin, ces études jettent un oeil nouveau sur le rôle de Vpu dans ce processus puisque nos résultats suggèrent que Vpu doive recruter d’autres partenaires cellulaires, mis à part β-TrCP, pour induire la dégradation de CD4. / HIV-1 has developed many mechanisms leading to the down-regulation of its cellular receptor, the CD4 molecule, in order to increase the release of infectious viral particles and to inhibit superinfection of the target cell. One of these mechanisms is the HIV-1 Vpu-mediated degradation of newly synthesized CD4 at the level of endoplasmic reticulum (ER). Vpu must interact with CD4 and recruit the cellular ubiquitin ligase SCFβ-TrCP, via its binding to β-TrCP, in order to induce CD4 degradation. Because CD4 has to be retained in the ER to allow Vpu to induce its degradation via the ubiquitin-proteasome system, it has been suggested that this process involves a mechanism reminiscent of a cellular degradation pathway involved in the proteolysis of unfolded proteins called ERAD (endoplasmic reticulum-associated degradation). The ERAD degradation usually involves the dislocation of proteins from the ER to the cytoplasm in order to induce their poly-ubiquitination and subsequent degradation by the proteasome. We demonstrated that Vpu induces the poly-ubiquitination of CD4 in human cells. Our results also suggest that CD4 has to be dislocated in order to be degraded by the proteasome in presence of Vpu. Furthermore, the expression of a transdominant negative mutant of the ATPase p97, that is involved in the dislocation of ERAD substrates, inhibits completely the Vpu-mediated CD4 degradation process. Finally, our results demonstrated that the ubiquitination of putative ubiquitin acceptor residues (lysines) in the cytosolic tail of CD4 is not essential but the mutation of these lysines slowed down the process of CD4 degradation induced by Vpu. This results suggests that ubiquitination of CD4 cytosolic tail could represent an important step during Vpu-mediated CD4 degradation. Ubiquitin is usually attached on lysine residues in the targetted protein. However, the ubiquitination on non-lysine residues (S, T and C) has also been demonstrated. We demonstrated that the mutation of all cytosolic potential ubiquitination sites (K, C, S and T) of CD4 abolishes Vpu-mediated degradation. In addition, the presence of cysteines in the cytosolic tail of CD4 appeared sufficient to render CD4 sensitive to Vpu in absence of lysine, serine or threonine. In order to explain these results, we propose a model in which CD4 cytosolic tail ubiquitination is necessary for its degradation and where ubiquitination sites are selected non specifically by the ubiquitin ligase recruited by Vpu. Finally, we observed that co-expression of a phosphorylation mutant of Vpu unable to interact with β-TrCP (Vpu S52,56/D) appears to stabilize ER-retained CD4 molecules. In addition, other Vpu mutants seem able to recruit β-TrCP and CD4 without inducing CD4 degradation. These results suggest that Vpu association with CD4 and β-TrCP is essential but not sufficient for CD4 degradation. Consequently, these results raised the possibility that other cellular factors could be recruited by Vpu in order to induce CD4 degradation. The results presented here allowed us to better define the mechanism underlying Vpu-mediated CD4 degradation. In addition, these results allowed us to elaborate a model in which the ubiquitin ligase SCFβ-TrCP show some flexibility in the choice of ubiquitination sites in order to induce CD4 degradation. Finally, theses studies shed a new light on the role of Vpu in the CD4 degradation process because our results suggest that Vpu could recruit, in addition of β-TrCP, other cellular partners in order to induce CD4 degradation.

