<p>  </p> <p>Cyclic dinucleotides (CDNs) such as the bacterial CDNs (cyclic-di-AMP, cyclic-di-GMP and 3’3’cyclic GMP-AMP) and mammalian CDN, 2’3’-cGAMP, are essential immune response second messenger signaling molecules. These CDNs act via Stimulator of interferon genes (STING)-TANK Binding Kinase 1 (TBK1)-Interferon Regulatory Factor 3 (IRF3) pathway. However, data from our lab and others indicate that beyond the classical STING-TBK1-IRF3 pathway, CDNs also regulate other signaling axes related to both inflammatory and non-inflammatory pathways. But, a global view of how these CDNs affect signaling in diverse cells or through non-STING pathways is lacking. There is also paucity of data on CDN modulated kinases and no global assessment of phosphorylation events that follow cyclic GMP AMP synthase (cGAS)-STING axis stimulation in immune cells. Herein, I have used a proteomics approach to determine signaling pathways regulated by bacterial CDNs, c-di-GMP and c-di-AMP in human gingival fibroblasts such as pathways related to nucleotide excision repair (NER) which ordinarily do not channel through STING (Chapter 3). Additionally, with the use of phosphoproteomics and bioinformatics, this project accomplished a system-wide phosphorylation analyses of T cells treated with 2’3’cGAMP and showed that 2’3’cGAMP impact various, yet unreported critical kinases (E.g. LCK, ZAP70, ARG2) and signaling pathways important for T cell function (Chapter 4). Asides known interferon signaling, these differentially phosphorylated kinases were involved in T cell receptor (TCR) signaling, myeloid cell differentiation, cell cycle regulation, and regulation of double strand break repair. </p> <p>Another area of interest addressed by this project is the discovery of novel antibacterial agents against multi-drug resistant (MDR) bacteria. Thus, in Chapters 5 and 6, I show the identification, antibacterial activity and characterization of <strong>HSD1835</strong> and <strong>HSD1919 </strong>as novel SF5 and SCF-containing membrane active compounds, highly potent against preformed MDR biofilms with fast bactericidal activity against persister bacteria. Plus, an exciting addition to the fight against MDR bacteria in Chapter 7, the discovery of <strong>HSD1624</strong> and analogs, which are able to re-sensitize MDR and colistin resistant bacteria such as <em>Pseudomonas aeruginosa</em> from a colistin MIC of 1024 μg/mL to 0.03 μg/mL (64000-fold reduction). Ultimately, these compounds could be translated into anti-biofilm and, anti-MDR bacteria therapeutics, preventing repeated surgeries due to infections, and saving lives. </p>

La prématurité et la restriction de croissance fœtale (RCF) sont deux pathologies néonatales fréquentes, qui ont en commun des difficultés d'adaptation à la naissance, avec le développement d'une tubulopathie chez le prématuré, et le développement d'une hypertension artérielle (HTA) a l'âge adulte. L’objectif de ce travail était d’évaluer l'implication des voies de signalisation corticostéroïdes rénales dans la survenue de ces complications dans un modèle murin de prématurité induite par des lipopolysaccharides, et un modèle de RCF par exposition périnatale a la dexaméthasone. Dans ce travail nous avons montré que ces deux pathologies programment la survenue d’une HTA à l’âge adulte chez les mâles, associée à des altérations franches de la signalisation corticostéroïde rénale en période périnatale et une augmentation de la sensibilité rénale aux glucocorticoïdes à l’âge adulte. Dans le modèle de prématurité, nous avons identifié la transmission transgénérationelle d’anomalies de régulation de la pression artérielle chez les mâles jusqu’à la 3ème génération de souris, associée à une hypométhylation du promoteur de GILZ et une augmentation d’expression de GILZ. Notre étude a permis l’identification de potentiels mécanismes moléculaires impliqués dans la programmation fœtale de l’HTA, sur plusieurs générations, ce qui pourrait aboutir à une meilleure prise en charge des patients nés prématurés ou avec une RCF, et de leurs descendants. / Preterm birth and fetal growth restriction (IUGR) are prevalent neonatal diseases, which both induce poor perinatal adaptation, including the development of tubulopathy in premature infants, and the development of high blood pressure in adults. The objective of this work was to evaluate the involvement of renal corticosteroid signaling pathways in the development of these complications in a lipopolysaccharide-induced mouse model of