Le génome est sans cesse menacé dans sa structure par des stress génotoxiques d’origine endogène (stress oxydant, blocage de la réplication…) ou exogène (irradiations, produits chimiques, métaux lourds…). La voie de réponse aux dommages de l’ADN coordonne un réseau d’événements en cascades qui incluent les points de surveillance du cycle cellulaire, la réplication/réparation/recombinaison de l’ADN et la mort cellulaire programmée. Ces voies collaborent pour assurer la transmission fidèle du génome et empêcher la prolifération des cellules qui aurait accumulé des altérations génétiques. En général, des défauts dans une de ces voies entraînent un changement de sensibilité aux agents génotoxiques, une instabilité génétique et une prédisposition au cancer. La recombinaison homologue (RH) est une voie essentielle pour la réparation de l’ADN ; elle joue un rôle fondamental dans le maintien de la stabilité du génome. Le stress oxydant, résultant d’une augmentation de la concentration intracellulaire de ERO, est une des causes majeures de dommages aux lipides, aux protéines et à l’ADN et pour cela, il représente un défi pour la survie cellulaire et pour la stabilité du génome. Les ERO apparaissent physiologiquement lors de la respiration cellulaire ou résultent d’un stress environnemental, comme l’exposition aux radiations UV ou agents chimiques oxydants. Elles contribuent à certains processus comme la croissance cellulaire, l’activité et au repliement des protéines, la sénescence et la mort cellulaire programmée. Il est important de souligner qu’un état redox altéré est souvent associé à un fonctionnement anormal des cellules, comme il est observé pour les cellules cancéreuses et les cellules sénescentes. L’objectif de ce projet a été d’analyser l’interface entre les voies DDR et SAPK (Stress activated protein kinase) évolutivement conservées et d’en étudier les conséquences sur la stabilité du génome en utilisant comme organisme modèle la levure à fission. Nos résultats montrent que la voie SAPK joue un rôle sur la RH en promouvant la phosphorylation de Rad52, une protéine impliquée dans différentes sous-voies de la RH. Nous avons aussi montré que Rad52 est phosphorylée sur différents acides aminés, parmi lesquels certains sont les cibles de kinases inconnues qui n’ont aucun lien avec la voie SAPK. Nous avons observé que Rad52 est phosphorylée soit après un stress oxydant, soit dans des cellules génétiquement sujettes à des perturbations de la RH. Nous avons identifié deux sites de phosphorylation de la protéine Rad52, dont un seul est dépendant de la voie SAPK. En étudiant la phosphorylation de Rad52 dans les cellules invalidées pour la voie SAPK ou mutées pour un des sites de phosphorylation de Rad52, nous avons pu montrer que la RH est modulée par la voie SAPK même en absence de insulte externe. Notre travail ouvre le chemin vers une nouvelle compréhension des mécanismes fondamentaux du maintien de l’intégrité du génome. / Genomes are routinely submitted to injuries from either endogenous stress (oxidative stress, DNA replication block…) or from exogenous sources (radiations, chemicals, heavy metals…). The DNA damage response (DDR) coordinates a network of pathways including cell cycle checkpoints, DNA replication/repair/recombination, and programmed cell death, ensuring faithful genome transmission and preventing from the proliferation of cells bearing genetic alterations. Defect in one of these pathways generally results in altered sensitivity to genotoxins, genetic instability and cancer predisposition. Homologous recombination (HR) is an essential DNA repair pathway playing pivotal role to maintain genome stability.Oxidative stress, resulting from increased intracellular concentration of ROS, is one of the major causes of lipid, protein and DNA damage, and therefore a challenge for cell survival and genome stability. ROS are generated physiologically as by-products of cellular respiration, or as result of environmental stresses, such as exposure to solar UV radiations or to oxidant chemicals, and they actively participate in processes such as cellular growth, protein activity and folding, senescence and programmed cell death. It is noteworthy that an altered redox homeostasis is often associated to abnormally functioning cells, such as cancer and senescent cells. The aim of this project was to study the interface between two major evolutionarily conserved pathways, DDR and SAPK (Stress activated protein kinase) and the consequences on genome stability using fission yeast as a model organism. We report data showing that SAPK pathway impinges on HR by promoting phosphorylation of Rad52, a key protein involved in all sub-pathways of HR. We also revealed that Rad52 is phosphorylated at multiple different sites some of which are substrate for unidentified kinases unrelated to the SAPK pathway. Rad52 phosphorylation occurs either after oxidative stress or in cells genetically prone to HR perturbation. We identified two sites of phosphorylation, one of which is dependent on functional SAPK pathway. By studying Rad52 phosphorylation in cells mutated in the SAPK pathway or mutated at the Rad52 site of phosphorylation, we showed that HR is modulated by SAPK even in the absence of external insults. Our work pave the way to a novel understanding of fundamental mechanisms required for genome integrity maintenance.

