L'autophagie est un mécanisme de dégradation lysosomale qui joue un rôle important dans l'immunité innée et adaptative. La relation entre le Virus de l'Immunodéficience Humaine de type 1 (VIH-1) et l'autophagie est complexe. En effet, l'équipe a montré que l'enveloppe virale du VIH-1 (Env), exprimée à la surface des cellules infectées, induit une autophagie massive dans les cellules T CD4 bystanders non infectées. Au contraire, lorsque ces cellules s'infectent de façon productive, l'autophagie est inhibée, suggérant qu'une ou plusieurs protéines virales soient capables de bloquer ce processus. L'objectif de ce travail de thèse a été de rechercher ces protéines virales et leur mécanisme d'action. Un crible double hybride en levure a permis de mettre en évidence que plusieurs protéines du VIH-1 sont capables d'interagir avec des protéines autophagiques (Atg), et plus spécifiquement que la protéine virale Vif (Virion infectivity factor) interagit directement avec la protéine LC3, protéine essentielle au processus autophagique. Cette interaction a été confirmée en système in vitro et in vivo (GST pull-down et immunoprécipitation). Plusieurs mutants des protéines Vif et LC3 ont été réalisés pour déterminer les domaines de liaison. La partie C-terminale de Vif ainsi que la glycine C-terminale de LC3, responsable de la conjugaison au phosphatidylethanolamine (PE), semblent être les domaines impliqués dans cette interaction. Une des principales fonctions de Vif connues est de dégrader, via le protéasome, les facteurs cellulaires APOBEC (Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like) et en particulier la protéine APOBEC3G (A3G). Les résultats montrent que Vif est impliquée dans le blocage de l'autophagie induite par l'enveloppe indépendamment de son action sur A3G. / Autophagy is a lysosomal degradation pathway involved in the innate and adaptative immunity. The relationship between the Human Immunodeficiency Virus type 1 (HIV-1) and autophagy is complex. The team has demonstrated that autophagy is induced in uninfected CD4 T cells after contact with infected cells expressing HIV-1 envelope glycoproteins (Env), leading to apoptosis. In contrast, when these cells are productively infected, autophagy is repressed, suggesting that one or several viral proteins are able to block this process. The aim of the thesis was to search these viral proteins and to determine their mechanism of action. A two-hybrid screen has revealed that several HIV-1 viral proteins are able to interact with autophagic proteins (Atg). In particular, Vif (Virion infectivity factor) interacts directly with LC3, a protein involved in the formation of autophagosomes. This interaction has been confirmed in vitro and in vivo (GST pull-down and immunoprecipitation). Several mutants of Vif and LC3 have been done to analyze the binding domains. The C-terminal part of Vif and the C-terminal glycine of LC3, responsible for the conjugation to PE, seem to be involved in the interaction between Vif and LC3.Vif plays an important role during HIV-1 infection. One of its main functions is to degrade, by the ubiquitin-proteasome system, the antiviral factors called APOBECs (Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like) and in particular APOBEC3G (A3G). Our results demonstrate that Vif is involved in the blockade of Env-mediated autophagy independently of its role on A3G.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-10-29T13:31:11Z No. of bitstreams: 1 Beatriz Rocha Simone Dias Vacúolos....pdf: 43303992 bytes, checksum: 310fce5b6b7557207067a51be3ca3c64 (MD5) / Made available in DSpace on 2014-10-29T13:31:11Z (GMT). No. of bitstreams: 1 Beatriz Rocha Simone Dias Vacúolos....pdf: 43303992 bytes, checksum: 310fce5b6b7557207067a51be3ca3c64 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A Leishmania é um parasito intracelular obrigatório que vive e se multiplic adentro dos vacúolos parasitóforos em macrófagos no hospedeiro vertebrado. Apesar dos vacúolos induzidos por diferentes espécies de Leishmania apresentarem semelhanças bioquímicas, esses compartimentos apresentam diferenças significativas nos seus tamanhos. Os vacúolos parasitóforos induzidos por Leishmania mexicana e Leishmania amazonensis apresentam grandes dimensões e contêm uma grande quantidade de amastigotas, enquanto que os induzidos por Leishmania major e Leishmania donovani são pequenos e com pouco espaço ao redor das amastigotas. Estudos recentes demonstraram que compartimentos induzidos por microrganismos intracelulares são capazes de interagir com a via autofágica e esta pode controlar ou promover o estabelecimento da infecção a depender da natureza do microrganismo. Até o momento, poucos estudos foram realizados para avaliar o papel da autofagia na biogênese e maturação dos vacúolos parasitóforos induzidos por Leishmania. Recentemente, foi demonstrado que em macrófagos de camundongos BALB/c, a indução de autofagia provoca um aumento na carga parasitária de L. amazonensis, no entanto, não