La polyarthrite rhumatoïde (PR) est une maladie auto-immune chronique. Elle est caractérisée par une inflammation persistante touchant de multiples petites articulations, causant douleurs, rougeurs, gonflements et déformations. Des études menées auprès de patients et d’animaux ont démontré que certains auto-anticorps, cytokines et enzymes tissue-déstructives sont des médiateurs importants dans le développement de la PR. Au cours des deux dernières décennies, les traitements de fond (DMARDs en anglais) ont été démontrés très efficaces pour traiter la PR. D'autre part, des effets secondaires ont été rapportés pour ces traitements, par exemple l'augmentation du risque d'infections opportunistes. L’objectif de ce travail est d’acquérir des connaissances sur le rôle du TL1A (TNF-like molécule 1 A; TNFSF15) et son partenaire Nob1 (Pno1 ; YOR145c) dans la pathogenèse de la PR afin de découvrir de nouveaux médicaments contre ces molécules dans l'avenir. TL1A est un membre de la famille du TNF. Il déclenche des signaux co-stimulateurs via le récepteur de mort 3 (DR3) et induit la prolifération ainsi que la production des cytokines pro inflammatoires par les lymphocytes. Des données multiples suggèrent l'implication de la cascade TL1A-DR3 dans plusieurs maladies auto-immunes. Donc, nous avons proposé les hypothèses suivantes:1) la production locale de TL1A dans les articulations est un composant d’un cercle vicieux qui aggrave la PR; 2) dans la PR, la production de TL1A dans les organes lymphoïde augmente la production d’auto-anticorps pathogénique. Au cours de ce travail, nous avons démontré que la TL1A aggrave la maladie chez les souris où l’arthrite a été induite par le collagène (AIC). Par ailleurs, nous avons constaté que l’expression de TL1A est élevée dans les tissus atteints de PR ainsi que dans les ganglions lymphatiques drainant de la souris AIC. Mécaniquement, nous avons découvert que la TL1A est induite par le TNF-α et IL-17 produits par les cellules T in vitro. Ces résultats montrent directement que les TL1A-DR3 jouent un rôle essentiel dans la pathogenèse de la PR. De plus, afin de poursuivre notre étude, la TL1A a été génétiquement supprimée dans les souris (TL1A KO). Nous avons montré que les souris TL1A KO n’ont aucune anomalie apparente et aucun dysfonctionnement du système immunitaire dans des conditions normales. Cependant, ces souris manifestent des AIC améliorées et une réduction significative des niveaux d'anticorps, anti-collagène du type II i dans le sérum. Nous avons trouvé que les ganglions lymphatiques de drainage (dLNs) de souris KO étaient plus petites avec une cellularité inférieure comparativement aux souris WT de 14 jours après l’immunisation. De plus, nous avons découvert que le DR3 a été exprimé par les cellules plasmatiques dans l’étape de la différenciation terminale et ces cellules surviennent mieux en présence de TL1A. La conclusion de cette étude apporte des nouvelles connaissances sur le rôle de TL1A qui amplifie les réponses humorales d’AIC. Nous avons suggéré que TL1A pourrait augmenter la réponse d’initiation d'anticorps contre collagène II (CII) ainsi que prolonger la survie des cellules plasmatiques. Une autre molécule qui nous intéresse est Pno1. Des études antérieures menées chez la levure ont suggéré que Pno1 est essentielle pour la néogénèse du protéasome et du ribosome Le protéasome étant crucial pour la différenciation terminale des cellules plasmatiques pendant les réponses humorales chez les mammifères, nous avons donc supposé que Pno1 joue un rôle dans la production d'anticorps pathogenique dans la PR via la voie du protéasome. Nous avons donc généré des souris génétiquement modifiées pour Pno1 afin d’étudier la fonction de Pno1 in vivo. Cependant, une mutation non-sens dans le Pno1 provoque une létalité embryonnaire à un stade très précoce chez les souris. D'autre part, une réduction de 50% de Pno1 ou une surexpression de Pno1 n’ont aucun effet ni sur le fonctionnent des cellules T et B, ni sur les activités du protéasome ainsi que sur la réponse humorale dans l’AIC. Ces résultats suggèrent que Pno1 est une molécule essentielle sans redondance. Par conséquent, il n’est pas une cible appropriée pour le développement de médicaments thérapeutiques. En conclusion, nos études ont révélé que la TL1A n’est pas essentielle pour maintenir les fonctions du système immunitaire dans des conditions normales. En revanche, il joue un rôle critique dans la pathogenèse de la PR en favorisant l'inflammation locale et la réponse humorale