Diante do quadro atual de previsões de mudanças climáticas, estudos a respeito das possíveis respostas dos organismos a estas alterações são importantes. Com a finalidade de prever e verificar se estas serão de fato deletérias ou se os organismos são capazes de lidar com elas sem alterações na sua fisiologia, e consequentemente na estrutura do ambiente, E. brasiliensis foi utilizada como modelo para estudar possíveis impactos do aumento da temperatura e acidificação dos oceanos na sua fisiologia. Para isso, espécimes foram expostos a 9 possíveis combinações de temperatura (24ºC, 28ºC e 30ºC) e pH (8.0, 7.7 e 7.3) em diferentes intervalos de tempo (1, 3, 12, 24 e 48 h). Amostras de gônadas e fluido celomático foram coletadas para avaliar a expressão das proteínas de estresse HSP70, AIF-1 e p38-MAPK, e a variação no número e viabilidade dos celomócitos. Nossos resultados mostram que o modelo é sensibilizado pelas mudanças no ambiente, através da hiper-regulação das proteínas de estresse. O cenário considerado mais extremo (30°C + pH7.3) ocasionou a morte de 100% dos organismos após 24horas. E o segundo cenário mais severo (30°C + pH7.7) desencadeou o desenvolvimento de ulceração de pele. Os efeitos são mais pronunciados nos celomócitos e a acidificação da água parece ter efeitos antagônicos com a temperatura nos celomócitos e sinérgicos nas gônadas. Embora a resposta tenha sido sistêmica, o grau e a dinâmica foram distintos em relação às diferentes amostras e estresses. Podendo causar modificações na resposta imune dos organismos e consequentemente na sobrevivência da espécie a longo prazo. / Under the current Climate Change context, studies about the potential responses of the organisms to their changing environment are of extreme importance. Recent studies point out the synergy of temperature and ocean acidification altogether. In this study, we used the sea star E. brasiliensis to assess the physiological effects of rising temperature, seawater acidification and the interaction of both factors. Independent individuals (N=225) were exposed to 9 possible combinations of temperature (24ºC, 28ºC and 30ºC) and pH (8.0, 7.7 and 7.3), for 1, 3, 12, 24 and 48 h. We compared the stress produced by these treatments measuring the expression of heat shock proteins (HSP70), the production of the allograft inflammatory factor (AIF−1) and the activation of mitogen kinases (MAPKs) at both gonad and celomic fluid. Furthermore, we assessed the quantity and quality of coelomocytes. Our results demonstrated that E. brasiliensis is vulnerable to the interaction of temperature and acidification. All the stress proteins evaluated were upregulated. The extreme scenario (30°C + pH7.3) caused the death of 100% of organisms after 24 hours, while the second most severe scenario (30°C + pH7.7) triggered skin ulceration. Nevertheless, we found that water acidification produces antagonistic effects to the temperature in coelomocytes and synergistic effects in gonad cells. Furthermore, these effects were more pronounced in the coelomocytes than in the gonads. The systemic response found in this study suggest that the interactive effects of elevated temperatures in conjunction with ocean acidification may endanger the survival of this species, and it could compromise the ecosystem functioning at long term.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Mercury (Hg) consists in a heavy metal with toxicological relevance whose contamination is mainly attributed to anthropogenic activities, leading to environmental contamination. It is known that an induction in oxidative stress caused by increased production of reactive oxygen species and/or decrease in antioxidant enzymes activity and a modulation of phosphorylation of proteins that participate in processes of cellular signaling transduction, as Mitogen-Activated Protein Kinases (MAPKs) represent molecular mechanisms implicated in Hg toxicity in rodents and other vertebrates. Studies from the literature have been pointing Drosophila melanogaster as an organism with considerable potential to be used as a bio indicator organism to monitoring the presence of environmental contaminants, presenting biochemical and behavioral effects in response to contaminants similarly to those observed in mammals. Thus, our results showed that HgCl2, when incorporated through the diet with sucrose in D. melanogaster, causes a significant decrease in viability of flies from 24h in a concentration dependent manner. Moreover, after 48h of exposure, the flies treated with Hg 100μM presented deficits in locomotor performance, decrease in the activity of enzymes: acetylcholinesterase (AChE), glutathione peroxidase (GPx) and Superoxide dismutase (SOD), without alteration in catalase activity (CAT). In this study, gene expression of D. melanogaster was evaluated by the method of Real-Time PCR. It was observed that mRNA levels of SOD, CAT and NF-E2-associated factor 2 (Nrf2) were