é capaz de aumentar a carga parasitária de L. major. Além disso, estudos indicam que vacúolos parasitóforos de L. mexicana adquirem macromoléculas do citoplasma da célula hospedeira por meio de microautofagia. Uma vez que L. amazonensis integra o mesmo complexo que L. mexicana, nossa hipótese é que vacúolos parasitóforos induzidos por L. amazonensis interagem com a via autofágica.Assim, o presente estudo tem como bjetivo verificar e comparar a participação da autofagia na infecção por L. amazonensis ou L. major em macrófagos murinos. Para este fim, avaliamos quanto a características autofágicas, os vacúolos parasitóforos induzidos por L. amazonensis ou L. major em macrófagos de camundongo CBA e analisamos a influência da superexpressão de LC3 sobre a sobrevivência de L. amazonensis ou L. major em macrófagos infectados. Inicialmente, macrófagos de camundongos CBA foram infectados com L. amazonensis ou L. major e incubados com ysoTracker, marcador de compartimentos lisossomais, ou DQ-BSA, marcador de compartimentos degradativos. Além disso, foi avaliada a presença de LAMP, proteína lisossomal, e LC3, proteína específica de autofagossomo, na membrana destes vacúolos. Em seguida, a co- localização dos parasitos com os vacúolos parasitóforos contendo estes marcadores foi quantificada. Nossos resultados demostraram um maior percentual de co-localização tanto do LysoTracker como doDQ-BSAcom parasitos em vacúolos no interior de macrófagos infectados com L. major em comparação com aqueles infectados com L. amazonensis. No entanto, não houve diferença no percentual de co-localização de LAMP com L. major ou L. amazonensis e foi observado um maior percentual de co-localização do LC3 com parasitos em macrófagos infectados com L. amazonensis em comparação com aqueles infectados com L. major. Posteriormente, avaliamos o efeito da superexpressão da LC3 na infecção por L. amazonensis ou L. major. Células de linhagem macrofágica RAW foram transfectadas com o plasmídeo contendo a sequência codificante para a LC3 e infectadas com L. amazonensis ou L. major. Nós observamos uma reduçãono percentual de infecção por L. amazonensis e L. major nas células RAW- pmRFP-LC3 em comparação às controle. Essa diminuição na infecção se deu por inibição da fagocitose de L. amazonensis e L. major pois os parasitos continuam a interagir com a membrana das células RAW-pmRFP-LC3, mas não são internalizadas. Em conjunto, estes dados demonstram que os vacúolos parasitóforos de L. amazonensis e L. major interagem com compartimentos da via autofágica de forma distinta e que a superexpressão de LC3 reduz a fagocitose de L. amazonensis e L. major por células RAW, o que resulta na redução da infecção / Leishmania is an intracellular parasite that lives and multiplies within parasitophorous vacuoles in macrophages in the vertebrate host. Despite the fact that vacuoles induced by different species of Leishmania present biochemical similarities, these compartments have significant differences in their sizes and composition. The parasitophorous vacuoles induced by Leishmania mexicana and Leishmania amazonensis are large and contain a large number of amastigotes, while vacuoles induced by Leishmania major and Leishmania donovani are small and tight. Recent studies have demonstrated that depending on the type of intracellular microorganism, the induced compartments can interact with the autophagic pathway and control or promote the establishment of infection. To date, few studies have been conducted to evaluate the role autophagic process plays in the biogenesis and maturation of parasitophorous vacuoles induced by Leishmania. Recently, it has been demonstrated that in macrophages of BALB/c mice, the induction of autophagic causes an increase in parasitic load of L. amazonensis, but not L. major. Furthermore, other studies indicate that L. mexicana-induced parasitophorous vacuoles acquire macromolecules from the cytoplasm of the host cell through microautophagy. Once L. amazonensis belongs to the same complex that L. mexicana, our hypothesis is that L. amazonensis-induced parasitophorous vacuoles interact with the autophagic pathway. Thus, the present study aims to evaluate and compare the role autophagic process plays in Leishmania infection. We evaluated L. amazonensis- or L. major-induced parasitophorous vacuoles regarding their autophagic characteristics and we analyzed the influence of the overexpression of LC3 on the survival of parasites in infected macrophages. Initially, macrophages of CBA mice were infected with L. amazonensis or L. major and incubated with a marker of lysosomal compartments, LysoTracker, or a marker of degrading compartments, DQ-BSA. In addition, we evaluated the presence of the lysosomal membrane protein, LAMP-1, and a protein specific of autophagossomes, LC3 in the membrane of these vacuoles. Then, the colocalization of parasites with the marker labeled-compartments was quantified. Our results demonstrated a higher percentage of colocalization of both