Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ceilândia, Pós-Graduação em Ciências e Tecnologias em Saúde, 2016. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2016-09-08T14:50:10Z No. of bitstreams: 1 2016_MarcusAuréliodaCostaTavaresSabino.pdf: 1902510 bytes, checksum: 9746d9bbcc04914de08edc18633f28c0 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-11-08T11:12:37Z (GMT) No. of bitstreams: 1 2016_MarcusAuréliodaCostaTavaresSabino.pdf: 1902510 bytes, checksum: 9746d9bbcc04914de08edc18633f28c0 (MD5) / Made available in DSpace on 2016-11-08T11:12:37Z (GMT). No. of bitstreams: 1 2016_MarcusAuréliodaCostaTavaresSabino.pdf: 1902510 bytes, checksum: 9746d9bbcc04914de08edc18633f28c0 (MD5) / Nos animais sensíveis à privação de O2, é observada a injúria tecidual durante a hipóxia e o reoxigenação, tendo como causa principal o estresse oxidativo gerado por radicais livres derivados do oxigênio. Uma das estratégias encontradas por uma ampla variedade de animais à escassez de oxigênio é responder a este insulto com aumento das defesas antioxidantes, e assim evitar o dano oxidativo causado pelos radicais livres, fenômeno nomeado de preparo para o estresse oxidativo (POS). Nossa pergunta é saber se o fenômeno bioquímico do POS acontece na natureza, em especial, no ambiente entre marés, no qual mudanças cíclicas de retirada e oferta de oxigênio acontecem durante a baixa e alta das marés. Nestas condições de baixa e alta da maré em uma praia de Penha (Santa Catarina, Brasil), foi estudado o metabolismo redox envolvendo glutationa (GSH), em análise de corpo inteiro nos mexilhões da espécie Mytilaster solisianus, em três diferentes coletas (chamadas de I, II e III). Os animais mostraram suportar o esperado desequilíbrio redox durante as situações de exposição aérea (de até quatro horas na coleta I e II, e até 9 horas na coleta III) e reimersão no campo, pois os parâmetros antioxidantes endógenos relacionados ao metabolismo de GSH estiveram inalterados durante o ciclo das marés (i.e. Eq-GSH, GSH, GSSG e GSSG/Eq-GSH). Todavia na coleta III, houve uma queda dos níveis totais de Eq-GSH após duas horas de exposição aérea em relação ao grupo de animais pré-imersos. Na coleta III houve uma forte variação da temperatura do ar ao longo da exposição aérea (de 19 para 28° C), apesar disso, o indicador de desequilibrio redox não mudou (razão GSSG/Eq-GSH), mostrando que estes animais têm alto grau de adaptabilidade neste ambiente, no qual os fatores abióticos estão em constante mudança. _________________________________________________________________________________________________ ABSTRACT / In animals sensible to oxygen privation is seen injury in their tissues during hypoxia and reoxygenation, and the main cause is the oxidative stress generated by oxygen-derived free radicals. One of the strategies found by a widespread variety of animals to survive oxygen lack is respond with increase of antioxidant defenses, and thus avoiding the oxidative damage caused by free radicals, a phenomenon named preparation for oxidative stress (POS). Our question address if biochemical phenomenon of POS happens in nature, in special, in the intertidal environment, where changes cyclical of lack and supply oxygen happens during low and high tide. In these conditions of low and high tide in beach of Penha (Santa Catarina, Brazil), was studied redox metabolism of glutathione (GSH), in body whole analysis in the mussel specie Mytilaster solisianus in three differents expeditions (named of I, II e III). The animals shown endure to the expected redox imbalance during aerial exposure (until four hours in expedition I e II, and until 9 hours in expedition III) and reimmersion in field, because the endogenous antioxidant parameters related to the metabolism of the glutathione remained unchanged during tidal cycle (i.e GSH-eq, GSH, GSSG and GSSG/GSH-eq ratio). However in expedition III, there was a drop in glutathione pool (GSH-eq) after two hours of aerial exposure when compared to group of animals pre-emersion. In expedition III there was a strength variation in air temperature (19 to 28° C) during aerial exposure, despite this, the index of redox imbalance remained unchanged (GSSG/GSH-eq) in expedition III, showing that mussels have high level of adaptability in this environment, in which abiotic factors are in continuous changes.