contre des auto-antigènes. Par conséquent, une inhibition de la TL1A pourrait être une stratégie thérapeutique pour le traitement de la PR. Au contraire, Pno1 est essentiel pour la fonction normale des cellules. Une délétion totale pourrait entraîner des conséquences graves. Il n’est pas une cible appropriée pour développer des médicaments de la PR. / Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation of multiple small joints, which manifests pain, redness, swelling, and deformation. Studies with patients and animal models have found that autoantibodies, cytokines and tissue-destructive enzymes are important mediators of the pathogenesis of RA. In the past two decades, biologic disease-modifying antirheumatic drugs (DMARDs) have achieved great success in the treatment of RA. On the other hand, they are also associated with adverse effect like increasing the chance of opportunistic infections. The aim of present work was to investigate the roles of TNF-like molecule 1A (TL1A; TNFSF15) and partner of Nob1 (Pno1; YOR145c) in the pathogenesis of RA for developing novel drugs based on these molecules in the future. TL1A is a member of the TNF superfamily. It triggers costimulatory signals though death receptor 3 (DR3) and induces the proliferation and pro-inflammatory cytokine production in lymphocytes. Multiple lines of evidence suggest the implication of TL1A-DR3 signaling in several autoimmune diseases. Therefore, We hypothesized that 1) local TL1A production in the joints is a component of a vicious circle aggravating RA; 2) in RA, TL1A production in lymphoid organs enhances pathogenic autoantibody production. We demonstrated that the TL1A aggravates disease in murine collagen-induced arthritis (CIA). Moreover, we found elevated TL1A expression in RA-affected tissues, as well as in the draining lymph nodes (dLNs) of CIA mice. Mechanistically, we discovered that TL1A induces TNF-α and IL-17 production by T cells in vitro. These findings provided direct evidence that TL1A-DR3 signaling plays a critical role in the pathogenesis of RA. TL1A knockout (TL1A KO) mice were generated to further our study. We showed that TL1A KO mice have no visual anomaly, and no malfunction of immune system under a normal circumstance. However, they display ameliorated CIA and significantly reduced anti-Collagen II antibody levels in sera. We found that the draining lymph nodes (dLNs) from KO mice were smaller in size and lower in cellularity compared with their WT counterparts 14 days after immunization. Furthermore, we discovered that terminally differentiated plasma cells express DR3 and they survive better in the presence of TL1A. Our findings in this study present novel knowledge about the role of iii TL1A promoting the humoral responses in CIA; we suggest that TL1A could elevate the initial Ab response against Collagen II (CII), as well as prolong the survival of plasma cells producing such pathogenic Abs. Another molecule we were interested in present study is Pno1. Previous studies conducted in yeast suggest that Pno1 is essential to the proteasome and ribosome neogenesis. Since proteasome is crucial for the terminal differentiation of plasma cells during the humoral response in mammals, we hypothesized that Pno1 plays a role in the pathogenic Ab production in RA by affecting the proteasome assembly. For this purpose, we generated pno1 gene- modified mice to investigate the function of Pno1 in vivo. However, null-mutation in pno1 causes embryonic lethality in mice at a very early stage. On the other hand, a half amount reduction or overexpression of Pno1 is neither harmful nor useful to the T and B cell function, proteasome activities as well as humoral immune responses in CIA. These findings suggest that Pno1 is a vital molecule with no redundancy and is absolutely required for cell function, but animals can function normally with a small fraction of the normal Pno1 expression level. Thus, it might not be an appropriate target for developing therapeutic drugs. In conclusion, our studies suggest that TL1A seems not essential in maintaining the immune functions under normal circumstances, but plays critical roles in the pathogenesis of RA by promoting local inflammation and humoral immune responses against autoantigens. Therefore, inhibiting TL1A could be a propitious therapeutic strategy for treating RA. In contrast, Pno1 is vital to the normal cell function, and its disruption could cause disastrous consequences. Thus, it might not be a good drug target for treating RA.