LysoTracker and DQ-BSA with parasites in vacuoles within macrophages infected with L. major in comparison with those infected with L. amazonensis. However, there was no difference in the percentage of colocalization of LAMP with L. major or L. amazonensis. We also observed a higher percentage of LC3-co-localizing with parasites in macrophages infected with L. amazonensis in comparison with those infected with L. major. Subsequently, we evaluated the effect of overexpression of LC3 in macrophages infected with L. amazonensis or L. major. RAW cells were transfected with the plasmid containing the coding sequence for the LC3 (RAW-pmRFP-LC3) and then were infected with L. amazonensis or L. major stationary phase promastigotas. A reduction was observed in the percentage of infected RAW-pmRFP-LC3 cells with L. amazonensis and L. major compared to control cells. This decrease in the percentage of infected cells is due to the inhibition of phagocytic ability of RAW-pmRFP-LC3 cells, since the parasites continue to interact with cell membrane, but is not internalized. Together, these findings show that L. amazonensis- and L. major-induced parasitophorous vacuoles interact differently with compartments of the autophagic pathway and that the overexpression of LC3 reduces phagocytosis of both L. amazonensis and L. major by RAW-pmRFP-LC3 cells resulting in the reduction of infection.

La macroautophagie est un processus cellulaire qui permet la dégradation et le recyclage de constituants cytoplasmiques par formation de vésicules à double membrane, les autophagosomes qui fusionnent ensuite avec les lysosomes. Ce processus intervient dans divers processus physiologiques tels que le développement, la longévité, la mort cellulaire et dans des pathologies humaines comme des cancers ou maladies neurodégénératives. Mes travaux de thèse ont révélé l’existence de rôles séquentiels et spécifiques des protéines autophagiques, LGG-1 et LGG-2, homologues d’Atg8/LC3 chez le nématode Caenorhabditis elegans. Cette étude a été réalisée dans l’embryon précoce sur une population particulière d’autophagosomes responsables d’un processus physiologique stéréotypé : la dégradation des mitochondries paternelles au moment de la fécondation. Nous avons montré que LGG-1 est recruté au niveau des autophagosomes précoces et permet le recrutement de LGG-2 qui intervient plus tardivement dans le processus autophagique pour permettre la fusion des autophagosomes avec les lysosomes. De plus, la fonction de LGG-1 peut être complémentée par son homologue humain témoignant de l’intérêt du système modèle C. elegans pour l’analyse des homologues d’Atg8.Par ailleurs, des études récentes ont démontré que la protéine autophagique LC3 était recrutée au cours de la phagocytose des corps apoptotiques. Ce processus a été appelé LAP pour LC3-associated phagocytosis. Par des approches génétiques et cellulaires, utilisant la microscopie optique et électronique, j’ai montré qu’il existait une implication différente de protéines autophagiques LGG-1 et LGG-2 dans la dégradation des corps apoptotiques chez C. elegans. La protéine LGG-2, spécifiquement, joue un rôle dans la cellule phagocytaire afin de dégrader le corps apoptotique. Ces travaux suggèrent également une implication de l’autophagie dans le corps apoptotique pour permettre la phagocytose. / Macroautophagy is a major ubiquitous catabolic process which allows the bulk degradation and recycling of cytoplasmic constituents by formation of double membrane vesicles called autophagosomes which then fuse with lysosomes. This process is involved in a large variety of physiological processes such as development, anti-aging, cell death and in human pathologies like cancers or neurodegenerative diseases. My thesis work revealed the existence of sequential and specific roles of autophagic proteins LGG-1 and LGG-2, homologs of Atg8/LC3 in Caenorhabditis elegans. In this study, we focused on a particular population of autophagosomes involved in a physiological process in early embryos: the degradation of paternal mitochondria during fertilization. We showed that LGG-1 is recruited at the early autophagosomes and allows LGG -2 recruitment which acts later in the autophagic process to allow the fusion of autophagosomes with lysosomes. Moreover, the function of LGG -1 can be complemented with its human homologs revealing the interest of the C. elegans model system for analyzing Atg8 homologs.Furthermore, recent studies have identified the recruitment of autophagic proteins during phagocytosis of apoptotic cells in the so called LC3-associated phagocytosis (LAP). By genetic and cellular approaches, using optical and electron microscopy, I showed that there is a different involvement of autophagic proteins, LGG-1 and LGG-2 in the degradation of apoptotic cells in C. elegans. LGG-2 protein, specifically, plays a role in phagocytic cell to degrade apoptotic corpses. Moreover, this work suggest a function of autophagy in the apoptotic corpses to allow phagocytosis.