Dissertação (mestrado)—Universidade de Brasília, Programa de Pós-Graduação em Biologia Molecular, 2014. / Submitted by Marília Freitas (marilia@bce.unb.br) on 2015-10-29T12:33:32Z No. of bitstreams: 1 2014_NestorFabianLeytonCastro.pdf: 3484643 bytes, checksum: 3f92622240d0f7e59822aca3974ed379 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-02-12T21:00:47Z (GMT) No. of bitstreams: 1 2014_NestorFabianLeytonCastro.pdf: 3484643 bytes, checksum: 3f92622240d0f7e59822aca3974ed379 (MD5) / Made available in DSpace on 2016-02-12T21:00:47Z (GMT). No. of bitstreams: 1 2014_NestorFabianLeytonCastro.pdf: 3484643 bytes, checksum: 3f92622240d0f7e59822aca3974ed379 (MD5) / A hipóxia ocorre quando as células são expostas a baixas tensões (< 5%) de oxigênio. Isto afeta o metabolismo, a expressão de genes, a secreção e resposta a hormônios e pode garantir a sobrevivência ou algumas vezes iniciar uma cascata apoptótica. O fator 1 induzível por hipóxia (HIF-1) humano regula a transcrição de inúmeros genes envolvidos na resposta à hipóxia e tem importante papel em processos fisiológicos e patológicos. Ele é um heterodímero formado pelas subunidades HIF-1α e HIF-1β, também chamada de transportador nuclear de arilhidrocarbono (ARNT). Em condições de normóxia, o HIF-1 é hidroxilado pelas prolilhidroxilases (PHDs) e marcado para degradação pelo proteasoma. Porém, quando as células se encontram sob hipóxia, as PHDs são inibidas e assim ocorre o acúmulo do HIF-1. Alterações no estado redox podem ter efeito na ativação do HIF-1 e foi verificado que condições de estresse oxidativo podem levar à ativação deste fator. O estresse oxidativo ocorre quando as moléculas antioxidantes e os mecanismos de defesa celular são incapazes de proteger a célula dos efeitos das espécies reativas de oxigênio (EROs) as quais atacam moléculas biológicas vitais, tais como lipídeos, DNA dentre outras. As EROs incluem os radicais livres derivados de oxigênio, entre essas o íon superóxido (O2●-), o radical hidroxila (●OH-), e outras moléculas que embora não possuam elétrons livres são muito reativas devido à sua instabilidade, como o peróxido de hidrogênio (H2O2). Para estudar os efeitos do HIF-1 sob condições de estresse oxidativo, escolhemos a levedura Saccharomyces cerevisiae, também conhecida como levedura de padeiro. A utilização da levedura S. cerevisiae tem provado ser muito útil como um modelo de sistema in vivo para estudar a ação de fármacos, a expressão de proteínas heterólogas e a função básica de fatores de transcrição humanos. Foram usadas três linhagens mutantes, EG103αβ a qual expressa as duas subunidades do fator de transcrição HIF-1, EG103αΔβ que expressa a subunidade beta truncada e a linhagem EG103M2 que possue o vetor vazio. O objetivo deste estudo foi avaliar a resposta ao estresse oxidativo dessas linhagens de leveduras para aprofundar o conhecimento sobre a regulação da atividade de HIF-1. O crescimento das linhagens foi investigado e os resultados mostraram que a linhagem EG103αβ teve um crescimento menor que as outras duas linhagens (EG103αΔβ e EG103M2) em normóxia. Quando tratada com várias concentrações de H2O2 o crescimento da linhagem EG103αβ diferiu do controle não tratado somente nas concentrações de 1 e 2 mM, quando houve uma diminuição do crescimento. Essa resposta foi similar àquela das outras duas linhagens. Então não foi uma resposta específica da linhagem EG103αβ, indicando que a expressão do HIF-1 não alterou a sensibilidade da resposta ao H2O2. Ao tratar as células com alguns agentes oxidantes como Menadiona (MD), hidroperóxido de cumene (CHP) e terc-butilhidroperóxido (t-BHP) os resultados mostraram que a linhagem EG103αβ apresentava uma susceptibilidade menor à MD na concentração 50 μM, em comparação com as outras linhagens. O uso do inibidor da catalase ATZ (10 mM) e H2O2 0,5 mM mostrou que a susceptibilidade da linhagem EG103αβ aumentou em relacão ao controle tratado somente com H2O2. Nas duas outras linhagens o aumento da susceptibilidade ocorreu com uma concentração menor de ATZ (2 mM) e H2O2 0,5 mM. Isto indica que talvez a linhagem EG103αβ tenha maiores concentrações de catalase em relação às outras duas linhagens. O uso do inibidor da síntese de glutationa, butionina-sulfoximina (BSO) não mostrou uma resposta diferenciada da linhagem EG103αΔβ à exposição ao H2O2. O uso do quelante de ferro, deferoxamina (DFO) também mostrou uma ação de proteção contra o dano oxidativo na linhagem EG103αβ, indicando um papel da ausência de ferro na proteção pelo HIF-1. Medidas dos níveis de glutationa (GSH-eq) e da enzima superóxido dismutase devem ser repetidas para conclusão definitiva de seu papel na linhagem EG103αβ. Em conclusão, os resultados indicam que a expressão das subunidades que compõem o HIF-1 humano em leveduras reduz seu crescimento, aumenta sua resistência à menadiona e talvez os níveis de catalase e faz com que um quelante de ferro tenha papel protetor nestas células. ______________________________________________________________________________________________ ABSTRACT / Hypoxia occurs when cells are exposed to low oxygen tensions (<5%). It affects the metabolism, gene expression, secretion and response to hormones and can ensure the survival or sometimes initiate an apoptotic cascade. The human hypoxia-inducible factor 1 (HIF-1) regulates the transcription of numerous genes involved in the response to hypoxia and plays an important role in physiological and pathological processes. HIF-1 is a heterodimer formed by the HIF-1α and HIF-1β subunits, the latter also called arylhydrocarbone nuclear transporter (ARNT). In normoxic conditions, HIF-1 