Thesis (Ph.D. in Experimental Pathology, Program in Cancer Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 144-158). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

De nos jours, parmi toutes les transformations chimiques dont disposent les chimistes organiciens, le réarrangement sigmatropique [3,3] constitue un outil puissant afin de créer une liaison C-C ou encore C-hétéroatome. Le réarrangement d’allyl cyanate en allyl isocyanate, jusqu’ici peu utilisé, est en train d’émerger, comme une nouvelle méthode efficace de préparation d’allylamines substituées. C’est dans ce contexte que s’inscrit mon travail de thèse qui a pour objectif d’étudier et d’utiliser le réarrangement sigmatropique [3,3] d’allyl cyanates diversement substitués pour la synthèse de molécules d’intérêt biologique. Dans une première partie, l’emploi d’allyl cyanates borylés nous a permis de synthétiser, de façon stéréocontrôlée, des ènecarbamates et des ènehydroxyurées cycliques à 7 chainons ainsi que des γ-butyrolactones. Cette méthodologie a été appliquée à la première synthèse totale de la (-)-Galbacin. Les ènecarbamates cycliques ont fait l’objet, en seconde partie, d’une étude structure-activité en tant qu’inhibiteur du protéasome humain. Dans une troisième partie, nous avons étudié le réarrangement d’allyl cyanates silylés ce qui nous a permis d’accéder à des α-amino allylsilanes énantioenrichis. En dernière partie, nous avons mis en évidence un oxo-réarrangement à partir d’allyl carbamates substitués par un groupement aryle. / Nowadays, among all chemical transformations in the organic chemist’s toolbox, [3,3] sigmatropic rearrangements represent a powerful method to create carbon-carbon or even carbon-heteroatom bonds. The allyl cyanate to allyl isocyanate rearrangement, underused so far, is becoming an attractive method to prepare substituted allylamines. In this context, I studied and applied in my thesis this [3,3] rearrangement on diversely substituted allyl cyanates for the synthesis of molecules of biological interest. First, we used borylated allyl cyanates to prepare, stereoselectively, cyclic seven membered enecarbamates and enehydroxyureas as well as γ-butyrolactones. This methodology was applied to the first total synthesis of (-)-Galbacin. Then, a library of cyclic enecarbamates was performed to study the structure-activity relationship on the human proteasome. Next, the study of silylated allyl cyanates allowed us to prepare some new enantioenriched α-amino allylsilanes. Finally, we brought to light an unexpected oxo-rearrangement from aryl substituted allyl carbamates.

Made available in DSpace on 2016-06-02T20:18:16Z (GMT). No. of bitstreams: 1 3973.pdf: 3412425 bytes, checksum: 4be152bd7ce18a298fe811d9024f0e59 (MD5) Previous issue date: 2011-11-28 / Universidade Federal de Minas Gerais / The quadriceps muscle weakness and atrophy after the anterior cruciate ligament (ACL) rupture, is present before and after its reconstruction, and are important limitations that restrict the return of subjects to activities of daily living and sports. The combination of reduced synthesis and increased levels of protein degradation results in a rapid loss of muscle protein. The rapid muscle proteolysis occurs in several animal models and in different conditions in humans, and are mainly associated to the ubiquitn proteasome system (UPS). Neuromuscular mechanisms are associated with atrophy and weakness of the quadriceps muscle after ACL rupture and reconstruction, but the molecular pathways involved in this process are unknown. The identification of these pathways may facilitate the establishment of therapeutic strategies to alleviate the muscle deficit. Thus, the objective of this thesis was to evaluate the effect of ACL transection of rats in the expression of genes of UPS (MuRF-1, Atrogina-1), as well as the expression of myostatin (involved in control of muscle mass) by the Real Time PCR (polymerase chain reaction) technique. It was evaluated the content of ubiquitinated proteins by Western blotting and the cross-sectional area of muscle fibers (CSA) of the vastus medialis (VM), rectus femoris (RF) and vastus lateralis (VL) of the quadriceps. Elevated Atrogin-1, MuRF-1 and myostatin mRNA levels were observed after ACL transection in all muscles assessed, as the reduction of CSA in VM and VL muscles. The results of this thesis contributed to the understanding of the mechanisms of quadriceps muscle atrophy after ACL transection. / A atrofia e fraqueza do quadríceps após a ruptura do ligamento cruzado anterior (LCA), presentes antes e após sua reconstrução, são importantes limitações que restringem o retorno dos indivíduos às atividades da vida