Huntington's disease, an autosomal dominant neurodegenerative disease, affects the cell in several toxical ways. One of them is accumulation of protein aggregates in cytoplasma, which could become a serious problem especially for long-lived cells such as neurons. Autophagy (macroautophagy) is an important catabolic pathway, crucial for cell survival. If fully functional, it should eliminate protein aggregates and reduce the toxic effect on the cell. However, recent works show that this pathway might be defective, most probably in the cytoplasmic cargo recognition. In my work I used a transgenic miniature pig model of Huntington's disease to verify the hypothesis of autophagical dysfunction in individuals suffering from Huntington's disease. I studied levels of autophagosomal markers - LC3 and p62 in mesenchymal stem cells after different autophagy stimulation treatments, and ammonium chloride was found the most effective. In addition I evaluated the effect of age of the animals on autophagic function, but no significant changes were identified, even if animal genotype was considered. Moreover I had an opportunity to study proteins levels in three porcine brain tissues - cortex, cerebellum and striatum. Even though there is no significant diference, we can observe a trend of LC3 II and p62 increase in...

Deregulation of autophagy plays an important role in the pathogenesis of diseases such as cancer, neuronal degenerative or cardiovascular disease. Autophagy is a process to engulf the cytoplasmic contents into autophagosome and deliver them for lysosomal degradation. Its major function is to clear unfolded protein or damage organelles for maintaining proper metabolic homeostasis and normal cell physiological activities. Autophagy and multivesicular bodies, MVBs, cooperate to regulate the turnover of intracellular macromolecule, defective organelles and signaling receptor. Endosomal sorting complex required for transport, ESCRT, is important for the formation of MVBs, which regulates membrane receptor recycling, protein sorting and vesicular trafficking. Tumor Susceptibility Gene 101(TSG101) is a member of ESCRT-I that plays an important role on MVBs formation and maintaining ESCRT function. Previous report indicated that autophagosome accumulation upon deprivation of TSG101, implying possible role of TSG101 during autophagic process. In this study, we observed the increase of TSG101 and autophagic marker proteins, such as LC3-II and ATG upon nutrient starvation. Furthermore, knockdown TSG101 in cervical carcinoma HeLa cell resulted in the elevation of LC3-II, ATG3 and ubiquitinated protein aggregates marker protein p62, which is congruous to other reports. However, in neuroblastoma SH-SY5Y cell, transfection of siRNA led to the decrease of LC-II and ubiquitinated protein level. These results indicated that TSG101 might be critical for autophagy and the maintenance of steady-state level of cellular ubiquitinated proteins. Ectopic upregulatory expression of HA-TSG101 led to the increase of LC3-II in both cell type. The elevation of ATG3 level is also observed in HeLa cell. Therefore, we speculated that TSG101 might be important for the formation of autophagosome, but our data did not exclude the possible role of TSG101 in regulation of the fusion of autophagosome and lysosome, because the increase of ATG3 indicated ectopic HA-TSG101 might facilitate the execution of autophagic flow. In addition, we have established GFP-LC3 expression cell lines. Our imaging data showed the colocalization of TSG101 and GFP-LC3 in both cytoplasm and nucleus that might be an interesting research topic for investigation the role of TSG101 in autophagic pathway.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

No description available.