is hydroxylated by prolylhidroxilases (PHDs) and marked for degradation by the proteasome. However, when cells are under hypoxia, the PHDs are inhibited and HIF-1 accumulates. Changes in the redox state may have an effect on HIF-1 activation and oxidative stress conditions have been to lead to the activation of this factor. Oxidative stress occurs when the antioxidant molecules and cellular defense mechanisms are unable to protect the cell from the effects of reactive oxygen species (ROS) which attack vital biological molecules such as lipids, DNA and others. ROS include free radicals derived from oxygen, among these the superoxide ion (O2●-), o hydroxyl radical (●OH-), and other molecules that although not having free electrons are very reactive due to its instability, such as hydrogen peroxide (H2O2). To study the effects of HIF-1 under conditions of oxidative stress, we chose the yeast Saccharomyces cerevisiae, also known as Baker's yeast. The use of S. cerevisiae has proven to be very useful as an in vivo model system to study the action of drugs, the expression of heterologous proteins and the basic function of human transcription factors. The three mutant strains, EG103αβ which expresses the two subunits of HIF-1 transcription factor, EG103αΔβ, expressing the truncated beta subunit and EG103M2 strain that possesses an empty vector were used. The objective of this study was to evaluate the oxidative stress response of the yeast strains to increase knowledge about the regulation of HIF-1 activity. The growth of the strains was investigated and the results showed that the strain EG103αβ had a slower growth than the other two strains (EG103αΔβ and EG103M2) under normoxia. When treated with various concentrations of H2O2 the growth of EG103αβ differed from the untreated control only at concentrations of 1 and 2 mM, when there was a decline in growth. This response was similar to that of the other two strains. So it was not a specific response of EG103αβ, indicating that HIF-1 expression did not change the sensitivity response to H2O2. By treating the cells with certain oxidizing agents such as Menadione (MD), cumene hydroperoxide (CHP) and tert-butylhydroperoxide (t-BHP) the results showed that the strain EG103αβ has lower susceptibility to 50 uM MD concentrations, compared to the other strains. The use of the catalase inhibitor ATZ (10 mM) and 0.5 mM H2O2 showed that the susceptibility of EG103αβ increased compared to the control treatment only with H2O2. In the other two strains increased susceptibility was observed with a smaller concentration of ATZ (2 mM) and 0.5 mM H2O2. This indicates that EG103αβ has higher catalase levels in comparison to the other two strains. The use of the inhibitor of glutathione synthesis, buthionine sulfoximine (BSO) showed that EG103αΔβ responded similarly to the other two strains. The use of the iron chelator, deferoxamine (DFO) showed a protective effect against oxidative damage in EG103αβ, indicating a role of iron absence in the protection by HIF-1. Measures of glutathione levels (GSH-eq) and superoxide dismutase should be repeated for definitive conclusion of their role in the EG103αβ lineage. In conclusion, the results indicate that the expression of the subunits that make up the human HIF-1 reduces yeast growth, increases its resistance to menadione and perhaps levels of catalase and makes an iron chelator a protective agent in these cells.

Les traitements des cancers des VADS reposent sur la chirurgie, la radiothérapie, et la chimiothérapie. Malgré ces traitements, la survie globale des patients à 5 ans est de l’ordre de 50%. Les causes d’échec thérapeutique sont dues au profil de résistance des tumeurs. Le ciblage de l’axe EGFR/mTOR/HIF-1 par une combinaison de rapalogues et d’anti-EGFR a montré son efficacité sur certaines tumeurs solides. L’objet de ce travail de thèse a été de caractériser l’impact d’une combinaison de drogues à faibles doses sur des lignées cellulaires des VADS au moyen d’une approche pharmacologique et moléculaires. Nos résultats montrent que la combinaison de la rapamycine (5nM) au cetuximab (2,5μg/ml) diminue la survie clonogénique des cellules et permet une inhibition du facteur de transcription HIF-1α. Cette combinaison de drogue améliore l’efficacité de la radiothérapie. En revanche, l’induction de HIF-2a induite par le traitement provoque la résistance des cellules aux traitements par, et une rechute rapide des tumeurs in vivo. L’inhibition de HIF-2 permet une inhibition de la survie cellulaire d’environ 100% dans un modèle résistant. / Management of HNSCC relies on surgery, radiotherapy, and chemotherapy. Despite these treatments, the 5 years overall survival of patient is lower than 50%. Main causes of therapeutic failure are due to the profile of resistance of tumors. The efficacy of a combination rapalogues and anti-EGFR therapies in targeting the EGFR/mTOR/HIF-1 axis in solid tumors was shown previously. In this PhD work, we have evaluated the impact of a low-dose drug combination on head and neck cancer cells lines with a pharmacological and molecular approach. We show that the combination of rapamycine (5nM) and cetuximab (2,5μg/ml) efficiently inhibits the HIF-1 transcription factor and impairs cell clonogenic survival. The efficacy of radiation therapy is improved by this drug combination. However, cell resistance to the treatment is acquired via the induction of HIF-2 in our resistant model cell line. This induction is associated with more tumor relapse in tumors mice xenograft. The inhibition of HIF-2 achieves a dramatic drop of cell clonogenic survival to < 1%.