diária e desportivas. A rápida proteólise muscular que ocorre nos diversos modelos animais e em diferentes condições em humanos se deve, principalmente, ao sistema ubiquitna proteassoma (UPS). Mecanismos neuromusculares são associados à atrofia e fraqueza do quadríceps após a ruptura e reconstrução do LCA, porém as vias moleculares envolvidas nesse processo são desconhecidas. A identificação dessas vias pode favorecer o estabelecimento de estratégias terapêuticas para amenizar o déficit muscular. Assim, o objetivo dessa tese foi avaliar o efeito da transecção do LCA de ratos na expressão dos genes pertencentes ao UPS (MuRF-1, a Atrogina-1), e também da Miostatina (envolvida no controle da massa muscular) pela técnica de PCR (reação em cadeia da polimerase) em tempo real no quadriceps. Avaliou-se ainda o conteúdo de proteínas ubiquitinadas por Western Blotting e a área de secção transversa das fibras musculares (AST) dos músculos Vasto Medial (VM), Reto Femoral (RF) e Vasto Lateral (VL). Níveis elevados de Atrogina-1, MuRF-1 e Miostatina ocorreram após transecção do LCA em todos os músculos avaliados assim como a redução da AST nos músculos VM e VL. Os resultados desta tese contribuiram para a compreensão dos mecanismos de atrofia do quadríceps após a transecção do LCA.

Le gène Gec1/Gabarapl1 a été identifié au sein de notre laboratoire comme un gène régulé par les estrogènes. Il appartient à la famille Gabarap incluant les gènes Gabarap, gabarap/2 et Gabarapl3 qui codent des protéines présentant de fortes homologies de séquences. L'étude fonctionnelle de Gabarapl 1 a montré que cette protéine est impliquée dans le transport des récepteurs et particulièrement les récepteurs Gabaₐ et des κ-opioïdes via son interaction avec la tubuline et la protéine NSF. Cependant, il a été décrit que certaines protéines de la famille Atg8 sont impliquées dans l' autophagie, un mécanisme de dégradation et de survie cellulaire, qui se caractérise par la formation de doubles membranes appelées autophagosomes. Les objectifs de mon travail étaient, d'une part, de caractériser le rôle de la protéine GABARAPL1 au cours de !'autophagie et, d'autre part, de caractériser son expression dans des lignées et tissus cancéreux et sa régulation en réponse à des composés anti-cancéreux. Tout d'abord, nous avons montré que Gabarapl1 est clivée par la protéase Atg4B au niveau de sa glycine 116 avant sa conjugaison à des phopholipides. Cette forme modifiée, lipidée, est localisée à la surface des autophagosomes et des lysosomes. Nous avons ensuite montré que Gabarapl1 est faiblement exprimée dans de nombreuses lignées cancéreuses, que son expression est altérée dans les méningiomes et qu'elle est régulée par des inhibiteurs du protéasome. Ces travaux ont montré, pour la première fois, que la protéine Gabarapl1 est associée à des vésicules autophagiques et permettront de poser les hypothèses de nos futurs travaux. / The Gec1 / Gabarapl1 gene was identified in our laboratory as an early estrogen regulated gene. Gabarapl1 belongs to the Gabarap family, also including Gabarap, Gabarapl2 and Gabarapl3 genes, that encode proteins which present high sequence homology with each other. A functional study of the Gabarapl 1 protein showed that this protein is involved in the transport of receptors such as the Gabaₐ and κ-opioid receptors via its interaction with tubulin and NSF. It has been reported that the Atg8 family proteins are involved in autophagy, a mechanism of degradation and cell survival that is charactenzed by the formation of double membranes called autophagosomes. The aims of my research were, firstly, to characterize the role of the Gabarapl1 protein during autophagy and, secondly, to study its expression in cancer cell lines and cancerous tissues and its regulation in response to anti-cancer drugs. First, we showed that Gabarapl1 is cleaved in the cells by the protease Atg4B at its 116 glycine residue prior to its conjugation to phospholipids. This modified form, lipidated, is located on the surface of autophagosomes and lysosomes. We then showed that Gabarapl1 expression is reduced in many cancer cell lines, and that its expression is also altered in meningiomas. Finally, we showed that Gabarapl1 expression is regulated by proteasom€: inhibitors. Thus, our results demonstrated for the first time that the Gabarapl1 protein is associatec with autophagie vesicles and allow us to propose hypothesis for future work