A desnutrição proteica (DP) pode ocasionar alterações na matriz extracelular (MEC) de diferentes órgãos e tecidos, inclusive o hematopoético, com comprometimento funcional. Estudos do nosso laboratório demonstraram, em modelo murino de DP, aumento da expressão proteica de fibronectina (FN) no estroma medular ósseo in vivo, principalmente na região subendosteal (local de fixação da célula tronco progenitora hemopoética). Já in vitro, no estroma medular ósseo, observou-se tanto o aumento quanto a diminuição de FN e a presença de suas isoformas. Essas alterações de FN parecem estar envolvidas com a hipoplasia da medula óssea (MO) em camundongos desnutridos. As modificações quantitativas de FN podem ser devidas: (i) à ação das metaloproteinases de matriz (MMP) responsáveis pela degradação das proteínas da MEC; (ii) aos inibidores de metaloproteinases (TIMP) que regulam a degradação da MEC; (iii) às alterações transcricionais, reguladas pela via de AKT/mTOR, que controla os splicing alternativos na FN, resultando em isoformas dessa proteína; (iv) a processos pós-transcricionais modulados por LC3, que aumenta a tradução do RNAm de FN. Assim, o objetivo deste estudo foi elucidar os mecanismos que alteram o turnover de FN no estroma medular ósseo em modelo murino de DP. Utilizamos camundongos, C57BL/6J machos, adultos, separados em dois grupos: controle e desnutrido, alimentados, ad libitum, com ração contendo 12% e 2% de proteína, respectivamente. Após cinco semanas de indução à desnutrição os camundongos foram eutanasiados, e coletado o material biológico. Avaliamos: o estado nutricional, o hematológico, a histologia da MO femoral bem como a determinação imunohistoquímica da FN, MMP-2 e MMP-9, determinação da expressão de FN e suas isoformas em células totais da MO, o estabelecimento do estroma medular ósseo in vitro, por 28 e 35 dias de cultivo. A partir das culturas foram avaliadas a expressão de RNAm de FN e suas isoformas, MMP-2, MMP-9, TIMP-1, TIMP-2, AKT, mTOR e LC3α e β, quantificação de MMP-2, MMP-9, TIMP-1, TIMP-2,TNFα, TGFβ e IL-1β e determinação de LC3β e proteínas da via de AKT/mTOR. Não observamos alterações na expressão do RNAm de FN e suas isoformas ex vivo e in vitro, mas um aumento da deposição de FN na MO.Também não observamos modificações na imunolocalização de MMP-2 e MMP-9 na MO e na atividade dessas proteínas no sobrenadante de culturas de células estromais in vitro, mas houve aumento da expressão do RNAm de MMP-9 em 28 dias de cultivo. Não detectamos alterações na expressão de RNAm e na concentração de TIMP-1 e TIMP-2 no sobrenadante das culturas. Houve redução significativa de TNFα e TGFβ no sobrenadante das culturas de 28 dias. Observamos aumento da expressão do RNAm de mTOR em culturas de 28 dias e LC3α e LC3β em 35 dias de células estromais. Encontramos menor fosforilação de PI3K, AKT, PTEN, mTOR e mTOR total e aumento de LC3β em culturas de 28 dias, mas redução de LC3β em 35 dias. Em função dos dados inferimos que a DP conduz a alterações da FN que não estão relacionadas à ação de MMPs e TIMPs e sim a modificações de LC3β e da via de AKT/mTOR. / Protein malnutrition (PM) can lead changes in extracellular matrix (ECM) from several organs and tissues, including hematopoietic, with functional impairments. Research from our laboratory demonstrated, in a murine model of protein malnutrition, increase in proteic expression of fibronectin (FN) in vivo bone marrow stroma, principally in subendosteal region (attachment site of hematopoietic stem/progenitor cell - HSPC). It was observed as both an increase and a decrease in the presence of FN and its isoforms in vitro bone marrow stroma. These FN changes seem to be related to bone marrow (BM) hypoplasia in malnourished mice. Quantitative FN changes may be due to: (i) action of matrix metalloproteinases (MMP) responsible for ECM proteins degradation; (ii) tissue inhibitors of metalloproteinases (TIMP) that regulate ECM degradation; (iii) transicional changes regulated by AKT/mTOR pathway, which controls alternative splicing in FN, resulting in isoforms from this