preterm birth, and a dexamethasone-induced model of IUGR. In this work, we have shown that these two pathologies program the development of hypertension in former preterm and IUGR male mice, associated with strong alterations of renal corticosteroid signaling in the perinatal period, and an increase in renal sensitivity to glucocorticoids in adulthood. Moreover, we have identified a transgenerational inheritance of altered blood pressure regulation induced by preterm birth, in males, up to the 3rd generation of mice, associated with GILZ promoter hypomethylation and increased GILZ expression.Our study has identified potential molecular mechanisms involved in the fetal programming of hypertension, over several generations. These findings could facilitate better management of patients born prematurely

Le placenta humain est un organe indispensable au bon déroulement de la grossesse. La villosité choriale est l’unité structurale et fonctionnelle du placenta. Elle est constituée essentiellement de cellules trophoblastiques. Les cytotrophoblastes extra-villeux (CTEV) présentent des propriétés invasives et assurent l’ancrage du placenta dans l’endomètre maternel. De plus, une apoptose physiologique assure le renouvellement des cytotrophoblastes tout au long de la grossesse.Le Facteur Préimplantatoire (PIF) est un peptide de 15 acides aminés, sécrété par des embryons viables. Le PIF exerce un effet autocrine positif sur le développement embryonnaire. Le PIF est également impliqué dans le contrôle de l’immunité et de l’inflammation dans divers types cellulaires.Au cours de ce travail de thèse, nous nous sommes intéressés aux rôles du PIF dans le développement placentaire humain. Dans un premier temps, nous avons caractérisé l’expression protéique du PIF dans des placentas humains de 1er et 3ème trimestre de grossesse.Nous avons montré que i) l’expression du PIF diminue au cours de la grossesse et ii) le PIF est majoritairement exprimé dans les CTEV.Dans un second temps, nous avons mis en évidence que le PIF i) favorise l’invasion trophoblaste et ii) inhibe l’apoptose des CTEV en régulant la voie de signalisation de p53.Par ailleurs, des altérations de l’invasion et de l’apoptose trophoblastiques sont associées à des pathologies de la grossesse telles que la pré-éclampsie et le retard de croissance intra-utérin. Ainsi, dans un dernier temps nous avons montré que l’expression du PIF est diminuée dans des placentas humains de 3ème trimestre issus de grossesses pathologiques par comparaison avec des grossesses normales.L’ensemble de ces résultats démontrent que le PIF est un nouvel acteur de la placentation humaine. De plus, le PIF pourrait être considéré comme un nouveau biomarqueur des pathologies de la grossesse. / Human placentation is a critical step in the establishment of a successful pregnancy. The chorionic villus constitutes the structural and functional unit of the placenta. The extravillous trophoblast (EVT) is a placental cell type that differentiates from the highly proliferative cytotrophoblast located at the base of the anchoring villous. EVT have invasive properties, essential for placenta anchoring in the endometrium and uterine artery remodeling. Moreover, programmed cell death is an active process required for normal trophoblastic cell turnover during pregnancy.PreImplantation Factor (PIF) is a 15-amino-acid peptide secreted by developing embryos. PIF exerts autotrophic and protective effects on the embryo. PIF is also implicated in the control of immune and inflammatory processes in various cell types.In this work, we aimed to determine the direct effects of PIF on human placental development.In a first part, we characterized PIF protein expression in first and third trimester human placentas. We showed that PIF protein expression i) decreased over the course of the pregnancy and ii) was higher in EVT compared to villous trophoblast.In a second part, we showed that PIF i) enhanced pro-invasive capacities and ii) prevented cell death by regulating p53 signaling pathway in human EVT.Dysregulation of trophoblastic invasion and apoptosis have been associated with pregnancy pathologies. Thereby, in a last part, we showed that PIF protein expression was lower in placentas from preeclampsia and intra-uterine growth restriction as compared with non-pathological placentas. Altogether, we highlighted for the first time, that PIF is a new positive regulator of placental functions. PIF could be considered as a novel biomarker of a favorable outcome of pregnancy.