Positionnée en aval des voies PI3K/AKT et RAS/MAPK, la protéine kinase mTOR joue un rôle déterminant dans le développement et la progression tumorale des cancers colorectaux où elle est fortement surexprimée. Par ailleurs, les cancers colorectaux comme toutes les tumeurs solides, ont un microenvironnement hypoxique. L’adaptation des cellules tumorales à l’hypoxie est notamment régulée par la voie PI3K/AKT/mTOR ainsi que par les facteurs de transcription HIFs dont l’expression protéique et l’activité transcriptionnelle est en partie régulée par mTOR. Dans cette étude, nous avons montré que l’inhibition verticale et complète de l’axe PI3K/AKT/mTOR/HIF-1α par l’utilisation combinée d’irinotecan à faible dose et d’inhibiteurs catalytiques de mTOR inhibe significativement la prolifération cellulaire de lignées coliques humaines, la croissance tumorale et le développement de métastases de xénogreffes de tumeurs coliques dérivées de patients.En parallèle, une étude de cohorte de tumeurs coliques humaines de stade III par Tissue Micro Array montre que les facteurs HIFs sont fortement exprimés dans l’épithélium et le stroma de cancers du côlon de stade III, qu’une faible expression nucléaire de HIF-1α dans les cellules épithéliales confère une mauvaise survie aux patients et qu’elle a une valeur prédictive de moins bonne réponse au traitement 5-FU. / Downstream of the PI3K/AKT and RAS/MAPK pathways, mTOR protein kinase plays a decisive role in the development and tumor progression of colorectal cancers. Furthermore, the microenvironment of colorectal cancers is hypoxic. The adaptation of the tumor cells to hypoxia is regulated by the PI3K/AKT /mTOR pathway as well as by the HIFs transcription factors whose protein expression and transcriptional activity is partially regulated by mTOR. In this study, we showed that the vertical and complete inhibition of the PI3K / AKT / mTOR /HIF-1α axis by the combined use of low-dose irinotecan and mTOR catalytic inhibitors significantly inhibits human colon cancer cell proliferation, as well as the growth and metastatic development of xenografted human colon tumors. In parallel, a Tissue Micro Array study on a cohort of stage III human colic tumors shows that the HIFs are strongly expressed in the epithelium and stroma of the tumors and a low nuclear expression of HIF-1α in epithelial cells provides with poor survival to patients and has a predictive value of worse response to 5-FU treatment.

The tumor-selective viral replication capacity and pro-apoptotic effects of oncolytic reovirus have been reported to be dependent on the presence of an activated RAS pathway in several solid tumor types. However, the mechanisms of selective anticancer efficacy of the reovirus-based formulation for cancer therapy (Reolysin, pelareorep) have not been rigorously studied in soft tissue sarcomas (STS). Here we report that Reolysin triggered a striking induction of the anti-angiogenic chemokine interferon-gamma-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) in both wild type and RAS mutant STS cells. Further analysis determined that Reolysin treatment possessed significant anti-angiogenic activity irrespective of RAS status. In addition to CXCL10 induction, Reolysin dramatically downregulated the expression of hypoxia inducible factor (HIF)-1 alpha, HIF-2 alpha and inhibited vascular endothelial growth factor (VEGF) secretion. CXCL10 antagonism significantly diminished the anti-angiogenic effects of Reolysin indicating that it is a key driver of this phenomenon. Xenograft studies demonstrated that Reolysin significantly improved the anticancer activity of the anti-angiogenic agents sunitinib, temsirolimus, and bevacizumab in a manner that was associated with increased CXCL10 levels. This effect was most pronounced following treatment with Reolysin in combination with temsirolimus. Further analysis in additional sarcoma xenograft models confirmed the significant increase in CXCL10 and increased anticancer activity of this combination. Our collective results demonstrate that Reolysin possesses CXCL10-driven anti-angiogenic activity in sarcoma models, which can be harnessed to enhance the anticancer activity of temsirolimus and other agents that target the tumor vasculature.