protein; (iv) post-transcriptional processes modulated by LC3 that increases FN mRNA translation. Therefore, the aim of this study was to elucidade the mechanisms that changes the FN turnover in bone marrow stroma in a murine model of PM. C57BL/6J, adult and male mice were used and divided into two groups: control and malnourished, fed ad libitum with ration containing 12% and 2% of protein, respectively. After five weeks of induction malnutrition, mice were euthanized and the biological material was collected. We evaluated: nutritional and hematologic status, the femoral BM histology, immunohistochemistry determination of FN, MMP-2 and MMP-9, the FN and its isoforms expression determination in total BM cells, establishment of in vitro bone marrow stroma for 28 and 35 days of culture. From the cultures were evaluated FN mRNA expressions and its isoforms, MMP-2, MMP-9, TIMP-1, TIMP-2, AKT, mTOR, LC3α and β, quantification of MMP-2, MMP-9, TIMP-1, TIMP-2,TNFα, TGFβ and IL-1β and determination of LC3β and AKT/mTOR proteins. No changes were observed, ex vivo and in vitro, in the expression of FN mRNA and its isoforms, but there was a FN deposition increase in BM. We did not observe modifications in MMP-2 e MMP-9 immunolocalization in BM and in these proteins activity in the supernatant of in vitro stromal cell culture, but there was an increase in MMP-9 mRNA expression after 28 days of culture. We did not detect changes in mRNA and in TIMP-1 and TIMP-2 expressions in the supernatant of cultures. There was significant reduction of TNFα and TGFβ in the cultures supernatant of 28 days. We observed an increase of mTOR RNAm in 28 days cultures and also LC3α and LC3β in stromal cells with 35 days. We found lower phosphorylation of PI3K, AKT, PTEN, mTOR e total mTOR and an LC3β increase in 28 days cultures, yet an LC3β reduction in 35 days. According to the data we conclude that PM leads to FN changes that are not related to MMPs and TIMPs actions, but the LC3β and AKT/mTOR pathway modifications.

A desnutrição proteica (DP) pode ocasionar alterações na matriz extracelular (MEC) de diferentes órgãos e tecidos, inclusive o hematopoético, com comprometimento funcional. Estudos do nosso laboratório demonstraram, em modelo murino de DP, aumento da expressão proteica de fibronectina (FN) no estroma medular ósseo in vivo, principalmente na região subendosteal (local de fixação da célula tronco progenitora hemopoética). Já in vitro, no estroma medular ósseo, observou-se tanto o aumento quanto a diminuição de FN e a presença de suas isoformas. Essas alterações de FN parecem estar envolvidas com a hipoplasia da medula óssea (MO) em camundongos desnutridos. As modificações quantitativas de FN podem ser devidas: (i) à ação das metaloproteinases de matriz (MMP) responsáveis pela degradação das proteínas da MEC; (ii) aos inibidores de metaloproteinases (TIMP) que regulam a degradação da MEC; (iii) às alterações transcricionais, reguladas pela via de AKT/mTOR, que controla os splicing alternativos na FN, resultando em isoformas dessa proteína; (iv) a processos pós-transcricionais modulados por LC3, que aumenta a tradução do RNAm de FN. Assim, o objetivo deste estudo foi elucidar os mecanismos que alteram o turnover de FN no estroma medular ósseo em modelo murino de DP. Utilizamos camundongos, C57BL/6J machos, adultos, separados em dois grupos: controle e desnutrido, alimentados, ad libitum, com ração contendo 12% e 2% de proteína, respectivamente. Após cinco semanas de indução à desnutrição os camundongos foram eutanasiados, e coletado o material biológico. Avaliamos: o estado nutricional, o hematológico, a histologia da MO femoral bem como a determinação imunohistoquímica da FN, MMP-2 e MMP-9, determinação da expressão de FN e suas isoformas em células totais da MO, o estabelecimento do estroma medular ósseo in vitro, por 28 e 35 dias de cultivo. A partir das culturas foram avaliadas a expressão de RNAm de FN e suas isoformas, MMP-2, MMP-9, TIMP-1, TIMP-2, AKT, mTOR e LC3α e β, quantificação de MMP-2, MMP-9, TIMP-1, TIMP-2,TNFα, TGFβ e IL-1β e