not altered by Hg (II) treatment when compared to the control group, however, Hsp83 expression was10 times increased. Additionally, from 6h of treatment with Hg (II) 100 μM it was verified an induction of lipid peroxidation and oxidative stress, these effects persisted until 48 h of treatment. In the present study, the amount of Hg in the treated and control flies was measured and expressed in μg Hg / g tissue. It was observed that the flies treated for 48h with HgCl2 (100 μM) presented higher levels of Hg than the control group (100 times increase). With regard to the modulation of signaling pathways by Hg (II) treatment, the metal caused an increase in phosphorylation levels of ERK and JNK without altering phosphorylation of p38 as well as the total content of these proteins. Hg (II) exposure caused an induction of PARP cleavage, indicating an induction of apoptotic cell death. In conclusion, the data obtained over this work draw attention to the Hg (II) toxicity and help to extend the knowledge about the mechanisms involved in the toxicity of this metal in invertebrates. In addition, this study points the antioxidant system and modulation of specific signaling transduction pathways as common mechanisms mediating the toxicity of Hg (II) in vertebrates and invertebrates. / O Mercúrio (Hg) é em um metal pesado de relevância toxicológica cuja contaminação ambiental decorre principalmente de atividades antropogênicas. Dentre os mecanismos moleculares envolvidos na toxicidade do Hg estão um aumento na produção de espécies reativas de oxigênio, diminuição na atividade de enzimas antioxidantes e modulação da fosforilação de proteínas que participam de vias de transdução de sinal celular, entre estas as Proteínas Cinases Ativadas por Mitógenos (MAPKs). Estudos apontam Drosophila melanogaster como um organismo que apresenta um considerável potencial para atuar como um bioindicador de contaminantes ambientais, apresentando alterações bioquímicas e comportamentais similares a efeitos observados em mamíferos. Deste modo, nossos resultados demonstraram que o HgCl2, quando incorporado na dieta junto com sacarose em D. melanogaster, causa significativa diminuição da viabilidade das mesmas a partir de 24h de modo dependente de concentração utilizada do composto. Além disso, após 48h de exposição ao Hg(II) as moscas tratadas com concentrações a partir de 100μM obtiveram prejuízos no desempenho locomotor, diminuição na atividade das enzimas: Acetilcolinesterase (Ache), Glutationa Peroxidase (GPx) e Superóxido Dismutase (SOD), não alterando a atividade da Catalase (CAT). Na análise dos genes de D. melanogaster, através do método de Real time-PCR, observou-se que os níveis NF-E2-relacionados fator 2 (Nrf2) não foram alterados em comparação ao grupo controle, sendo que os níveis de HSP83 foram aumentados em 10 vezes. Além disso, a partir de 6h de tratamento as moscas demonstraram indução na peroxidação lipídica e estresse oxidativo efeito este que persistiu até as 48h de tratamento. Na medição dos níveis de Hg, expressa em μg de Hg/g de tecido, observou-se que as moscas tratadas por 48h com HgCl2 apresentaram 100 vezes maiores níveis de Hg em comparação ao grupo controle. Com relação à análise da modulação de MAPKs pelo tratamento com HgCl2 observou-se aumentada fosforilação de JNK e ERK, sem alterar a fosforilação de p38MAPK, bem como o conteúdo total destas proteínas. HgCl2 levou a uma elevação na clivagem da proteína PARP, indicativo da indução de morte celular do tipo apoptótica. Os dados obtidos neste trabalho ressaltam a toxicidade do Hg(II) e estendem o conhecimento sobre seus mecanismos de ação para um modelo animal de invertebrado, além disso, o presente estudo aponta o sistema antioxidante e vias de transdução de sinal celular como mecanismos comuns envolvidos na toxicidade deste metal em vertebrados e invertebrados..

Made available in DSpace on 2014-07-29T15:13:44Z (GMT). No. of bitstreams: 1 Tese2011_Luciana_Bradstetter.pdf: 2039765 bytes, checksum: fc789f9ab9e7cf36309b7d69f8c21304 (MD5) Previous issue date: 201-11-18 / Mechanic obstruction of blood vessels that irrigate the intestine leads to ischemia and intense reduction of oxygenation and tissue perfusion. If oxygenated blood flow returns abruptly to tissues before cellular death reperfusion injury occurs; this can be initiated by several mechanisms resulting in inflammatory response. Intestinal ischemia/reperfusion (I/R) injury has been widely associated to cell necrosis, although another distinct biochemical and morphological type of cell death, called apoptosis, is involved. Such conditions are mediated by signaling molecules in cellular surface, which lead to long term changes in gene expression. In the majority of cells, MAP (Mitogen Activated Protein) kinases (MAPK), which are part of a group of cytoplasmic enzymes, will transmit mitogenic and cell differentiation signs. Three MAPK that have been identified in