The drug discovery process is challenging, time-consuming, and costly, with drug target identification being an essential step in developing effective therapies. Drug repurposing offers a strategy for identifying new uses for existing drugs, aiming to simplify the process. Machine learning models and network analysis methods have demonstrated promise in both drug target identification and repurposing, providing powerful tools for analyzing complex biological data. This thesis will explore the applications of neural networks and multilayer biological networks for drug repurposing opportunities and network inference problems applied to signaling pathways. A novel machine learning and network-based workflow is presented for identifying drug targets for cystinosis, a rare disease that causes progressive kidney disease, currently lacking effective therapies to prevent the kidney failure. This approach permits to recapitulate the disease mechanisms in the context of renal tubular physiology and identify candidate drug targets for further validation using a cross-species workflow and disease-relevant screening technologies. While machine learning approaches have shown promise, they often need more mechanistic understanding, which is necessary for robust drug target identification and repurposing strategies. Mechanistic models provide crucial insights into the underlying biological mechanisms, complementing machine learning techniques. However, inferring mechanistic signaling networks from omics data poses challenges due to non-identifiability, resulting in multiple valid solutions consistent with the data. After that, the focus shifts towards quantifying signaling model diversity through solver-agnostic solution sampling with CORNETO, an ongoing effort that aims to unify network inference problems via constrained optimization. Mechanistic signaling networks can be inferred from omics data and prior knowledge using combinatorial optimization and mathematical solvers to find the optimal network. However, this problem is in general, non-identifiable, and several solutions may be equally valid. Ignoring the existence of these alternative solutions leads to an incomplete picture of the hypothesis space of consistent mechanistic signaling networks. To alleviate this issue, an algorithm to explore the space of alternative solutions and to conduct sensitivity analysis on the optimal solution is implemented and presented. These algorithms are applied to data from pancreatic cancer cell lines treated with kinase inhibitors to study cellular responses to drug perturbations by inferring mechanistic signaling networks from omics data.

Die Regulation der Nahrungsaufnahme erfolgt zentral im Hypothalamus wo eine Vielzahl von G-Protein-gekoppelten Rezeptoren exprimiert werden die an der Gewichtsregulation beteiligt sind. Periphere hormonelle Signale aktivieren ihre korrespondierenden Rezeptoren im Nucleus arcuatus (ARC) oder im Nucleus paraventricularis (PVN) und modifizieren dadurch sowohl das anorexigene System, z.B. über die Stimulation des Melanocortin-4-Rezeptors (MC4R) im PVN, als auch das orexigene System mit dem Neuropeptide Y (NPY) sowie dem Agouti-related Protein (AgRP). Im Zuge einer systematischen Interaktionsstudie wurden verschiedene GPCRs, die entweder mit dem MC3R oder dem MC4R auf dem gleichen Neuron koexprimiert werden und nachweißlich die Appetit- und Gewichtregulation beeinflussen, untersucht. Basierend auf den Ergebnissen von Sandwich-ELISA und FRET- (Fluoreszenz-Energie-Transfer)Studien konnte eine Interaktion des MC3R mit dem Growth hormone secretagogues Rezeptor (GHSR) bestimmt werden, die beide auf den NPY/AgRP-Neuronen des ARC lokalisiert sind. Der MC3R gehört zu den Gαs bindenden Rezeptoren wohingegen GHSR über den Gαq vermittelten Signaltransduktionsweg signalisiert. Es konnte eine Erhöhung der induzierten cAMP-Spiegel infolge der Stimulation des MC3R sowohl mit α-, als auch β- und γ-MSH für die Koexpression von MC3R mit GHSR im Vergleich zum MC3R Homodimer ermittelt