Muitos genes têm sido indicados como responsivos ao estresse genotóxico, mas devido à necessidade de validação, a busca por marcadores gênicos continua. Vários genes são relacionados ao sistema ubiquitina-proteassomo (UPS), o qual é responsável pela remoção seletiva de proteínas, sendo que falhas no UPS têm sido relacionadas a doenças neurodegenerativas e ao câncer. Assim, o presente trabalho teve como objetivo a busca e confirmação de marcadores gênicos de resposta ao estresse genotóxico, por meio do estudo da expressão transcricional e protéica dos genes ERN1, EIF2AK3, GADD153 e TRAF2, visando à confirmação das respostas em linhagens de fibroblastos (GM07492A e AS405) e de glioblastoma (U87MG), sob tratamentos com peróxido de hidrogênio (H2O2) e Bleomicina (Blm). Foram utilizados o Ensaio Cometa, a análise de expressão gênica transcricional por qPCR em tempo real e de expressão gênica ao nível protéico (imunofluorescência). Os resultados mostraram que os tratamentos empregados foram capazes de induzir danos no DNA, sendo que a sensibilidade ao tratamento e a capacidade de recuperação das linhagens foi variável dependendo do agente testado. A análise de expressão gênica mostrou que GM07492A apresentou indução dos genes ERN1 e GADD153 após tratamento com H2O2 (resposta precoce, zero e 2 h) e Blm (durante todo pós-tratamento). A linhagem AS405 exibiu indução de ERN1 e GADD153 para H2O2, enquanto que para Blm foram induzidos os genes EIF2AK3 e GADD153. Para U87MG, a indução de EIF2AK3 pelo H2O2 ocorreu de modo tardio, enquanto GADD153 mostrou-se induzido após ambos os tratamentos. A proteína ERN1 apresentou expressão discreta e pontual, inclusive nos pontos onde não houve indução transcricional, indicando uma expressão basal. Essa proteína se expressou em GM07492A no tratamento com Blm em zero hora, diferentemente de AS405. Para U87MG tratada com H2O2 observou-se discreta expressão de ERN1, sendo mais evidente para Blm. Quanto à proteína GADD153, esta foi expressa em fibroblastos nos vários tempos analisados. No entanto, U87MG mostrou expressão nuclear apenas nas células tratadas, sendo mais evidente para H2O2 comparativamente à Blm. Assim, as alterações observadas nos perfis de expressão gênica são compatíveis com a indução de danos no DNA, indicando o envolvimento de genes do UPS nas respostas celulares ao estresse genotóxico. Em conjunto, os resultados estimulam uma avaliação mais detalhada desses genes como marcadores de resposta ao estresse e evidencia a sua importância no cenário da via UPS. / Many genes have been reported as responsive to genotoxic stress, but due to the need of validation, the search for genetic markers still continues. Several genes are related to the ubiquitin-proteasome system (UPS), which is responsible for the selective removal of proteins, and UPS failures have been associated to neurodegenerative diseases and cancer. Thus, this study aimed the search and confirmation of genetic markers that were responsive to genotoxic stress. For this, we evaluated the transcriptional or protein expression of the genes ERN1, EIF2AK3, GADD153 and TRAF2, seeking confirmation of responses in fibroblast cell lines (GM07492A and AS405) and glioblastoma (U87MG) under treatment with hydrogen peroxide (H2O2) and bleomycin (BLM). We used the Comet Assay, the transcriptional analysis of gene expression by quantitative real-time PCR and protein expression byimmunofluorescence. The results showed that the treatments employed were able to induce DNA damage, and that cell sensitivity to treatments and recovery capability of cell lines varied according to the tested agent. The gene expression analysis showed that GM07492A presented induction of ERN1 and GADD153 genes after treatment with H2O2 (early response, zero and 2 h) and Blm (throughout the post-treatment). The cell line AS405 showed induction of GADD153 and ERN1 after H2O2, whereas with Blm the genes induced were EIF2AK3 and GADD153. For U87MG, the induction of EIF2AK3 by H2O2 occurred at a later stage, while GADD153 was promptly induced after both treatments. The protein ERN1 showed discreet and punctual expression, even at time point without transcriptional induction, indicating a basal expression. This protein was expressed in GM07492A by treatment with Blm at zero hour, differently of AS405. For U87MG treated with H2O2, ERN1 showed a slight expression, being more evident for Blm. Regarding GADD153, protein expression was observed in fibroblasts at all time point. However, U87MG showed nuclear expression only in cells treated with H2O2, being more evident that in BLM-treated cells. Thus, the observed changes in gene expression profiles are consistent with the induction of DNA damage, which indicates the participation of UPS genes in cellular responses to genotoxic stress. Together, the results encourage further evaluation of these genes as markers of stress response, demonstrating its importance in the UPS acting scope.