mammalian cells participate in the signaling pathway: ERK, p38 and JNK. Activated p38 and JNK are usually related to apoptosis, while activated ERK 1/2 (P44/42) have a protection function inhibiting apoptosis. With the purpose of minimizing reperfusion injury after liver transplant, Carolina Rinse Solution (CRS) was designed by North Carolina University, USA. Although this solution has been used in horses to attenuate I/R injury in horse intestine, little is known about the eventual tissue protection mechanism. The present study aimed to investigate the effects of topical and intraluminal CRS in I/R injury, and the activation profile of p38 and ERK 1/2 MAPK, in rabbit jejunum. Fifteen New Zealand rabbits were allocated to three groups: Sham-operated (A), Ischemia and reperfusion (B) and CRS (C). Groups B and C were subjected to one hour of jejunal ischemia and two hours of reperfusion. In group C, ten minutes prior to reperfusion, the bowel lumen was filled with CRS, and the segment was immersed in CRS until reperfusion onset. Changes such as degeneration, necrosis, edema, hemorrhage, PMN infiltration and margination were not significantly different between groups B and C, showing that topical and intraluminal CRS did not attenuate deleterious effects of I/R in small intestinal of rabbits. I/R stimulated the phosphorylation of p44/42 MAPK and p38 MAPK pathways in some layers of jejunum. Progressive activation of p44/42 MAPK was chiefly localized to the crypts of Lieberkühn, circular and longitudinal muscle layers, whereas p38 MAPK was prominently activated in myenteric plexus and both muscle layers. All layers that did respond to I/R insult with activation increase of ERK 1/2 and p38, in all groups, showed low baseline phosphorylation levels as compared to those that did not react to the insult. The results of this work indicate that topical and intraluminal CRS does not interfere in ERK 1/2 and p38 activation profile in rabbit jejunum subjected to I/R. / A obstrução mecânica de vasos sanguíneos que irrigam o intestino leva à isquemia, com redução na oxigenação e perfusão teciduais. Se o fluxo sanguíneo oxigenado retorna aos tecidos bruscamente antes da morte celular, ocorre a chamada injúria de reperfusão, a qual pode ser iniciada por vários mecanismos, levando a uma resposta inflamatória. A injúria de isquemia e reperfusão (I/R) tem sido amplamente associada à necrose celular, apesar de um tipo bioquímico e morfológico distinto de morte celular, chamada de apoptose, também esteja envolvida. Tais processos são mediados por moléculas de sinalização na superfície das células, as quais levam a mudanças na expressão dos genes. Na maioria das células, as MAP (Mitogen Activated Protein) quinases (MAPK), as quais são parte de um grupo de enzimas citoplasmáticas, transmitem os sinais mitogênicos e de diferenciação. Três MAPK que foram identificadas em células de mamíferos participam do mecanismo de sinalização: ERK, p38 e JNK. As MAPK p38 e JNK ativadas normalmente estão associadas à apoptose, enquanto ERK 1/2 (P44/42) ativada tem função de inibição da apoptose. Com o propósito de minimizar a injúria de reperfusão após o transplante de fígado, a solução de Carolina Rinse (CRS) foi desenvolvida pela Universidade da Carolina do Norte nos EUA. Essa solução tem sido utilizada para minimizar os efeitos da I/R em intestino de eqüinos, mas pouco se sabe do seu possível mecanismo de ação protetora. O presente estudo objetivou investigar os efeitos do uso tópico e intraluminal da CRS na injúria de I/R e no perfil de ativação das MAPK p38 e ERK 1/2 no jejuno de coelhos. Quinze coelhos da raça Nova Zelândia foram alocados em três grupos: Instrumentado (A), Isquemia e reperfusão (B) e CRS (C). Os grupos B e C foram submetidos a uma hora de isquemia e duas horas de reperfusão de jejuno. No grupo C, dez minutos antes da reperfusão, o lumen do segmento foi preenchido com CRS e o mesmo foi imerso em CRS até o início da reperfusão. Mudanças como degeneração, necrose, edema, hemorragia, infiltrado e marginação de PMN não foram significativamente diferentes entre os grupos B e C, o que mostra que o uso tópico e intraluminal da CRS não atenuou os efeitos deletérios da I/R no intestino delgado de coelhos. A I/R estimulou a fosforilação das MAPK p44/42 e p38 em algumas camadas do jejuno. A ativação progressiva de p44/42 ocorreu principalmente nas criptas de Lieberkühn e nas camadas musculares circular e longitudinal, enquanto a MAPK p38 foi ativada principalmente no plexo mioentérico e em ambas as camadas musculares. Todas as camadas que responderam ao insulto da I/R com aumento da ativação de ERK 1/2 e p38, em todos os grupos, apresentaram baixos níveis basais de fosforilação, quando comparados àquelas que não responderam ao insulto. Os resultados desse estudo indicam que o uso tópico e intraluminal da CRS não interfere no perfil de ativação de ERK 1/2 e p38 no jejuno de coelhos submetidos à I/R.

Diante do quadro atual de previsões de mudanças climáticas, estudos a respeito das possíveis respostas dos organismos a estas alterações são importantes. Com a finalidade de prever e verificar se estas serão de fato deletérias ou se os organismos são capazes de lidar com elas sem alterações na sua fisiologia, e consequentemente na estrutura do ambiente, E. brasiliensis foi utilizada como modelo para estudar possíveis impactos do aumento da temperatura e acidificação dos oceanos na sua fisiologia. Para isso, espécimes foram expostos a 9 possíveis combinações de temperatura (24ºC, 28ºC e 30ºC) e pH (8.0, 7.7 e 7.3) em diferentes intervalos de tempo (1, 3, 12, 24 e 48 h). Amostras de gônadas e fluido celomático foram coletadas para avaliar a expressão das proteínas de estresse HSP70, AIF-1 e p38-MAPK, e a variação no número e viabilidade dos celomócitos. Nossos resultados mostram que o modelo é sensibilizado pelas mudanças no ambiente, através da hiper-regulação das proteínas de estresse. O cenário considerado mais extremo (30°C + pH7.3) ocasionou a morte de 100% dos organismos após 24horas. E o segundo cenário mais severo (30°C + pH7.7) desencadeou o desenvolvimento de ulceração de pele. Os efeitos são mais pronunciados nos celomócitos e a acidificação da água parece ter efeitos antagônicos com a temperatura nos celomócitos e sinérgicos nas gônadas. Embora a resposta tenha sido sistêmica, o grau e a dinâmica foram distintos em relação às diferentes amostras e estresses. Podendo causar modificações na resposta imune dos organismos e consequentemente na sobrevivência da espécie a longo prazo. / Under the current Climate Change context, studies about the potential responses of the organisms to their changing environment are of extreme importance. Recent studies point out the synergy of temperature and ocean acidification altogether. In this study, we used the sea star E. brasiliensis to assess the physiological effects of rising temperature, seawater acidification and the interaction of both factors. Independent individuals (N=225) were exposed to 9 possible combinations of temperature (24ºC, 28ºC and 30ºC) and pH (8.0, 7.7 and 7.3), for 1, 3, 12, 24 and 48 h. We compared the stress produced by these treatments measuring the expression of heat shock proteins (HSP70), the production of the allograft inflammatory factor (AIF−1) and the activation of mitogen kinases (MAPKs) at both gonad and celomic fluid. Furthermore, we assessed the quantity and quality of coelomocytes. Our results demonstrated that E. brasiliensis is vulnerable to the interaction of temperature and acidification. All the stress proteins evaluated were upregulated. The extreme scenario (30°C + pH7.3) caused the death of 100% of organisms after 24 hours, while the second most severe scenario (30°C + pH7.7) triggered skin ulceration. Nevertheless, we found that water acidification produces antagonistic effects to the temperature in coelomocytes and synergistic effects in gonad cells. Furthermore, these effects were more pronounced in the coelomocytes than in the gonads. The systemic response found in this study suggest that the interactive effects of elevated temperatures in conjunction with ocean acidification may endanger the survival of this species, and it could compromise the ecosystem functioning at long term.

Células dendríticas são as principais células apresentadoras de antígeno e apresentam alterações em pacientes com câncer. As vias de sinalização ERK 1/2 e p38 MAPK participam da diferenciação de DCs derivadas de monócitos (Mo-DCs). A exposição ao sobrenadante tumoral (ST) da linhagem MCF-7 levou à diminuição de CD1a e aumento de CD14 (frequência), além do aumento de IL-6 e IL-10. A inibição da via ERK1/2 MAPK corrigiu a expressão de CD14 e corrigiu parcialmente a produção das citocinas. A inibição da via p38 MAPK corrigiu a expressão de CD1a e CD14 e diminuiu parcialmente a produção das citocinas. Identificamos a proteína de choque térmico HSP27. A exposição à HSP27 não levou às alterações observados quando as células foram expostas ao ST. Por fim, em Mo-DCs de pacientes com câncer de mama o tratamento com o inibidor da p38 MAPK diminuiu a expressão de CD86 e HLA-DR. Portanto, os resultados deste trabalho sugerem que a inibição da via p38 MAPK não parece ser uma abordagem interessante na manipulação de Mo-DCs de pacientes com carcinoma ductal invasivo de mama. / Dendritic cells are the main presenting cells and present alterations in cancer patients. The signaling pathways p38 and ERK1/2 MAPK participate of monocyte-derived dendritic cells (Mo-DCs) differentiation. Exposition to MCF-7s supernatant (TS) decreased CD14 and CD1a expression (frequency) while enhanced IL-6 and IL-10 production. Inhibition of ERK1/2 MAPK reverted CD14 expression and partially reverted cytokines production. Inhibition of p38 MAPK reverted CD1a and CD14 expression and partially reverted cytokines production too. We identified the heat shock protein HSP27. Exposition to HSP27 did not cause the observed alterations seen when the cells were exposed to TS. Lastly, treatment of Mo-DCs from breast cancer patients with the p38 inhibitor decreased CD86 and HLA-DR expression. Therefore, the data presented in this study suggest that p38 MAPK inhibition does not appear to be an interesting approach in the manipulation of Mo-DCs from breast cancer patients.

Le mélanome malin (MM) métastatique, un des cancers les plus intrinsèquement résistants aux agents anti-cancéreux et présentant une forte capacité à développer des résistances acquises, constitue un défi thérapeutique. La meilleure compréhension des mécanismes impliqués dans cette chimiorésistance permettrait d'identifier des cibles thérapeutiques ou de guider le choix du traitement pour une meilleure efficacité. Les travaux réalisés durant cette thèse se sont focalisés sur l'identification de nouveaux déterminants moléculaires de la résistance acquise du MM vis-à-vis (i) des vinca-alcaloïdes (VAs, chimiothérapie classique), (ii) des inhibiteurs de MAP kinases (iMAPK, thérapie ciblée). Pour la première étude, un modèle de lignées cellulaires de MM résistantes aux VAs (CAL1R-VAs) a été établi (exposition continue, 12 mois, de la lignée parentale CAL1-wt à la VCR, la VDS ou la VRB : CAL1R-VCR, CAL1R-VDS et CAL1R-VRB respectivement). La comparaison des profils d'expression a permis de distinguer deux groupes de lignées cellulaires (CAL1R-VCR et CAL1R-VDS ; CAL1R-VRB et CAL1-wt), suggérant une résistance différentielle du MM aux VAs : d'une part à la VCR et à la VDS, d'autre part à la VRB. L'analyse des données transcriptomiques par une démarche associant successivement trois méthodes - RMA (Robust Multi-array Average), RDAM (Rank Difference Analysis of Microarrays) et MGSA (model-based gene set analysis) – a permis d'identifier des fonctions cellulaires altérées lors de la sélection des lignées CAL1R-VAs, et donc potentiellement à l'origine de la résistance de ces lignées. Des analyses fonctionnelles in vitro ont permis de confirmer l'implication des lysosomes et de la réponse au stress du réticulum endoplasmique (RE) dans la résistance différentielle des cellules CAL1 aux VAs. Ainsi, une sous-expression des cathepsines B et L (bioinformatique) et une réduction du volume du compartiment acide (in vitro) ont été observées spécifiquement dans le premier groupe de lignées (CAL1R-VCR et CAL1R-VDS), suggérant une sensibilité réduite de ces lignées à la voie lysosomale de l'apoptose. Par ailleurs, l'inhibition de la voie de réponse au stress du RE par l'acide tauroursodésoxycholique (TUDCA) a induit une sensibilisation différentielle de l'ensemble des lignées CAL1 aux VAs, suggérant l'implication de cette voie dans la résistance différentielle primaire et acquise aux VAs. De plus, l'inhibition de la réponse au stress du RE a induit une sensibilisation d'une autre lignée cellulaire de MM, MDA-MB-435, à la VCR et à la VDS mais pas à la VRB. Ainsi, la voie de réponse au stress du RE semble impliquée dans la résistance différentielle du MM aux VAs. Ce mécanisme pourrait mettre en jeu l'autophagie, dont le flux était significativement augmenté dans le premier groupe de lignées. La même démarche d'analyse transcriptomique a été appliquée pour l'étude des mécanismes moléculaires de résistance acquise du MM aux iMAPK. Des lignées cellulaires de MM résistantes aux trois iMAPK majeurs ont été établies par exposition continue de la lignée parentale A375-wt, portant la mutation activatrice BRAF V600E, au vémurafenib (VMF, inhibiteur de BRAF), dabrafenib (DBF, inhibiteur de BRAF), et trametinib (TMT, inhibiteur de MEK): A375R-VMF, A375R-DBF et A375R-TMT respectivement. La comparaison des profils transcriptomiques n'a pas permis de regrouper les lignées résistantes entre elles, suggérant que les mécanismes de résistance au VMF, au DBF ou au TMT sont différents. Ces mécanismes ne seraient donc communs ni à la voie ciblée (MAPK), ni à la cible moléculaire (BRAF ou MEK). L'identification des fonctions cellulaires altérées procurera un rationnel pour l'étude mécanistique de nouveaux déterminants de la résistance du MM aux iMAPK. / Malignant melanoma (MM), one of the most intrinsically resistant cancers to anticancer agents and presenting a strong ability to develop acquired resistance, remains a therapeutic challenge. A better understanding of the mechanisms involved in MM chemoresistance should provide therapeutic targets or guide therapeutic choice for improved efficiency. This thesis has focused on the identification of new molecular determinants of MM acquired resistance to (i) vinca alkaloids (VAs, conventional chemotherapy), and to (ii) MAP kinases inhibitors (MAPKi, targeted therapy). In the first study, MM cell lines resistant to VAs (CAL1R-VAs) were established (continuous exposure, 12 months, of CAL.1-wt parental line to the VCR, VDS or VRB: CAL1R-VCR, CAL1R- VDS and CAL1R-VRB respectively). Comparison of expression patterns led to distinguish two groups of cell lines (CAL1R-VCR and CAL1R-VDS; CAL1R-VRB and CAL.1-wt), suggesting a differential resistance of MM to VAs: one the one hand to VCR and VDS, on the other hand to VRB only. The analysis of transcriptome data by a process involving successively three methods - RMA (Robust Multi-array Average), RDAM (Rank Difference Analysis of Microarrays) and MGSA (model-based gene set analysis) – allowed the identification of functions altered during the resistant cell line selection, and therefore potentially involved in resistance mechanisms of these cell lines. In vitro functional analyzes confirmed the involvement of the lysosomes and of the response to endoplasmic reticulum (ER) stress (unfolded protein response, UPR) in the differential resistance of CAL1 cells to VAs. Thus, an under-expression of cathepsins B and L (bioinformatics), and a reduction of the acidic compartment volume (in vitro) were specifically observed in the first cell group (CAL1R-VCR and CAL1R-VDS), suggesting a reduced sensitivity of these lines to the lysosomal pathway of apoptosis. Furthermore, UPR inhibition using tauroursodeoxycholic acid (TUDCA) induced a differential sensitization of all the CAL1 lines to VAs, suggesting the involvement of this pathway in the primary and acquired differential resistance to VAs. Moreover, TUDCA-inhibition of UPR induced sensitization another MM cell line, MDA-MB-435, to VCR and VDS but not to VRB. Thus, a UPR up-regulation could to be a significant mechanism of differential resistance of MM to VAs. This mechanism could involve autophagy, whose flow was significantly increased in the first group of lines. The same transcriptome analysis strategy was applied to study (ii) the molecular mechanisms of MM acquired resistance to MAPKi. MM cell lines resistant to the three major MAPKi were established by continuous exposure of the parental A375-wt line, carrying the activating mutation BRAF V600E, to vemurafenib (VMF, BRAF inhibitor), dabrafenib (DBF, BRAF inhibitor), or trametinib (TMT, MEK inhibitor): A375R-VMF, A375R-DBF and A375R-TMT, respectively. Comparison of transcriptomic profiles showed separate expression profiles, suggesting that the molecular mechanisms responsible for resistance to VMF, DBF or TMT were different. These mechanisms cannot therefore be common to the targeted pathway (MAPK) or to the molecular target (BRAF or MEK). The identification of the altered cellular functions will provide a rationale for mechanistic studies of new determinants of MM resistance to MAPKi.

Malgré les récentes avancées réalisées dans le traitement du cancer du côlon, la résistance des tumeurs est une cause fréquente de l'échec des chimiothérapies. Cette thèse a pour objectif d'identifier les mécanismes moléculaires impliqués dans la résistance à l'Irinotécan, un agent couramment utilisé dans le traitement des cancers colorectaux. Nous avons montré l'implication de la MAPK p38 dans la résistance à l'Irinotécan et en particulier avons démontré que les isoformes α et β étaient impliquées dans cette résistance. De plus, nous avons corrélé la faible phosphorylation de p38 dans des tumeurs coliques primaires de patient sensibles au traitement à l'Irinotécan par rapport aux patients non répondeurs. Dans la suite du projet nous avons étudié le rôle de p38 dans les processus autophagiques et leur impact dans la réponse à l'Irinotécan. Nous avons démontré que p38 induisait une autophagie qui mène à la survie des cellules cancéreuses déficientes pour p53, et que l'inhibition de l'autophagie sensibilisait ces cellules au traitement au SN38, métabolite actif de l'Irinotécan. Enfin de manière préliminaire, nous avons étudié le rôle de p38 dans l'augmentation du métabolisme lipidique dans des cellules déficiente pour p53. Ces travaux ouvrent de nouvelles voies de recherche pour l'identification des mécanismes impliqués dans la résistance aux traitements anticancéreux et pour le développement d'approches pharmacologiques pour contourner la résistance. / Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. This work was aimed to determine the molecular mechanisms involved in the resistance to Irinotecan, an anticancer agent widely used in colorectal cancer treatment. We have demonstrated that the α and β forms of p38 MAPK were involved in this resistance. Moreover, we have correlated less phospho-p38 in colon cancer primary tumor of patients sensitive to Irinotecan-based treatment, compared to non responder patients. During the project, we aimed to determine the role of p38 MAPK in the processes of autophagy in colorectal cancer cells, and their impact in Irinotecan cytotoxicity. We have shown that p38 induced survival autophagy in p53 deficient cells. Then, we have shown that autophagy inhibition increased the SN38 cytotoxicity (active metabolite of Irinotecan) in p53 deficient cell lines. Finally, we have studied the role of p38 MAPK in lipid metabolism in p53 deficient cells. All these findings highlight new ways of research to identify the molecular mechanisms involved in chemoresistance as well as new pharmacological approaches to overcome the resistance.