werden. Die Charakterisierung des neuen Signalisierungsverhaltens des Heterodimers unter der Verwendung verschiedener Inhibitoren zeigte eine Aktivierung von Gαi in Gegenwart der endogenen Agonisten beider Rezeptoren. Die Beobachtung unterschiedlicher Regulationsmuster nach der Kostimulation des Heterodimers in Abhängigkeit von α- oder γ-MSH jeweils in Anwesenheit von Ghrelin verweist auf komplexe Interaktionsmechanismen zwischen dem Melanocortin- und dem Ghrelin-Rezeptor innerhalb der hypothalamischen Gewichtsregulation. / Food intake is centrally regulated in hypothalamic nuclei where many GPCRs are expressed which are known to be involved in weight regulation.Peripheral hormonal signals activate their corresponding receptors in the arcuate nucleus (ARC) or paraventricular nucleus (PVN) and modulate the orexigenic (appetite-supressing) pathway mediated by stimulation of the melanocortin-4-receptor (MC4R) as well as the anorexigenic (appetite-stimulating) pathway including neuropeptide Y (NPY) and agouti-related protein (AgRP). In a systematic approach we investigated the interaction of a selective number of GPCRs which are co-expressed on the same neurons like MC3R or MC4R and know to play an essential role in hypothalamic weight regulation. Based on the results of a sandwich ELISA and fluorescence resonance energy transfer (FRET) approach we report the interaction of the MC3R and the growth hormone secretagogue receptor (GHSR) which are co-expressed on arcuate NPY/AgRP neurons. It is known that MC3R couple to the Gαs whereas GHSR couple to the Gαq signaling pathway. However, here the co-expression of MC3R and GHSR reveal a profoundly increase cAMP-accumulation after melanocortin (α-, β- and γ-MSH) challenge, that is higher compared to MC3R activation alone. In-depth characterization of the new signaling properties of the MC3R/GHSR heterodimer by different inhibitors revealed the activation of Gαi in the presents of both endogene agonists. The observation of different regulatory pattern after co-stimulation of the heterodimer depending on the endogenouse ligands (α- or γ-MSH) of MC3R reflect complex functional interaction mechanisms between melanocortin and ghrelin receptors within the hypothalamic signaling pathways of weight regulation.

Les polymorphonucléaires neutrophiles (PMNs) représentent une arme primordiale dans la défense contre divers agents pathogènes; notamment les bactéries, les champignons, les cellules tumorales de même que les cellules infectées par des virus. Cependant, certaines pathologies reliées à l’inflammation chronique soulèvent l’implication des neutrophiles notamment dans l’arthrite rhumatoïde. La réponse inflammatoire persistante générée par l’activation et la survie des neutrophiles engendre une destruction des tissus environnants suite à la sécrétion non contrôlée de leurs produits cytotoxiques. Même si l’activation chronique des neutrophiles est néfaste dans plusieurs pathologies, elle pourrait s’avérer un bon outil en cas de neutropénie, comme c’est souvent le cas les patients ayant reçu des traitements de chimiothérapie. Ce projet fait suite aux travaux doctoraux de Lagraoui (1999). Il vise à identifier le(s) facteur(s) du liquide synovial qui augmente la survie des neutrophiles ainsi que le mécanisme d’action impliqué dans ce processus. Similairement au facteur semi-pur isolés par Lagraoui (1999), le milieu conditionné concentré (MCC) augmente la survie des PMNs de 75% (39% ± 9.5 vs 68% ± 2.5, p<0.01). Suivant le séquençage du MCC parallèlement au facteur semi-pur actif, deux protéines ont été identifiées à la fois dans le MCC et dans le facteur semi-pur soient : l’albumine et la fétuine. Notre projet vise donc à comparer les effets de l’albumine et de la fétuine à ceux du GM-CSF dans l’optique d’une thérapie alternative au GM-CSF en tant qu’adjuvant de chimiothérapie. La présence d’albumine, de fétuine ou de GM-CSF chez les PMNs incubés 24 heures avec la Mutamycin® induit une diminution du nombre de cellules en apoptose par rapport à la