A atrofia está associada ao aumento da degradação protéica em doenças sistêmicas, sendo o sistema proteolítico ubiquitina proteassoma (SUP) uma das principais vias envolvidas. Contudo, pouco é conhecido sobre a contribuição do SUP à atrofia desencadeada pela insuficiência cardíaca (IC). Sabendo dos benefícios do treinamento físico aeróbico (TFA) e que os mecanismos moleculares envolvidos na atrofia na IC ainda não estão esclarecidos, nessa dissertação investigamos: 1) a contribuição do SUP para a atrofia associada à IC em 2 modelos experimentais: um modelo genético de camundongos com hiperatividade simpática (HS), e um modelo de infarto do miocárdio (IM) em ratos e 2) o efeito do TFA sobre a atrofia associada à IC e sobre o SUP. Na HS verificamos aumento da expressão das E3 ligases, da deubiquitinase USP28, das proteínas ubiquitinadas e da atividade do proteassoma no sítio quimiotripsina, sendo que o TFA reduziu a expressão dos componentes alterados. No IM, observamos disfunção cardíaca não associada à IC, porém, com aumento da expressão de Atrogin-1; enquanto o TFA não produziu efeitos significantes. Dessa forma, os dados sugerem a participação do SUP na atrofia desencadeada pela IC na HS e, que o TFA previne a atrofia por reduzir a expressão/atividade de alguns componentes do SUP; e, que no IM, o aumento da expressão de Atrogin-1 precedeu a perda de massa muscular / Skeletal muscle atrophy is associated with increased protein degradation in systemic diseases, which seems to be mainly related to ubiquitin-proteasome system (UPS). However, little is known about UPS contribution to the heart failure-induced muscle atrophy (HF-MA). Likewise, aerobic exercise training (AET) has been established as an adjuvant therapy for HF and molecular mechanisms underlying HF-MA has not been clarified yet. The objectives of the study were: 1) to verify UPS contribution for HF-MA in 2 experimental models: sympathetic hyperactivity-induced HF (α2A/α2CARKO) in mice, and myocardial infarction model (MI) in rats and 2) AET effects on HF-MA and UPS. In α2A/α2C ARKO mice, we observed activation of UPS characterized by increased mRNA levels of E3 ligases Atrogin-1 and E3-a, deubiquitinating enzyme USP28, increased levels of ubiquitinated proteins and chymotrypsin-like proteasome activity. AET prevented HF-MA in the α2A/α2C ARKO by reducing of UPS activity. In MI model, rats displayed cardiac dysfunction and exercise intolerance with no signs of atrophy. However, Atrogin-1 mRNA and protein levels were increased. Therefore, alterations in Atrogin-1expression might precede atrophy and HF in this model. In conclusion, our data provide evidence for skeletal muscle anti-atrophic effect upon AET in α2A/α2C ARKO that is related, at least in part, to a reduced UPS

Orientador: Maria Cristina Cintra Gomes Marcondes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T06:14:31Z (GMT). No. of bitstreams: 1 Ventrucci_Gislaine_D.pdf: 735812 bytes, checksum: 9ccc59181ffe68918015bc1467e83a50 (MD5) Previous issue date: 2005 / Resumo: Caquexia, presente na maioria dos hospedeiros com câncer, é um estado caracterizado pela perda involuntária de peso. Pacientes com caquexia apresentam expectativa de vida muito reduzida e menor qualidade de vida. Nos pacientes com câncer-caquexia há intensa mobilização de substratos dos tecidos da carcaça do organismo hospedeiro ocorrendo, preferencialmente, depleção de proteína muscular em função da redução da síntese e/ou aumento da degradação protéica no músculo. Este aumento da taxa de proteólise muscular tem como função prover aminoácidos para síntese de glutamina, bem como para a demanda de síntese das células neoplásicas. Trabalhos da literatura mostram que o desenvolvimento de câncer dá-se de forma mais agressiva e severa quanto mais jovem for o paciente. Pacientes acometidos pelo