L’hypoxie tumorale est l’une des principales causes de l’agressivité des glioblastomes (GB). Plusieurs études attestent de l’implication de l’isoforme HIF-1α (hypoxia inducible factor-1α) dans la progression de ces tumeurs et dans leur résistance à la radiothérapie (RT). Plus récemment, il a été établi que l’isoforme HIF-2α régule la capacité tumorigénique des cellules souches de GB (CSG). Cependant, le rôle de ce facteur dans la croissance des cellules de GB différenciées et leur réponse à la RT est moins documenté. Dans ce contexte, l’objectif de ce travail a été de renforcer ces connaissances à l’échelle préclinique en utilisant deux approches d’ARN interférence (ARNi) pour moduler l’expression de HIF-2 : cibler directement HIF-2α ou cibler HAF (hypoxia associated factor), un facteur impliqué dans le switch de HIF-1α vers HIF-2α. Les résultats obtenus sur un modèle orthotopique de cellules humaines de GB (U251-MG) différenciées montrent que l’invalidation de HAF conduit à un fort ralentissement de la croissance de ces tumeurs mais indépendamment de HIF-1α ou HIF-2α. L’effet de l’invalidation de HIF-2α serait, quant à lui, dépendant de l’environnement tumoral. En effet, la diminution d’expression de HIF-2α dans les cellules U251 ne modifie pas la croissance tumorale dans un modèle de greffe sous-cutanée, alors que celle-ci favorise la croissance tumorale lorsque les cellules de GB sont implantées en intracérébral. Par comparaison aux tumeurs contrôles, ces tumeurs sont plus invasives et mieux perfusées. In vitro, l’inhibition de l’expression de HIF-2α n’a aucun effet sur la survie des cellules U251 alors qu’elle diminue la mort apoptotique de ces cellules exposées aux rayons X.L’ensemble des données présentées dans cette étude suggère que HAF et HIF-2α pourraient réguler la capacité tumorigénique des cellules de GB différenciées, tout comme observé pour les CSG. En outre, ces résultats soulignent la nécessité de prendre en compte le microenvironnement cellulaire afin de mieux comprendre le comportement de la tumeur dans son environnement hypoxique. / Hypoxia is one of the main causes of glioblastoma (GB) aggressiveness. Various studies attest on the involvement of the HIF-1α isoform (hypoxia inducible factor-1α) in the progression of these tumors and in their resistance to radiation therapy (RT). More recently, it was established that the HIF-2α isoform regulates the tumorigenic capacity of GB stem cells (GSC). However, the role of this factor in the growth of differentiated GB cells and their response to RT is less documented. In this context, the goal of this work was to strengthen this knowledge at the preclinical level by using two RNA interference (RNAi) strategies to modulate the expression of HIF-2: one directly targets HIF-2α, the other one targets HAF (hypoxia associated factor), a factor involved in the switch of HIF-1α to HIF-2α. Our results obtained on an orthotopic model of differentiated human GB (U251-MG) cells showed that the invalidation of HAF leads to a strong slowdown in tumor growth but independently of HIF-1α or HIF-2α. On the other hand, the effect of HIF-2α silencing seems dependent on the tumor environment. Indeed, the extinction ofHIF-2α expression in U251 cells does not modify tumor growth in a subcutaneous model, whereas it promotes tumor growth when GB cells are intracerebrally grafted. Compared to control tumors, these tumors are more invasive and highly perfused. In vitro, the inhibition of HIF-2α expression has no effect on GB cell survival whereas decreasing the X-rays induced apoptotic death.Collectively, these data suggest that HAF and HIF-2α could regulate the tumorigenic capacity of differentiated GB cells, like it does in CSGs. In addition, these results highlight the need to take into account the cellular microenvironment to better understand the behavior of the tumor in its hypoxic environment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20974号 / 医博第4320号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 増永 慎一郎, 教授 高田 穣, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hypoxia-inducible factor-1 (HIF-1) est un facteur de transcription hétérodimérique ubiquitaire responsable de l’activation de nombreux gènes essentiels à l’adaptation des cellules suite à une diminution de la disponibilité en oxygène. En raison de l’induction du facteur de croissance de l’endothélium vasculaire (VEGF), une puissante