Mutamycin® (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). L’effet de l’albumine dépend de la voie de la kinase PI3 mais également celle la kinase ERK, alors que celle de la fétuine dépend de la kinase PI3. Similairement l’EPO, l’albumine et la fétuine supporte la différentiation des HSCs en précurseurs érythrocytaires de type BFU-E. Dans un modèle murin de chiomioprotection, l’albumine augmente la concentration cellulaire rapport au groupe contrôle des leukocytes de la rate (66 ±8 x106c/ml vs 81 ±16 x106c/ml) et du sang (3.6 ±0.4 x106c/ml vs 5.7 ±2.3 x106c/ml). Donc, in vitro, l’albumine et la fétuine sont comparables au GM-CSF au niveau fonctionalité et mécansimes d’action. Cependant, vu leur manque de spécificité, l’application thérapeutique en tant qu’adjuvant de chiomiothérapie de l’albumine et la fétuine est peu prometteuse. Par contre, les maladies dégénératives et les évènements ischémiques pourraient s’avérer de bonnes cibles thérapeutiques, principalement pour l’albumine. / Circulating polymorphonuclear neutrophils (PMN) possess a short half-life and are constantly renewed by the bone marrow to ensure the first-line of defense. Therefore, homeostasis must be maintained through a well-regulated process of apoptosis. Survival of PMN can be regulated by several cytokines as well as conditioned media (CM). Although PMN are crucial for protection against microorganisms, activated neutrophils can lead to severe tissue damage in diseases characterized by chronic inflammation. Indeed, in rheumatoid arthritis (RA), activated PMN contribute to tissue damage by releasing a number of destructive agents. On the other hand, chronic activation of PMN could prevent opportunistic infections present in immunosuppressed patients. This project addresses the isolation and mechanism of action of synovial liquid components on the survival of neutrophils based on previous work (Lagraoui, 1999). Following tangential flow filtration (MW cut off: 30 and 50 kDa), concentrated CM enhanced the viability (75%) of 24-hour cultured human neutrophils isolated from peripheral blood of healthy volunteers (39% ± 9.5 vs 68% ± 2.5, p<0.01) as seen in Lagraoui (1999) previous work. N-terminal protein sequence analysis of the concentrated CM and fractionated conditioned media from previous work revealed 2 known proteins contained in both analysis: albumin, and fetuin. In view of the importance of neutrophiles in immune defense, we compared the benefits of albumin and fetuin to those of granulocytes macrophages-colony stimulating factor (GM-CSF), a growth factor used as an adjunct to cancer chemotherapy. Albumin and fetuin were tested by the AnnexinV-FITC/7-AAD method and displayed an inhibition of neutrophil apoptosis of two to three folds relative to control value. Moreover, albumin (A : 200μM) and fetuin (F : 200μM) rescue human PMN from mutamycin-induced apoptosis, comparable to GM-CSF (GM : 10ng/ml); (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). Albumin also induces cellular signaling pathways activation via PI3-K and ERK, whereas fetuin acts through PI3-K pathway only. They induce the differentiation of HSCs into erythrocytes progenitors BFU-E. In immunosuppressed mice, albumin protects white blood cells depletion induced by cytotoxic agent from spleen and blood. Considering all the benefits of albumin and fetuin, their targeting as an adjunct to cancer chemotherapy could be disappointing in view of their lack of specificity. On the other hand, their multiple benefits could have a major impact on neurodegenerative disorders and ischemic events.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