determinação de LC3β e proteínas da via de AKT/mTOR. Não observamos alterações na expressão do RNAm de FN e suas isoformas ex vivo e in vitro, mas um aumento da deposição de FN na MO.Também não observamos modificações na imunolocalização de MMP-2 e MMP-9 na MO e na atividade dessas proteínas no sobrenadante de culturas de células estromais in vitro, mas houve aumento da expressão do RNAm de MMP-9 em 28 dias de cultivo. Não detectamos alterações na expressão de RNAm e na concentração de TIMP-1 e TIMP-2 no sobrenadante das culturas. Houve redução significativa de TNFα e TGFβ no sobrenadante das culturas de 28 dias. Observamos aumento da expressão do RNAm de mTOR em culturas de 28 dias e LC3α e LC3β em 35 dias de células estromais. Encontramos menor fosforilação de PI3K, AKT, PTEN, mTOR e mTOR total e aumento de LC3β em culturas de 28 dias, mas redução de LC3β em 35 dias. Em função dos dados inferimos que a DP conduz a alterações da FN que não estão relacionadas à ação de MMPs e TIMPs e sim a modificações de LC3β e da via de AKT/mTOR. / Protein malnutrition (PM) can lead changes in extracellular matrix (ECM) from several organs and tissues, including hematopoietic, with functional impairments. Research from our laboratory demonstrated, in a murine model of protein malnutrition, increase in proteic expression of fibronectin (FN) in vivo bone marrow stroma, principally in subendosteal region (attachment site of hematopoietic stem/progenitor cell - HSPC). It was observed as both an increase and a decrease in the presence of FN and its isoforms in vitro bone marrow stroma. These FN changes seem to be related to bone marrow (BM) hypoplasia in malnourished mice. Quantitative FN changes may be due to: (i) action of matrix metalloproteinases (MMP) responsible for ECM proteins degradation; (ii) tissue inhibitors of metalloproteinases (TIMP) that regulate ECM degradation; (iii) transicional changes regulated by AKT/mTOR pathway, which controls alternative splicing in FN, resulting in isoforms from this protein; (iv) post-transcriptional processes modulated by LC3 that increases FN mRNA translation. Therefore, the aim of this study was to elucidade the mechanisms that changes the FN turnover in bone marrow stroma in a murine model of PM. C57BL/6J, adult and male mice were used and divided into two groups: control and malnourished, fed ad libitum with ration containing 12% and 2% of protein, respectively. After five weeks of induction malnutrition, mice were euthanized and the biological material was collected. We evaluated: nutritional and hematologic status, the femoral BM histology, immunohistochemistry determination of FN, MMP-2 and MMP-9, the FN and its isoforms expression determination in total BM cells, establishment of in vitro bone marrow stroma for 28 and 35 days of culture. From the cultures were evaluated FN mRNA expressions and its isoforms, MMP-2, MMP-9, TIMP-1, TIMP-2, AKT, mTOR, LC3α and β, quantification of MMP-2, MMP-9, TIMP-1, TIMP-2,TNFα, TGFβ and IL-1β and determination of LC3β and AKT/mTOR proteins. No changes were observed, ex vivo and in vitro, in the expression of FN mRNA and its isoforms, but there was a FN deposition increase in BM. We did not observe modifications in MMP-2 e MMP-9 immunolocalization in BM and in these proteins activity in the supernatant of in vitro stromal cell culture, but there was an increase in MMP-9 mRNA expression after 28 days of culture. We did not detect changes in mRNA and in TIMP-1 and TIMP-2 expressions in the supernatant of cultures. There was significant reduction of TNFα and TGFβ in the cultures supernatant of 28 days. We observed an increase of mTOR RNAm in 28 days cultures and also LC3α and LC3β in stromal cells with 35 days. We found lower phosphorylation of PI3K, AKT, PTEN, mTOR e total mTOR and an LC3β increase in 28 days cultures, yet an LC3β reduction in 35 days. According to the data we conclude that PM leads to FN changes that are not related to MMPs and TIMPs actions, but the LC3β and AKT/mTOR pathway modifications.

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