Le cancer colorectal représente la troisième forme de cancer la plus fréquente au Canada. Malgré les récents développements, aucun traitement curatif n’est disponible pour les patients diagnostiqués à un stade avancé de la maladie. Plusieurs évidences supportent un rôle important des prostaglandines dans cette maladie. En effet, une surexpression des récepteurs de type EP et FP est remarquée. Ces derniers ainsi que les molécules de signalisations intracellulaires qu’ils activent représentent donc de nouvelles cibles pour traiter ce cancer. Nous et d’autres groupes avons démontré que les protéines G monomériques sont des petits interrupteurs moléculaires importants dans la signalisation intracellulaire engagée par les récepteurs dans des cellules saines mais qu’un dérèglement de celles-ci est associé au cancer. L’objectif principal de ce projet de recherche vise donc à identifier les voies de signalisations par lesquelles le récepteur FP contribue aux capacités invasives des cellules tumorales d’origine colorectale. Notre hypothèse est que la protéine ARF6, une des 6 isoformes des ARFs, et la protéine RhoA, agissent pour coordonner l’activation des voies de signalisations associées à la migration et l’invasion cellulaire. Nos résultats ont indiqué que la stimulation des cellules HEK293 exprimant de façon stable le récepteur FP (HA-FP) ainsi que les cellules SW480, une lignée invasive de cancer colorectal, par le PGF2α augmentait les niveaux d’activation d’ARF6 mais également de la protéine RhoA. De plus, d’autres médiateurs et intermédiaires associés à la réorganisation du cytosquelette comme la cofiline et la chaine légère de la myosine (MLC) ont été hautement phosphorylés suite à la stimulation par la prostaglandine. Ces observations sont associées avec une augmentation des fibres de stress dans les cellules. Nous avons tenté de déterminer si l’inhibition de ces protéines G affectait la capacité du PGF2α à activer ces intermédiaires de signalisation ainsi que certains effets biologiques. Ainsi, nos expériences contribueront à identifier de nouvelles cibles thérapeutiques potentielles pour le traitement du cancer colorectal. / Colorectal cancer is the third most common form of cancer in Canada. Despite recent developments, no curative treatment is available for patients diagnosed at an advanced stage of the disease. Several evidences support an important role of prostaglandins in this disease. Indeed, overexpression of EP-like and FP receptors is noticed. These and intracellular signaling molecules that are activated following receptor stimulation, represent new targets for treating cancer. We and others have demonstrated that monomeric G proteins are important molecular switches coordinating intracellular signaling cascades initiated by receptors in normal cells but that disruption thereof is associated with cancer. The main objective of this research project aims to identify the signaling pathways by which the FP receptor contributes to the invasive capacity of tumor cells of colorectal origin. Our hypothesis is that the proteins ARF6, one of the 6 ARF isoforms, and RhoA protein, act to coordinate the activation of signaling pathways associated with cell migration and invasion. Our results show that stimulation of HEK293 cells that stably express the FP receptor (HA-FP) and SW480 cells, a lineage of invasive colorectal cancer that endogenously express FP receptor, by PGF2α, increased the level of activation of ARF6, but also that of RhoA. In addition, other mediators and intermediates associated with cytoskeleton reorganization such as cofilin and the myosin light chain (MLC) were found highly phosphorylated following stimulation by prostaglandin PGF2α. These observations are associated with an increase of stress fibers in cells. Finally, we have examinated whether inhibition of ARF6 and RhoA, affected the ability of PGF2α to activate these effectors and some biological effects. Thus, our results will contribute to identify new potential therapeutic targets for the treatment of colorectal cancer.

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga / The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

No description available.

No description available.

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga / The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

Fibroblasts are the principal cellular component of the connective tissue. They are a heterogeneous group of cells which contribute to the structure of connective tissue and wound healing by their ability to produce extracellular matrix (ECM). Fibroblasts and cells derived from them are involved in many pathological processes such as formation of malignant tumors and fibrosis. Tumor progression which finally leads to metastasis is a serious biomedical problem. There is a growing body of the recent evidence showing an important role of the tumor stroma and its interaction with cancer cells in cancer progression. Tumor stroma comprises mainly of myofibroblasts and their products, namely ECM, soluble factors, and enzymes. Myofibroblasts contribute more or less to all steps of cancer progression. Furthermore myofibroblasts play a key role in fibrosis, another serious human disease which is not efficiently treatable and which is associated with cancer progression. These facts made us to search for molecular means capable of eliminating the myofibroblastic phenotype. We succeeded to entirely dedifferentiate primary myofibroblasts by concomitant inhibition of TGFβ signaling and perturbation of MAPK signaling in a chick model that we have introduced. Malignant fibroblasts form sarcomas. ECM is the first...

Le tonus myogénique (TM) est la capacité des artères de résistance à se contracter suite à une élévation de la pression intraluminale. Il permet la protection des capillaires contre une augmentation excessive de pression, et régule les débits sanguins locaux. L'implication du TM dans le développement de certaines pathologies fait de lui une cible thérapeutique intéressante. Cela nécessite une identification précise des protagonistes moléculaires. La voie RhoA/Rho-kinase joue un rôle important dans le TM en régulant la sensibilisation au calcium de l'appareil contractile. Nous nous sommes donc particulièrement intéressés à des éléments pouvant moduler ou être modulés par cette voie. Ainsi, nous avons mis en évidence que l'intervention de la voie RhoA/Rho-kinase dans le TM implique tout d'abord une localisation membranaire de RhoA, afin d'interagir avec la cavéoline-1 et d'activer ses effecteurs tels que Rho-kinase. De plus l'activation de RhoA par la pression est régulée par le récepteur Notch3 intervenant ainsi comme un mécanosenseur. Nos travaux montrent aussi le rôle du facteur de réponse au sérum (SRF) dans le TM via la régulation du cytosquelette et de l'activité des canaux mécanosensibles. Pour finir, l'inhibition de l'activité de RhoA, lors d'un traitement avec du Sildénafil, entraine une surexpression de RhoA qui est responsable d'une élévation du TM et d'une hypertension à l'arrêt du traitement. Cette thèse apporte de nouvelles connaissances sur l'implication de RhoA, Notch3 et SRF dans le TM permettant une meilleure compréhension de ce mécanisme. Cela offre de nouvelles cibles thérapeutiques pour combattre les pathologies impliquant des dysfonctions vasculaires.

Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.

No description available.

Les lymphocytes T (LT) perçoivent et intègrent en permanence des signaux solubles et cellulaires, conditionnant leur comportement et leur devenir. A l'état de repos, les propriétés des LT s'appuient sur un réseau moléculaire caractéristique, au sein duquel les facteurs de transcription FoxOs jouent un rôle majeur. En effet, ces derniers sont impliqués dans le maintien de la quiescence et de la capacité circulatoire des LT, de par le profil transcriptionnel qu'ils induisent. Nous avons identifié Fam65b comme une nouvelle cible transcriptionnelle de FOXO1. D'un point de vue fonctionnel, nous avons démontré que Fam65b régule négativement le seuil de prolifération des LT en réponse à une stimulation du récepteur à l'antigène (TCR) ou du récepteur aux chimiokines CCR7. In vivo, dans un modèle de souris transgénique pour le TCR, ces caractéristiques fonctionnelles se traduisent par une réponse secondaire plus efficace en absence de Fam65b. Physiologiquement, la moindre expression de Fam65b que nous avons observé dans les LT mémoires par comparaison aux LT naïfs corrèle avec leur plus grande réactivité. L'ensemble de ces résultats suggère que Fam65b pourrait être un marqueur fonctionnel des LT mémoires. Enfin, nous avons pu démontrer que les effets fonctionnels de Fam65b résultent d'une inhibition de l'activité de RhoA.Fam65b est donc un régulateur de la quiescence et de la migration des LT. De par son rôle de régulateur de l'activité de RhoA, Fam65b constitue un nouveau lien fonctionnel entre deux familles majeures, contrôlant la physiologie des LT : les Rho-GTPases et les FoxOs.