O Vírus Polioma (Py), induz a formação de múltiplos tumores em camundongos, e causa transformação maligna em cultura, levando à menor dependência de soro e ancoragem para crescimento. Dentre as proteínas codificadas pelo vírus polioma, o antígeno MT (Middle T), isoladamente, é capaz de transformar linhagens celulares imortalizadas e gerar tumores quando injetadas em camundongos, sendo que este caráter transformante está relacionado à sua capacidade de se ligar e modular a atividade de diversas proteínas, muitas das quais relacionadas com o controle do ciclo celular. Para melhor compreender os mecanismos moleculares da transformação maligna mediada por MT e do controle do ciclo celular, o objetivo deste trabalho foi identificar e caracterizar novas vias de transdução de sinal e novos genes regulados por MT. Para a busca de genes regulados pela atividade de MT, comparou- se o perfil da expressão gênica da linhagem que expressa MT (MTWT) com o da linhagem controle (PLJ) através de três metodologias: membranas de nylon comerciais (Atlas Mouse Cancer 1.2 - Clontech), bibliotecas subtrativas de cDNA construídas através da técnica de RDA (Análise de Diferença Representacional) e lâminas comercias de microarrays (CodeLink Bioarrays - GE Healthcare). As três metodologias permitiram a identificação de cerca de 1.200 genes-candidatos a serem regulados por MT, dos quais 84 genes foram selecionados para terem sua expressão diferencial analisada por Real-Time PCR. Para identificar possíveis vias de transdução de sinal relacionadas à regulação dos genes analisados utilizou-se, como template, além do cDNA proveniente das linhagens PLJ e MTWT, cDNAs de linhagens que expressam a proteína MT mutada nas tirosinas 315, 322, 250 e 315/322. Após confirmação por Real-Time PCR, dois genes foram selecionados para analise funcional: Dlk1 (Delta Like-1 homolog) que, apesar de não ter relação direta com o controle do ciclo celular, está ligado à formação de alguns tumores, e MN1 (Meningioma 1), um gene pouco caracterizado que já foi relacionado à formação de leucemias mielóides e meningiomas. - I - RESUMO Para caracterizar o papel de MN1 na linhagem PLJ, foram geradas linhagens PLJ que superexpressam RNAi de MN1. A análise dos parâmetros de crescimento, em substrato sólido e semi-sólido, da linhagem PLJ parental e PLJ expressando o RNAi de MN1 não apresentou alterações estatisticamente significativas. Para o estudo do efeito de Dlk1 na transformação mediada por MT, foram geradas linhagens MTWT que superexpressam Dlk1, o que levou a um aumento estatisticamente significativo na taxa de divisão celular, quando comparada com a linhagem controle (vetor vazio), indicando um possível papel de Dlk1 no sinal desencadeado por MT e no controle do ciclo celular. Visando identificar novas vias de regulação entre os genes analisados, os níveis de expressão gênica dos demais genes foram avaliados por Real-Time PCR e analisados através de correlação de Pearson, aplicada a cada par de genes. Após a análise, foram identificadas 64 possíveis vias de regulação, sendo que 13 destas foram descritas pela primeira vez no presente trabalho e as demais, por já estarem descritas na literatura, permitiram validar o modelo estudado. / Polyomavirus (Py) induces multiple tumors in mice and malignant transformation in culture, leading to a lower serum and anchorage dependence. Among the proteins coded by the polyomavirus genome, the MT antigen (Middle T) alone is able to transform immortalized cell lines and to generate tumors when injected into animals. This transforming ability is related to its capacity of bind to and modulate several proteins, many of which are related to cell cycle control. Aiming at a better understanding not only of the molecular mechanisms of malignant transformation mediated by MT, but, also, of the processes underlying cell cycle control, the objectives of this work were to identify and characterize novel genes and signal transduction pathways regulated by MT. For screening of the MT regulated genes, the gene expression profiles of the MT expressing MTWT cell line and of the PLJ control cell line were compared using three methodologies: commercial nylon membranes (Atlas Mouse Cancer 1.2 - Clontech), subtracted cDNA libraries constructed using the RDA methodology (Representational Difference Analysis) and commercial microarrays (CodeLink Bioarrays - GE Healthcare). These three methodologies allowed us to