crescimento de neoplasia maligna concomitante à gravidez sofrem da mesma agressividade desta doença, com um agravante maior tratam se de dois pacientes: mãe e feto. Neste trabalho analisamos os efeitos de uma dieta rica em leucina sobre o metabolismo protéico em animais jovens prenhes portadores ou não do carcinossarcoma de Walker 256. Ratas Wistar foram distribuídas em grupos experimentais de acordo com a inoculação ou não do carcinossarcoma de Walker 256 e submetidas ou não a dieta rica em leucina. Após 20 dias de experimento foi realizado ensaios com o músculo esquelético (gastrocnêmio) a fim de elucidar o mecanismo de catabolismo tecidual que ocorre durante o processo de câncer-caquexia. Os grupos apresentaram aumento da taxa de proteólise muscular e redução da taxa de síntese protéica muscular. Porém, o grupo inoculado com tumor e tratado com dieta rica em leucina apresentou aumento da síntese protéica e menor espoliação de proteína muscular quando comparado com o grupo inoculado com tumor e não tratado com leucina na dieta. A suplementação de leucina na dieta, uma vez que este aminoácido é utilizado como fonte energética pelo músculo esquelético pode prevenir a depleção da carcaça, preservar a massa protéica corpórea e impedir o estado caquético do animal / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Regulation of gene expression occurs at several levels in a cell. While control of transcription is often viewed as the main source of regulation, it is now clear that post-transcriptional processes are essential to fine-tune protein availability. The presence of AU-rich elements (ARE) in the 3’ untranslated region (3’UTR) of many important mRNAs exemplifies one such process. AREs alter the mRNA translation or degradation status by recruiting ARE-binding proteins (ARE-BP). ARE-BPs of the TTP/TIS11 family bind to their cognate ARE-RNAs using their conserved tandem zinc-finger domain and induce rapid decay of their targets. This allows for proper regulation of cell proliferation, cell death and inflammation. In this regard, TTP/TIS11 are main regulators of gene expression, and as such are put under strict transcriptional, post-transcriptional as well as several layers of post-translational control.<p>In this work, we aimed at elucidating the degradation mechanisms affecting TTP/TIS11. Using Drosophila as a model, we found that dTIS11 protein turnover is rapid due to continuous degradation by the proteasome. However, proteasomal recognition did not require ubiquitination of dTIS11 as non-ubiquitinable mutants were efficiently degraded by the proteasome. In addition, dTIS11 was digested by the 20S proteasome that lacks ubiquitin-recognition domains. Our results further indicate that intrinsically disordered regions (IDR) in dTIS11 may be responsible for proteasomal recognition. In fact, dTIS11 is predicted as disordered and possesses the main characteristics of intrinsically disordered proteins (IDP). We also identified dTIS11 N- and C-terminal domains as functional signals for degradation, potentially due to their destructuration. This ubiquitination-independent, disorder-dependent degradation process is conserved throughout evolution as dTIS11 mammalian counterpart, TTP, undergoes the same degradation by default pathway. In addition, we established that phosphorylation prevents degradation of TTP/TIS11 by the proteasome. <p>Together, our results pinpoint a new essential characteristic for TTP/TIS11 that may redefine the identity of these proteins. In addition, we unraveled a novel and conserved mechanism of regulation of TTP/TIS11. This control is essential for cell physiology as defects in this process can lead to defects in the inflammatory response, increased radiation-induced lung toxicity and tumorigenesis.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished