As proteína quinases C (PKC) pertencem à família das serina/treonina quinases, que vem sendo apontadas como importantes enzimas para os processos de proliferação e diferenciação das células tronco embrionárias (CTE), todavia, a função exata de cada isoforma dessa família ainda não está clara. Dados anteriores do nosso laboratório indicam que dentre as PKCs expressas em CTE, formas cataliticamente ativas da PKCβI são altamente expressas no núcleo das CTE murinas. Estas ao se diferenciarem expressam essa quinase no seu citoplasma ou deixam de expressar a mesma, e que a maioria dos alvos da PKCβI em CTE indiferenciada estão envolvidos em processos de regulação da transcrição de proteínas envolvidas em processos de proliferação/ diferenciação. Dando continuidade aos resultados anteriores do laboratório, no presente trabalho, com técnicas de proteômica e fosfoproteômica identificamos outros alvos nucleares da PKCβI em CTE indiferenciadas. Vimos que de fato inibindo-se a PKCβI diminuiu-se a fostorilação de fatores envolvidos com a indiferenciação das CTE. Dentre os alvos da PKCβI encontramos a proteína adaptadora, TIF1 que recruta proteínas remodeladoras de cromatina. Essa proteína é essencial para a manutenção do estado indiferenciado das CTE. In vitro a PKCβI foi capaz de fosforilar a TIF1β e inibindo-se a PKCβI por RNAi vimos uma diminuição na expressão da TIF1β e no fator de indiferenciação Nanog cuja expressão já foi demonstrada ser regulada pela TIF1β. Além disso vimos que inibindo-se a PKCβI com o peptídeo inibidor da PKCβI aumentou a expressão de proteínas reguladas pelo c-Myc. E que o RNAi para a PKCβI aumentou a expressão de proteínas que regulam a expressão do c-Myc. Não vimos nenhum efeito na fosforilação ou expressão do c-Myc após a inibição da PKCβI o que sugere que a PKCβI ative proteínas repressoras do c-Myc. Nossos estudos sugerem que a PKCβI regula a manutenção do estado indiferenciado das CTE regulando a expressão e atividade da Tif1β um possível alvo direto da PKCβI. Levando a modificações da cromatina e regulação da expressão de genes que mantém as CTE indiferenciadas. Outro ponto de regulação da PKCβI parece ser a nibição da atividade de c-Myc o que seria importante para a manutenção do estado indiferenciado visto que o c-Myc é um amplificador das vias de sinalização que mantém as células proliferando. Desta forma a PKCβI parece ter um papel central na regulação da expressão gênica de CTE à nível de modificações epigenéticas e a nível transcricional mantendo as CTE indiferenciadas. / The Protein kinase C (PKC) family of serine/treonine kinases, are being described as important enzymes for proliferation and diferentiation of embryonic stem cells (ESC), however, the exact function of the different isoenzymes of this family still is unclear. Previous data from our laboratory indicates that amongst the PKCs expressed in ESC, catalytically active forms of PKCβI are highly expressed in nucleus of murine ESC. When these cells differentiate this kinase can be found in the cytoplasm or not expressed at all, and that the majority of PKCβI targets in undifferentiated ESC are involved in the regulation of proteins involved in transcription of proteins involved in proliferation/ diferentiation. Continuing our previous work herewith using proteomics and phosphoproteomics techniques we identified other nuclear PKCβI targets in undifferentiated ESC. We indeed saw that inhibiting PKCβI decreased the phosphorylation of factors involved with maintainance of the undifferentiated state of ESC. Amongst the targets of PKCβI we found the adaptor protein, TIF1βI, that recruits cromatin remodeling proteins. This protein is essential for the maintenance of the undifferentiated state of ESC. In vitro PKCβI phosphorylated TIF1β and inhibiting PKCβI with RNAi decreased the expression of TIF1β and of the undifferentiation factor Nanog whose expression has been shown to be regulated by TIF1β. We also saw that inhibiting PKCβI with a peptide inhibitor increased the expression of proteins regulated by c-Myc, and that RNAi for PKCβI increased the expression of proteins that regulate the expression of c-Myc. We did not see any effect on the phosphorylation or expression of c-Myc after inhibition of PKCβI suggesting that PKCβI activates c-Myc repressor proteins. Our studies sugest that PKCβI regulates the maintenance of the undiferentiated state of ESC regulating the expression and activity of Tif1β a possibly a direct target of PKCβI, leading to chromatin modifications and regulation of genes that maintain ESC undiferentiated. Another form of regulation of PKCβI seems to be by inhibiting the activity of c-Myc which is importante to maintain ESC undifferentiated since c-Myc is na an amplifyer of signaling patheways that maintain ESC proliferating. Together PKCβI has a central role in the regulation of the gene expression of ESC at the level of epigenetic modifications and transcriptional regulation

As proteína quinases C (PKC) pertencem à família das serina/treonina quinases, que vem sendo apontadas como importantes enzimas para os processos de proliferação e diferenciação das células tronco embrionárias (CTE), todavia, a função exata de cada isoforma dessa família ainda não está clara. Dados anteriores do nosso laboratório indicam que dentre as PKCs expressas em CTE, formas cataliticamente ativas da PKCβI são altamente expressas no núcleo das CTE murinas. Estas ao se diferenciarem expressam essa quinase no seu citoplasma ou deixam de expressar a mesma, e que a maioria dos alvos da PKCβI em CTE indiferenciada estão envolvidos em processos de regulação da transcrição de proteínas envolvidas em processos de proliferação/ diferenciação. Dando continuidade aos resultados anteriores do laboratório, no presente trabalho, com técnicas de proteômica e fosfoproteômica identificamos outros alvos nucleares da PKCβI em CTE indiferenciadas. Vimos que de fato inibindo-se a PKCβI diminuiu-se a fostorilação de fatores envolvidos com a indiferenciação das CTE. Dentre os alvos da PKCβI encontramos a proteína adaptadora, TIF1 que recruta proteínas remodeladoras de cromatina. Essa proteína é essencial para a manutenção do estado indiferenciado das CTE. In vitro a PKCβI foi capaz de fosforilar a TIF1β e inibindo-se a PKCβI por RNAi vimos uma diminuição na expressão da TIF1β e no fator de indiferenciação Nanog cuja expressão já foi demonstrada ser regulada pela TIF1β. Além disso vimos que inibindo-se a PKCβI com o peptídeo inibidor da PKCβI aumentou a expressão de proteínas reguladas pelo c-Myc. E que o RNAi para a PKCβI aumentou a expressão de proteínas que regulam a expressão do c-Myc. Não vimos nenhum efeito na fosforilação ou expressão do c-Myc após a inibição da PKCβI o que sugere que a PKCβI ative proteínas repressoras do c-Myc. Nossos estudos sugerem que a PKCβI regula a manutenção do estado indiferenciado das CTE regulando a expressão e atividade da Tif1β um possível alvo direto da PKCβI. Levando a modificações da cromatina e regulação da expressão de genes que mantém as CTE indiferenciadas. Outro ponto de regulação da PKCβI parece ser a nibição da atividade de c-Myc o que seria importante para a manutenção do estado indiferenciado visto que o c-Myc é um amplificador das vias de sinalização que mantém as células proliferando. Desta forma a PKCβI parece ter um papel central na regulação da expressão gênica de CTE à nível de modificações epigenéticas e a nível transcricional mantendo as CTE indiferenciadas. / The Protein kinase C (PKC) family of serine/treonine kinases, are being described as important enzymes for proliferation and diferentiation of embryonic stem cells (ESC), however, the exact function of the different isoenzymes of this family still is unclear. Previous data from our laboratory indicates that amongst the PKCs expressed in ESC, catalytically active forms of PKCβI are highly expressed in nucleus of murine ESC. When these cells differentiate this kinase can be found in the cytoplasm or not expressed at all, and that the majority of PKCβI targets in undifferentiated ESC are involved in the regulation of proteins involved in transcription of proteins involved in proliferation/ diferentiation. Continuing our previous work herewith using proteomics and phosphoproteomics techniques we identified other nuclear PKCβI targets in undifferentiated ESC. We indeed saw that inhibiting PKCβI decreased the phosphorylation of factors involved with maintainance of the undifferentiated state of ESC. Amongst the targets of PKCβI we found the adaptor protein, TIF1βI, that recruits cromatin remodeling proteins. This protein is essential for the maintenance of the undifferentiated state of ESC. In vitro PKCβI phosphorylated TIF1β and inhibiting PKCβI with RNAi decreased the expression of TIF1β and of the undifferentiation factor Nanog whose expression has been shown to be regulated by TIF1β. We also saw that inhibiting PKCβI with a peptide inhibitor increased the expression of proteins regulated by c-Myc, and that RNAi for PKCβI increased the expression of proteins that regulate the expression of c-Myc. We did not see any effect on the phosphorylation or expression of c-Myc after inhibition of PKCβI suggesting that PKCβI activates c-Myc repressor proteins. Our studies sugest that PKCβI regulates the maintenance of the undiferentiated state of ESC regulating the expression and activity of Tif1β a possibly a direct target of PKCβI, leading to chromatin modifications and regulation of genes that maintain ESC undiferentiated. Another form of regulation of PKCβI seems to be by inhibiting the activity of c-Myc which is importante to maintain ESC undifferentiated since c-Myc is na an amplifyer of signaling patheways that maintain ESC proliferating. Together PKCβI has a central role in the regulation of the gene expression of ESC at the level of epigenetic modifications and transcriptional regulation

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCβI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCβI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCβI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a β-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCβI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e β-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na β-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCβI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCβI em CTE indiferenciadas. / Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCβI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCβI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCβI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and β-tubulin, as a phosphorylation site of α-tubulin by PKCβI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and β-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in β-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCβI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCβI in undifferentiated ESC.

L’apoptose est une forme de mort cellulaire essentielle au développement et au maintien de l’homéostase chez les animaux multicellulaires. La machinerie apoptotiq ue requiert la participation des caspases, des protéases conservées dans l’évolution et celle des organelles cytoplasmiques. Les lysosomes subissent des ruptures partielles, labilisation de la membrane lysosomale (LML), qui entraînent l’activation des cathepsines dans le cytoplasme de cellules cancéreuses humaines en apoptose induite par la camptothecin (CPT), incluant les histiocytes humains U-937. Ces modifications lysosomales se manifestent tôt durant l’activation de l’apoptose, concomitamment avec la perméabilisation de la mitochondrie et l’activation des caspases. Une étude protéomique quantitative et comparative a permis d’identifier des changements précoces dans l’expression/localisation de protéines lysosomales de cellules U-937 en apoptose. Lors de deux expériences indépendantes, sur plus de 538 protéines lysosomales identifiées et quantifiées grâce au marquage isobarique iTRAQ et LC-ESIMS/ MS, 18 protéines augmentent et 9 diminuent dans les lysosomes purifiés de cellules en cours d’apoptose comparativement aux cellules contrôles. Les candidats validés par immuno-buvardage et microscopie confocale incluent le stérol-4-alpha-carboxylate 3- déhydrogénase, le prosaposin et la protéine kinase C delta (PKC-d). Des expériences fonctionnelles ont démontrées que la translocation de PKC-d aux lysosomes est requise pour la LML puisque la réduction de son expression par ARN interférents ou l’inhibition de son activité à l’aide du rottlerin empêche la LML lors de l’apoptose induite par la CPT. La translocation de PKC-d aux lysosomes conduit à la phosphorylation et l’activation de la sphingomyelinase acide lysosomale (ASM), et à l’accroissement subséquent du contenu en céramide (CER) à la membrane lysosomale. Cette accumulation de CER endogène aux lysosomes est un évènement critique pour la LML induite par la CPT car l’inhibition de l’activité de PKC-d ou de ASM diminue la formation de CER et