molécule pro-angiogénique, HIF-1 joue un rôle particulièrement important au niveau des cellules vasculaires et dans la formation de nouveaux vaisseaux sanguins. Dernièrement, plusieurs études ont clairement démontré que la sphingosine-1-phosphate (S1P) est également un facteur pro-angiogénique majeur. Relâché dans le sérum principalement par les plaquettes activées, ce phospholipide bioactif vital lie et stimule des récepteurs spécifiques des cellules endothéliales (ECs) et musculaires lisses vasculaires (VSMCs), engendrant ainsi une variété de réponses cellulaires cruciales et essentielles dans la régulation du système vasculaire dont la prolifération, la migration et la survie. D’autres études ont clairement démontré que des stimuli non-hypoxiques peuvent aussi mener à l’activation de HIF-1 en conditions normales d’oxygénation. Puisque HIF-1 et S1P jouent un rôle central au niveau de l’angiogenèse et de la biologie des cellules vasculaires et qu’ils sont tout deux impliqués dans la pathogénèse de maladies comme l’athérosclérose et le cancer, cette thèse visait à déterminer le rôle potentiel de la S1P dans l’induction et l’activation de HIF-1 au niveau vasculaire et à identifier les mécanismes moléculaires conduisant à cette activation. Brièvement, nous montrons que le traitement des ECs et des VSMCs avec la S1P induit fortement l’expression de la protéine HIF-1αla sous-unité active de HIF-1. Le complexe nucléaire ainsi formé est actif transcriptionnellement et se lie spécifiquement à la séquence promotrice de ses gènes cibles. Nous démontrons également que la stabilisation protéique, indépendante de pVHL (protéine von Hippel-Lindau), est le mécanisme principal à l’origine de cette induction et ce, suite à l’activation spécifique du récepteur S1P2. Finalement, l’expression de gènes dépendants de HIF-1, apportée par la S1P, est fortement diminuée suite à l’utilisation d’ARN interférants ciblant la protéine HIF-1α. Nous croyons que les résultats de ces travaux, qui identifient S1P comme étant un nouvel et puissant activateur de HIF-1, auront un impact certain sur différents aspects de la biologie vasculaire. / Hypoxia-inducible factor-1 (HIF-1) is a ubiquitous heterodimeric transcription factor responsible for the activation of many genes essential for adaptation to low oxygen conditions. Among these genes, HIF-1 strongly induces vascular endothelial growth factor (VEGF), a potent angiogenic molecule. Therefore, HIF-1 plays a crucial role in vascular cell biology and the formation of new blood vessels. Recent studies have clearly shown that sphingosine-1-phosphate (S1P) is also a key player in the angiogenic process. Released into circulation mainly upon platelet activation, this bioactive phospholipid binds to and activates specific receptors located on vascular endothelial (ECs) and smooth muscle cells (VSMCs). This leads to the stimulation of a wide range of essential vascular cell responses like proliferation, migration and survival, which are crucial in the regulation of the vascular system. Other studies have shown that non-hypoxic stimuli can also activate HIF-1 in oxygenated conditions. Since S1P and HIF-1 are both important regulators of vascular cell biology and especially angiogenesis and that they are also both implicated in the pathogenesis of different diseases like atherosclerosis and cancer, the goal of the present thesis was to determine whether S1P can modulate the vascular induction and activation of HIF-1 and to identify the molecular mechanisms underlying this activation. Briefly, we show that treatment of ECs and VSMCs leads to a strong induction of HIF-1α protein levels through the specific activation of the S1P type-2 receptor in a time and dose-dependant manner. We also demonstrate that the S1P-dependant HIF-1 nuclear complex formation, achieved through pVHL-independent (protein von Hippel-Lindau) stabilization of HIF-1α, is transcriptionally active and specifically binds to hypoxia-responsive elements. Moreover, S1P activates the expression of genes known to be closely regulated by HIF-1 and this induction could be blocked by the use of RNA interference oligonucleotides targeting HIF-1α protein. Thus, this work identifies S1P as a novel and potent non-hypoxic activator of HIF-1. We believe that understanding the role played by HIF-1 in S1P gene regulation will have a strong impact on different aspects of vascular biology.

Les facteurs de transcription induits par l’hypoxie (HIF) sont responsables de la transcription de nombreux gènes impliqués dans la réponse à l’hypoxie. En plus de réguler de nombreux processus cellulaires et physiologiques, ces facteurs sont impliqués dans plusieurs pathologies. Hétérodimères constitués d’une sous-unité β constitutive et d’une sous-unité α sensible à l’oxygène, ces facteurs sont majoritairement régulés par l’hydroxylation et la dégradation de la sous-unité α. En situation d’hypoxie, ce mécanisme de dégradation est inhibé, ce qui favorise la formation de complexes HIF. Les travaux présentés dans cette thèse visent à élucider les mécanismes régulant l’activation de HIF en situation d’hypoxie ou de normoxie. Dans la section Résultats, vous retrouverez une section consacrée à l’activation de HIF par l’angiotensine II (AngII) chez les cellules musculaires lisses vasculaires. Plus précisément, le rôle de la transactivation de récepteurs à activité tyrosine kinase suivi de l’implication de HIF dans la biologie de ces cellules seront abordés. Dans un deuxième temps, un inhibiteur des métalloprotéases, le BiPS, vous sera présenté comme étant un puissant inducteur des protéines HIF-α. En effet, le BiPS est un puissant inhibiteur des enzymes responsables de la dégradation des protéines HIF-α. En outre, le BiPS permet l’activation des complexes HIF ainsi formés. Ces résultats inattendus pourraient avoir des répercussions importantes dans l’utilisation de cet agent à des fins angiostatiques dans le traitement du cancer en plus de présenter un nouvel agent ayant un potentiel thérapeutique important dans le traitement de pathologies ischémiques. Finalement, vous retrouverez une section consacrée à l’étude d’un nouveau répresseur de HIF, l’histone acétyltransférase HBO1. De façon étonnante, HBO1 réprime l’activité des complexes HIF par un mécanisme indépendant de la stabilisation des sous-unités α mais dépendant du remodelage de la chromatine. En conclusion, ces résultats mettent en lumière de nouveaux mécanismes de régulation de l’activité des facteurs de transcription HIF. Considérant les rôles physiologiques importants de ces complexes ainsi que leurs implications dans diverses maladies, ces résultats permettront d’accroître les connaissances disponibles quant aux fonctions de ces complexes et mèneront vers le développement d’outils thérapeutiques efficaces. / Hypoxia-inducible transcription factors (HIF) are decisive elements in the transcriptional regulation of numerous genes expressed in conditions of hypoxic stress. In addition to their roles in many physiological and cellular processes, HIF are also involved in diverse pathological situations. Obligate heterodimers composed of a constitutive β subunit and of an oxygen tension-regulated α subunit, these transcription factors are mainly regulated by the hydroxylation and subsequent degradation of the α subunit. In hypoxia, this degradation mechanism is inhibited, resulting in HIF complex formation and binding to specific DNA sequences. The work presented in this thesis aims to elucidate regulatory mechanisms involved in HIF activation during hypoxia or in normal oxygen conditions. In the Results section, you will find a study devoted to HIF activation by angiotensin II (Ang II) in vascular smooth muscle cells. Specifically, the role of receptor tyrosine kinase transactivation on HIF activation was evaluated along with a description of HIF-1’s role in smooth muscle cells biology. Next, an inhibitor of matrix metalloproteases, BiPS, will be presented as a novel and potent HIF activator. This unexpected effect may have important implications for the use of this compound for its angiostatic potential in cancer treatment. In addition, BiPS and derivative molecules could also have strong therapeutic potential in ischemic diseases. Finally, you will find a section devoted to the study of a new transcriptional repressor of HIF complexes, the histone acetyltransferase bound to ORC-1, HBO1. Surprisingly, HBO1 represses the activity of HIF complexes by a mechanism independent of the availability of the α subunits, but dependent on a chromatin remodelling event. In conclusion, this thesis highlights new regulatory mechanisms responsible for HIF activation. Considering the important physiological roles of HIF complexes and their implications in the pathogenesis of different diseases, these studies increase the available knowledge concerning the biological functions of these complexes and could contribute to the development of more effective and safe therapeutic tools.

A tuberculose (Tb) é uma doença infecciosa crônica ocasionada pelo bacilo Mycobacterium tuberculosis (Mtb). No espaço alveolar, o bacilo entra em contato com células do sistema imune inato como as células dendríticas (DCs), assim como células epiteliais (AEC). Na Tb, o fator de transcrição HIF-1? (Fator Induzido por Hipóxia 1 alfa) se encontra elevado em macrófagos infectados e células epiteliais alveolares adjacentes. Por isso, o objetivo deste trabalho foi investigar o papel de HIF-1? na resposta pró- inflamatória de AEC do tipo II (AEC-II) e na modulação da função de DCs em contato com AEC-II durante a infecção por Mtb. Células MLE-15 foram infectadas com Mtb H37Rv (MOI10) para avaliação da permissividade à infecção (microscopia eletrônica), crescimento dos bacilos (CFU) e ativação através da análise de citocinas (ELISA), nitrito (ensaio de Griess), TLR2, TLR9 e HIF-1? (qPCR e/ou Western blotting). A modulação DCs derivadas da medula óssea (BMDCs) por AEC-II, foi analisada de forma direta (contato) ou indireta (meio condicionado - CM) de MLE-15 não infectadas (CM - NIC) ou infectadas (CM - IC). Foram determinadas citocinas (ELISA), nitrito (Ensaio de Griess), expressão de HIF-1?, enzimas glicolíticas, moléculas co-estimuladoras e CCR7 (citometria de fluxo). Ensaio de migração foi realizando em câmera de boyden. A proliferação de células T CD4+ naive em co-cultura com BMDCs e a manutenção da Th17 foram avaliadas por citometria de fluxo. Eventualmente, BMDCs foram previamente infectadas com Mtb (MOI2) e, em seguida, estimuladas com CM-NIC ou CM-IC. Células MLE-15 mostraram-se permissivas à infecção, não conseguindo controlar o crescimento dos bacilos. A infecção induziu aumento da produção de IL-6, NO2-,CCL5, S100A9 e IFN- ?. Apesar do acúmulo inicial de HIF-1?, a expressão gênica caiu com o passar do tempo. A expressão gênica de TLR2 e TLR9 também estava aumentada. A regulação positiva10 de HIF-1? em células epiteliais induziu uma redução de IL-6 e NO, sem, no entanto, interferir significativamente no número de CFU. BMDCs estimuladas com CM - IC mostraram maior produção de IL-1?, IL-12, IL-6 e IL-10, maior expressão gênica de GLUT1 e HK2, além do acúmulo inicial de HIF-1?, que foi degradado em 24 horas, acompanhado de baixa expressão de iNOS. A expressão MHC-II, CD80, CD86 e CCR7 estava aumentada em BMDCs submetidas ao CM - IC, enquanto a indução do acúmulo de HIF-1? através do seu estabilizador, DMOG, foi capaz de reverter negativamente essa resposta. A maior maturação de BMDCs ocasionou maior proliferação de células TCD4+ naive, desfavorecendo a indução de células T CD4+ IFN?+. Entretanto, as citocinas produzidas favorecerem a manutenção de células TCD4+ produtoras de IL-17. No entanto, o fenótipo de maior maturação foi perdido em BMDCs infectadas estimuladas com CM - IC, aliado à baixa produção de TNF e alta produção de IL-10. Em conclusão, HIF-1? mostrou uma função anti-inflamatória, reduzindo a produção de moléculas pró- inflamatórias por AEC-II e regulando negativamente a maturação e a migração de DCs. Além disso, apesar de AEC-II infectadas por Mtb favorecerem a maturação e migração de DCs, o Mtb é capaz de subverter essa resposta / Tuberculosis (Tb) is a chronic infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). In the alveolar space, the bacillus contacts cells from the immune system, such as dendritic cells (DCs), as well as alveolar epithelial cells (AEC). In Tb, the transcription factor HIF-1? (Hypoxia-inducible factor 1- alpha) is accumulated in infected macrophages and adjacent alveolar epithelial cells. Therefore, the aim of this work was to investigate the role of HIF-1? in the proinflammatory response of type II AEC (AEC-II) and modulation of DCs function upon contact with AEC-II during Mtb infection. MLE-15 cells were infected with Mtb H37Rv (MOI10) to evaluate the permissiveness to infection (electron microscopy), killing ability (CFU) and cell activation through cytokine analysis (ELISA), nitrite (Griess assay), TLR2, TLR9 and HIF-1? (qPCR and / or Western blotting). Bone marrow derived DCs (BMDCs) modulation by AEC-II, were analyzed directly (contact) or indirectly (conditioned medium - CM) of uninfected (CM-NIC) or infected (CMIC) MLE-15. We determined cytokines (ELISA), nitrite (Griess Assay), HIF-1? expression, glycolytic enzymes, co-stimulatory molecules and CCR7 (flow cytometry). Chemotaxie assay was performed on Boyden camera. Proliferation of naive CD4 + T cells in co-culture with BMDCs and maintenance of Th17 were assessed by flow cytometry. Eventually, BMDCs were previously infected with Mtb (MOI2) and then stimulated with CM-NIC or CM-IC. MLE-15 cells were permissive to infection, failing to control the bacilli growth. Infection induced increased production of IL-6, NO2-, CCL5, S100A9 and IFN-?. Despite the initial accumulation of HIF-1?, gene expression dropped over time. The gene expression of TLR2 and TLR9 was also increased. Positive regulation of HIF-1? in epithelial cells induced a reduction of IL-6 and NO, but did not significantly interfere with the number of CFU. BMDCs stimulated with CM-IC showed higher production of IL-1?, IL-12, IL-6, and IL-10, greater GLUT1 and HK2 gene expression, in addition to the initial12 accumulation of HIF-1?, which was degraded within 24 hours, accompanied by low iNOS expression. The expression of MHC-II, CD80, CD86 and CCR7 was increased in BMDCs undergoing CM-IC, while the induction of HIF-1? accumulation through its stabilizer, DMOG, was able to negatively revert this response. The higher maturation of BMDCs resulted in a greater proliferation of naive CD4 + T cells, but hampered induction of CD4 + IFN? + T cells. However, cytokines produced favor the maintenance of IL-17 producing CD4 + cells. The phenotype of higher maturation was lost in Mtb-infected BMDC, accompanied by low TNF production and high IL-10 production. In conclusion, HIF-1? showed an anti-inflammatory function, reducing the production of proinflammatory molecules by AEC-II and negatively regulating the maturation and migration of DCs. In addition, although Mtb-infected AEC-II favor maturation and migration of DCs, Mtbinfection of DCs is capable of subverting this response