Clostridium difficile (CD) représente la première cause d'infections digestives nosocomiales dans les pays développés. Les infections à CD (ICD) induisent une inflammation intestinale importante qui se manifeste principalement par des colites pseudomembraneuses ainsi que par un taux de mortalité élevé. Les facteurs majeurs de virulence sont les toxines TcdA et TcdB. Dans la première partie des travaux de cette thèse, nous avons validé le rôle in vivo d'un autre facteur bactérien, les flagelles, dans un modèle murin d'ICD, en montrant que les flagelles induisent une réponse inflammatoire au niveau de la muqueuse caecale en synergie avec les toxines. Nous avons ensuite montré une régulation de de cette réponse par un microARN (miARN) exerçant un rôle anti-inflammatoire en modulant l'activation de la voie de signalisation de NF-KB. Le traitement des souris infectées par ce miARN réduit l'inflammation intestinale apportant la preuve du concept pour une nouvelle approche thérapeutique. / Clostridium difficile (CD) is the leading cause of nosocomial digestive infections in developed countries. CD infections (CDI) induce significant intestinal inflammation that is manifested primarily by pseudomembranous colitis and a high mortality rate. The major virulence factors are TcdA and TcdB toxins. In the first part of the work of this thesis, we validated the in vivo role of another bacterial factor, the flagella, in a mouse model of CDI, showing that the flagella induce an inflammatory response in ceacal mucosa in synergy with toxins. We then showed a regulation of this response by a microRNA (miRNA) exerting an anti-inflammatory role by modulating the activation of the NF-KB signaling pathway. The treatment of mice infected with this miRNA reduces intestinal inflammation providing proof of concept for a new therapeutic approach.

La régénération est un mode de développement, qui suite à un stresse physique permet de reformer à l’identique des structures biologiques initialement développer au cours de l’embryogénèse. De plus ce phénomène, plus ou moins important en fonction des organismes, est néanmoins répandu chez les métazoaires, suggérant ainsi une origine monophylogénique. D’où l’hypothèse d’un lien étroit entre la régénération et l’embryogénèse. En me basant sur cette hypothèse j’ai employé comme modèle pendant ma thèse, l’anémone de mer Nematostella vectensis. Ce modèle cnidaire offre effectivement l’opportunité unique de comparer la régénération d’un corps entier, dite extrême, à l’embryogénèse et ainsi étudier leurs liens au niveau moléculaire. Initialement établie entant que modèle d’embryologie permettant d’étudier l’évolution des réseaux de régulation génétique (RRG) orchestrant les moments clé de l’embryogénèse et s’imposer en tant que modèle d’étude de la régénération extrême. Tout d’abord, au cours de ma thèse j’ai participé à caractérisation tissulaire et cellulaire de la régénération de ce model afin d’en établir un répertoire de référence des étapes clés. En employant ce répertoire et le criblage de 80 d’inhibiteur de kinase, j’ai pu identifier plusieurs voies de signalisation régissant différente étape de la régénération, impliquant les MAPKs, JNK et ERK ainsi que plusieurs récepteurs de facteurs de croissances. Notamment ERK a également été décrit dans le processus de gastrulation chez Nematostella, dont j’ai contribué à l’établissement du RRG associé. C’est donc en me basant sur ce RRG et une base de donnée transcriptomic complète de la régénération de ce modèle, que j’ai pu établir le RRG en aval de ERK associé à la régénération. Par cette approche j’ai pu démontrer la relation au niveau moléculaire entre ces processus développementaux et surtout identifier des aspects spécifiques à la régénération. / Regeneration is a developmental process, which allow to regrow missing structures initially develop during embryogenesis, in response to an injury. Although, this ability to regenerate can be more or less dramatic depending on the organism, it is widely spread among metazoan. As such, suggesting a monophyletic origin and a tight link with embryogenesis has also been hypothesized. Based on this hypothesis, I used during my thesis the sea anemone Nematostella vectensis, a cnidarian model offers the unique opportunity to compare, whole body regeneration and embryogenesis to investigate their molecular links. In fact, Nematostella was established as an embryonic model to investigate evolution of gene regulatory network (GRN) underlying key stages e.g. gastrulation, but recently it has been a rising model to study whole body regeneration. I started to my thesis by carefully characterizing hallmarks of Nematostella regeneration starting from tissular to molecular level, establishing a comprehensive regeneration time line. By taking advantage of this tool, in association to the screening of 80 kinases inhibitors, I have identify several signaling pathways regulating various steps of regeneration in Nematostella, including the MAPK ERK, JNK and growth factor receptors. In parallel I participated to the study of ERK role during Nematostella gastrulation and the underpinning (GRN). Which offered a solid groundwork for the comparison with regeneration at the GRN level. Combining a candidate approach based on the embryonic GRN and a global transcriptomic analysis of regeneration, I have been able to bring evidence on the relationship between embryogenesis and regeneration and additionally to identify regeneration specific aspects.