la LML.Ces résultats révèlent un nouveau mécanisme par lequel la PKC-d active l’ASM qui conduit à son tour à l’accumulation de CER à la membrane lysosomale et déclenche la LML et l’activation de la voie lysosomale de l’apoptose induite par la CPT. En somme, ce mécanisme confirme l’importance du métabolisme des sphingolipides dans l’activation de la voie lysosomale de l’apoptose. / Apoptosis is a distinct form of regulated cell death which is essential for the development and homeostasis maintenance of multicellular animals. Apoptosis is an evolutionary conserved process involving a specific molecular pathway, known as the caspase cascade, and the different cytoplasmic organelles. A lysosomal pathway, characterized by partial rupture, labilization of lysosomal membranes (LML), and cathepsin activation in the cytoplasm, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In two independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labelling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-d). Functional experiments demonstrate that PKC-d translocation to lysosomes is required for LML, as silencing its expression with RNA interference or suppressing its activity with the inhibitor rottlerin prevents CPT-induced LLM. PKC-d translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase of ceramide (CER) content at lysosomes. The accumulation of endogenous CER at lysosomes is a critical event for CPT-induced LLM as suppressing PKC-d or ASM activity reduces both CPT-mediated CER generation at lysosomes and CPT-induced LLM.These findings reveal a novel mechanism by which PKC-d mediates ASM phosphorylation/activation and CER accumulation at lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. Taken together, these results confirm the importance of sphingolipid metabolism in the activation of the lysosomal pathway of apoptosis.

La scoliose idiopathique de l’adolescent (SIA) est une maladie dont la cause est encore inconnue, et qui génère des déformations complexes du rachis, du thorax et du bassin. La prévalence est de 4% dans la population adolescente au Québec. Cette pathologie affecte surtout les filles durant leur poussée de croissance pubertaire. Parmi plusieurs hypothèses émises, l’hypothèse neuroendocrinienne, impliquant une déficience en mélatonine comme agent étiologique de la SIA a suscité beaucoup d’intérêt. Cette hypothèse découle du fait que l’ablation de la glande pinéale chez le poulet produit une scoliose ressemblant sous plusieurs aspects à la pathologie humaine. La pertinence biologique de la mélatonine dans la scoliose est controversée, étant donné que la majorité des études chez l’homme n’ont pu mettre en évidence une diminution significative des niveaux de mélatonine circulante chez les patients scoliotiques. Nous avons démontré un dysfonctionnement dans la signalisation de la mélatonine au niveau des tissus musculo-squelettiques chez une série de patients atteints de SIA (Moreau & coll. 2004). Nous avons confirmé ce défaut chez un plus grand nombre de patients ainsi qu’en utilisant une nouvelle technologie (spectroscopie cellulaire diélectrique) n’ayant pas recours à un prétraitement des cellules donnant ainsi des résultats plus précis. Cette technique a montré la présence des mêmes groupes fonctionnels identifiés auparavant par la technique d’AMPc. Le dysfonctionnement de la signalisation de la mélatonine est dû à une phosphorylation accrue des protéines G inhibitrices. Ce défaut pourrait être causé par un déséquilibre de l’activité des kinases et phosphatases capables de réguler la phosphorylation des protéines Gi. Parmi ces kinases, PKCd a suscité initialement notre intérêt vu qu’elle peut phosphoryler les protéines Gi. Nous avons démontré que cette kinase interagit avec le récepteur de la mélatonine MT2 et que cette interaction varie selon le groupe fonctionnel auquel un patient SIA appartient. Par la suite nos travaux se sont dirigés vers la découverte d’effecteurs cellulaires régulés par la mélatonine et plus spécifiquement l’ostéopontine (OPN), compte tenu de son rôle présumé comme mécanorécepteur et dans certaines structures jouant un rôle dans la proprioception, le contrôle postural et la fonction vestibulaire. L’OPN a été identifiée initialement par sa surexpression au niveau protéique et de l’ARNm dans la musculature paraspinale uniquement chez les poulets scoliotiques. Nous avons également utilisé un autre modèle animal, la souris C57Bl/6 naturellement déficiente en mélatonine. Nous avons généré des souris bipèdes en amputant les membres antérieurs de souris OPN KO, des souris CD44 KO ainsi que des souris contrôles C57Bl/6. Nos résultats ont montré qu’aucune souris OPN KO (n=50) ou CD44 KO (n=60) ne développe la maladie, contrairement aux souris contrôles C57Bl/6 (n=50) dont 45% deviennent scoliotiques. Ces résultats nous ont poussés à investiguer le rôle de cette protéine dans l’étiopathogenèse de la maladie chez l’humain. Nos résultats ont montré une augmentation des niveaux circulants d’OPN chez les patients atteints de la SIA et que l’élevation en OPN corrélait avec la sévérité de la maladie. Nos études chez les enfants asymptomatiques nés de parents scoliotiques et qui sont plus à risque de développer la maladie ont aussi démontré des différences significatives au niveau des concentrations en OPN en comparaison avec les sujets sains. En effet, plusieurs enfants à risque présentaient des niveaux d’OPN supérieurs à 800ng/ml suggérant un plus grand risque de développer une scoliose indiquant aussi que l’augmentation des niveaux en OPN précède le début de la maladie. / Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis that affects a significant number of young teenagers, mainly females. Historically, several hypotheses were postulated to explain the aetiology of AIS. The neuroendocrine hypothesis involving a melatonin deficiency as the source for AIS has generated great interest. This hypothesis stems from the fact that experimental pinealectomy in chickens, and more recently in rats maintained in a bipedal mode, produces scoliosis. The biological relevance of melatonin in idiopathic scoliosis is controversial since no significant decrease in circulating melatonin level has been observed in a majority of studies. Analysis of melatonin signal transduction in musculoskeletal tissues of AIS patients demonstrated for the first time a defect occurring in a cell autonomous manner in different cell types isolated from AIS patients suffering of the most severe form of that disease. We confirmed this defect by analysing more AIS patients and by using a new technology (cellular dielectric spectroscopy) which gives more precise results because it allows the measurement and analysis of receptor activation without the need to pretreat cells. This technique showed the same functional classification into three functional groups as identified by cAMP technique. Melatonin signalling dysfunction is caused by phosphorylation of serine residues affecting the activity of G inhibitory (Gi) proteins normally associated with melatonin receptors present at the cell surface. This defect could be caused by an imbalance in the activity of kinases or phosphatases that can regulate Gi proteins phosphorylation. Among these kinases PKCd was initially of interest because it has been shown that it can phosphorylate Gi proteins. We showed that this kinase interacts with melatonin receptor MT2 and that this interaction varies from one functional group to another. Thereafter, we moved one step further to characterise downstream effector regulated by melatonin. This work has led to the identification of osteopontin (OPN) which is a relevant candidate because it can act as a mecanosensor and it is involved in proprioception, postural and vestibular control. OPN was initially identified in pinealectomized chickens where it was shown to be upregulated at protein and mRNA levels only in scoliotic ones. We also used another animal model, C57Bl/6 mice which are naturally deficient in melatonin. We generated bipedal mice by amputating forelimbs of OPN knock-out mice, CD44 knock-out mice as well as C57Bl/6 wild type mice. Our results showed that all bipedal mice OPN Knock-out or CD44 Knock-out did not develop scoliosis contrasting with C57Bl/6 wt mice where 45% develop scoliosis. These results prompted us to investigate the role of this protein in scoliosis etiopathogenesis in humans. We showed an increase in the OPN circulating levels in AIS patients and this elevation correlates with disease severity. Elevated plasma OPN levels were also found in the asymptomatic at-risk group (offspring of scoliotic patients), suggesting that these changes precede scoliosis onset.

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

L’invasion métastatique des tumeurs humaines est un mécanisme complexe qui repose sur l’acquisition de nouvelles fonctionnalités par les cellules tumorales. Les protéines ADAM et plus particulièrement la protéine ADAM9, grâce à leur domaine extracellulaire se composant d’une activité métalloprotéasique et disintégrine, possèdent des fonctions importantes et nécessaires au processus d’invasion. Cependant, les mécanismes de régulation de la protéine restent globalement méconnus dans la pathologie cancéreuse. L’objectif de ce travail a consisté à évaluer le rôle de l’expression d’ADAM9 dans les mécanismes d’agressivité tumorale tels que l’adhérence cellulaire, la migration cellulaire ou l’angiogénèse ainsi que l’étude des mécanismes de régulation de l’expression de cette protéine. Compte tenu du fait que le peroxyde d’hydrogène est connu pour induire l’expression d’ADAM9, les premiers travaux ont eu pour objectif d’établir le lien entre stress oxydant, ADAM9 et adhérence tumorale. L’exposition des cellules d’adénocarcinome pulmonaire au peroxyde d’hydrogène induit une augmentation dose dépendante de l’expression de la protéine ADAM9 transmembranaire et de sa forme sécrétée. Les études in vitro ont permis d’établir que les capacités d’adhérence et d’invasion tumorale induites par le stress oxydant sont principalement médiées par les deux isoformes de la protéine ADAM9. Par ailleurs, l’expression d’ADAM9 provoque un accroissement de la néo-angiogénèse par l’intermédiaire d’un accroissement non transcriptionel de la biosynthèse d’IL8. Cette cytokine proangiogénique va interagir avec le récepteur CXCR2 et va permettre la mise en place d’une néovascularisation in vitro. Le développement d’un modèle de xénogreffe de cellule d’adénocarcinome pulmonaire a permis de confirmer le rôle majeur d’ADAM9 dans les processus de dissémination métastatique et d’angiogénèse tumoraux. L’étude de la modulation pharmacologique d’ADAM9 a reposé sur deux stratégies pharmacologiques différentes : d’une part l’interaction directe avec les différentes isoformes d’ADAM9 au moyen d’un anticorps neutralisant et d’autre part une action sur les mécanismes de transduction cellulaire tels que la protéine SRC ou la protéine kinase C. Ce travail a permis de mieux comprendre l’implication de la protéine ADAM9 au cours du processus de cancérogenèse de part sa participation aux étapes majeures que sont la dissémination métastatique induite par le stress oxydant et l’angiogenèse / Tumor invasion is a complex mechanism that is based on the acquisition of tumor cells new functions. ADAM proteins, especially protein ADAM9, through their extracellular domain consisting of a disintegrin and metalloprotease activity, have important functions and processes necessary for invasion. However, the mechanisms regulating protein remain largely unknown in cancer pathology. The objective of this work was to evaluate the role of ADAM9 expression in tumor aggressiveness such as cell adhesion, cell migration and angiogenesis and to study the mechanisms regulating this protein expression. Given the fact that hydrogen peroxide is known to induce the expression of ADAM9 protein, the first work aimed to establish the relationship between oxidative stress, adhesion and tumor ADAM9 expression. Hydrogen peroxide induces a dose-dependent increase of both expression and activity of ADAM9 on adenocarcinoma pulmonary cells. Oxidative stress induced ADAM9 expression and activity are mainly supported by the secreted form of ADAM9 protein. In vitro studies have shown that capacity of adhesiveness and invasiveness induced by oxidative stress are mainly mediated by the two forms of ADAM9 protein. In addition, ADAM9 protein expression induces neoangiogenesis through increased production of interleukin 8. This proangiogenic cytokine that interacts with the CXCR2 receptor is able to stimulate neovascularization in vitro studies. Development of a lung adenocarcinoma xenograft model confirmed that ADAM9 protein have an important role on metastasis process and tumor angiogenesis. The study of pharmacological modulation of ADAM9 expression was based on two different pharmacological strategies: the first interacts with different isoforms of ADAM9 using a neutralizing antibody and the second strategy modulate cell transduction mechanism such as SRC protein or protein kinase C (PKC). This work aims to understand the involvement of ADAM9 protein during the process of carcinogenesis, such as tumor invasion induced by oxidative stress and neoangiogenesis

Le système ubiquitine-protéasome joue un rôle central dans le contrôle de la progression du cycle cellulaire par la dégradation régulée de nombreuses protéines. Dans ce système, Fbw7 (aussi appelée Fbxw7, hCdc4, hAgo, Sel-10), est l'une des protéines F-box qui sert d'adaptateur de substrats pour l'une des plus importantes familles d'ubiquitine ligases : les complexes SCF (Skp1/Cullin/ F-box). Fbw7 assure la dégradation de plusieurs régulateurs positifs du cycle cellulaire : la cycline E, cMyc, c-Jun, Notch, Aurora A, mTOR, MCL1. En conséquence, l'altération des fonctions de Fbw7 conduit à des défauts de prolifération cellulaire, de différenciation et à de l'instabilité génomique. La mutation de Fbw7 dans les cancers entraîne une dérégulation de l'expression périodique cycline E qui n'est alors plus restreinte à la transition G1/S du cycle cellulaire. Nos résultats montrent qu'une isoforme, Fbw7, est exprimée dans les œufs de xénope matures arrêtés en métaphase II mais n'est pas fonctionnelle, expliquant la présence de grande quantité de cycline E dans les œufs à cette phase mitotique. Nous montrons que Fbw7 est maintenue inactive sous forme poly-ubiquitylée suite à sa phosphorylation par une PKC jusqu'à la fin des cycles embryonnaires rapides, au moment où la cycline E est brutalement dégradée. Nous montrons que la régulation négative de Fbw7 par PKC est conservée au cours des cycles cellulaires somatiques des cellules humaines, et contribue à l'expression périodique de la cycline E. Ces résultats mettent en évidence un nouveau mécanisme critique pour la régulation de Fbw7 au cours du cycle cellulaire et suggèrent que les fonctions de Fbw7 peuvent être altérées par une dérégulation de PKC, un phénomène observé dans de nombreux types de tumeurs humaines. / The ubiquitin-proteasome system plays a central role in the control of cell cycle progression through the regulated degradation of numerous critical proteins. In this process, one key family of ubiquitin ligases are the SCF (Skp1/Cul-1/F-box) complexes, in which F-box-bearing proteins act as substrate-recruiting factors. Fbw7 (also known as Fbxw7, hCdc4, hAgo, Sel-10) is one such F-box protein. It controls the stability and thus the levels of several positive regulators of the cell cycle, including cyclin E, cMyc, c-Jun, Notch, Aurora A, mTOR, Mcl1. As a consequence of its biological roles, alterations of the functions of Fbw7 lead to defects in cellular proliferation, differentiation and genetic instability. As seen in cancers, mutation of Fbw7 leads to deregulation of cyclin E expression, which is no more restricted to the G1-S phase boundary of the cell cycle. Here we report that Fbw7, although expressed in mature Xenopus eggs arrested in metaphase II, is not functional, explaining why cyclin E can be stockpiled in this mitotic-like phase. We found that, in these eggs as well as in early Xenopus embryos, Fbw7 is maintained under a PKC-dependent poly-ubiquitylated state until the end of the early rapid cleavage cycles where cyclin E is abruptly degraded. Importantly, we show that this PKC-dependent negative regulation of Fbw7 is conserved during human somatic cell cycles, resulting into the periodic expression of cyclin E. These findings reveal a novel mechanism critical for the temporal regulation of Fbw7 and suggest that the key functions of Fbw7 can be altered by PKC dysregulation, a mechanism known to occur in many types of human tumours.

Des études menées au laboratoire avaient démontré un rôle critique de l’inflammasome NLRP3 dans l’asthme allergique en réponse à l’ovalbumine en absence d’adjuvant. Mes travaux de thèse ont porté sur le rôle de NLRP3 et de la caspase-1 dans un modèle murin d’inflammation pulmonaire induite par l’allergène d’acarien HDM. Nous avons montré un rôle régulateur de la caspase-1 dépendant de l’inflammasome NLRP3 et la molécule adaptatrice ASC mais pas de l’inflammasome NLRC4. Cette régulation de la réponse allergique se caractérise par une augmentation de l’infiltration des éosinophiles, de l’hyperréactivité bronchique et de la production des cytokines de type Th2 telles que l’IL-4, l’IL-5, l’IL-13 et l’IL-33 dans les poumons. Nous avons montré que les mécanismes responsables de cette régulation sont associés à l’IL-33 produite par les macrophages et que la neutralisation de l’IL-33 par administration locale de la protéine de fusion au récepteur ST2 (muST2-Fc) atténue les caractéristiques de l’asthme allergique. Ces résultats suggèrent que l’activation de la caspase-1 réduit la production d’IL-33 in vivo et régule ainsi la réponse l’inflammation pulmonaire induite par HDM et la réponse Th2. D’autre part, nous nous sommes intéressés au rôle de la Protéine Kinase C thêta (PKC-θ) dans ce même modèle d’inflammation pulmonaire. Nous avons démontré que PKC-θ joue non seulement un rôle protecteur dans l’asthme allergique mais également un rôle critique pour la prolifération et l’activation des cellules lymphoïdes innées (ILC2). D’autre part, l’inhibition de PKC-θ in vivo par administration orale de son inhibiteur spécifique C20 (BIX02656) atténue l’inflammation pulmonaire et la production d’IL-5 et d’IL-13. Nous suggérons que PKC-θ est impliquée dans la différenciation des Th2 et des ILC2 via un mécanisme dépendant des facteurs de transcription IRF4 et NFAT-1. Au total, mes travaux de thèse mettent en exergue deux molécules IL-33 et PKC-θ qui pourraient constituer des cibles thérapeutiques potentielles. / Studies from our laboratory have shown a critical role of NLRP3 inflammasome in response to ovalbumin allergen. In the present study we investigate the role of NLRP3 and caspase-1 in a mouse model of pulmonary inflammation induced by HDM. We have shown a regulatory role of caspase-1 dependant of the NLRP3 inflammasome and the adaptator molecule ASC but not NLRC4. The regulation of the allergic response is characterized by an increase of eosinophilia, bronchial hyperreactivity and Th2 cytokines production (IL-4, IL-5, IL-13 and IL-33) in lungs. We have shown that mechanisms responsible of this regulation are associated with IL-33 production by macrophages and that neutralization of IL-33 by local administration of a fusion protein of the ST2 receptor (muST2-Fc) reduce characteristics of asthma. These results suggest that caspase-1 activation reduce IL-33 production in vivo regulating lung inflammation and Th2 response induced by HDM. Moreover, we investigate the role of the Protein Kinase C theta (PKC-θ) in allergic airway inflammation. We have demonstrated that PKC-θ plays a protective role in allergic asthma but is critical for the activation and proliferation of innate lymphoid cells (ILC2). In addition, in vivo inhibition by oral administration of PKC-θ specific inhibitor C20 (BIX02656) reduces pulmonary inflammation with IL-5 and IL-13 production. We suggest that PKC-θ is implicated in Th2 and ILC2 differenciation by a mechanism dependant on transcription factors IRF4 and NFAT-1. Finally, my thesis projects describe IL-33 and PKC-θ as potential therapeutic targets for allergic lung inflammation.