identify about 1,200 candidate genes as being regulated by MT, from which 84 genes were selected to have their differential expression analyzed by Real- Time PCR. In an attempt to identify possible signal transduction pathways related to these genes, we used as templates, in addition to cDNAs from the PLJ and MTWT cell lines, cDNAs from cell lines which express MT mutated in tyrosines 315, 322, 250 and 315/322. After confirmation by Real-Time PCR, two genes were selected for further functional analysis: Dlk1 (Like-1 Delta homolog), which is related to the differentiation process and to formation of some tumors, and MN1 (Meningioma 1), a newly identified gene, which can induce leukemia and meningioma tumor formation. To characterize the functional role of MN1, we generated PLJ cell lines in which MN1 was knocked-down by RNAi. The growth characteristics in solid and semi-solid substrates of control PLJ and of - III - ABSTRACT MN1 knocked down PLJ cells did not present any statistically significant alteration. To study the effect of Dlk1 in MT mediated cell transformation, we generated MTWT cell lines overexpressing Dlk1, which induced a significant increase in cell division rate when compared to the empty vector control cell line, indicating a possible role of Dlk1 in the MT induced signals and in cell cycle control. Aiming at identifying new regulatory pathways among the genes analyzed, the Real-Time PCR results were subjected to analysis by Pearson correlation, applied to each pair of genes, allowing us to identify 64 possible regulatory pathways, 13 of which were described, for the first time, in the present work. The other pathways, which had previously been described in the literature, allowed us to validate the model studied.

L’eczéma allergique de contact (EAC) est une réaction inflammatoire aiguë médiée par les lymphocytes T (LT), survenant suite à l’exposition répétée de la peau avec une molécule allergisante présente dans l’environnement quotidien ou professionnel. Les molécules allergisantes sont des composés de faible poids moléculaire, appelés haptènes, qui activent les cellules dendritiques (DC). Les DC jouent un rôle essentiel dans la mise en place d’un EAC : elles acquièrent un phénotype mature, contrôlé par la voie des MAPK et la voie NF-B, leur permettant de présenter l’haptène aux LT afin d’initier ainsi une réponse immunitaire spécifique.Nous avons identifié au sein de la DC une nouvelle kinase, la protéine kinase CK2, indispensable à l’acquisition d’un phénotype mature et à la sécrétion de cytokines pro-inflammatoires clés dans l’orientation d’une réponse immunitaire. L’activité de la CK2 dans la DC est nécessaire à la génération d’une réponse Th1 en contrôlant la sécrétion d’IFN par les LT, et maintient une réponse Th17 préexistante. De plus, la CK2 permet à la DC de contrôler l’induction d’une réponse Th2 spontanée. Par ailleurs, la CK2 contrôle l’expression des gènes cibles de Nrf2, un facteur de transcription majeur dans la lutte contre le stress chimique induit par les haptènes. L’activation de Nrf2 met en jeu de nombreuses voies de signalisation, et nous avons mis en évidence c-Jun, facteur de transcription activé par les molécules allergisantes, comme un potentiel partenaire transcriptionnel de Nrf2. / Allergic contact dermatitis represents a severe health problem with increasing worldwide prevalence. It is a T cell-mediated inflammatory skin disease caused by chemicals present in daily or professional environment. Contact sensitizers are low molecular weight compounds termed haptens. These molecules are known to induce an up-regulation of phenotypic markers and cytokine secretion in dendritic cells (DCs), professional antigen-presenting cells, leading to the generation of effector T lymphocytes (LT).We identified a new kinase, termed CK2 (formerly casein kinase 2), as a key kinase in DCs in the acquisition of a mature phenotype and in the production of pro-inflammatory cytokines, involved in T cell polarization in response to contact sensitizers. CK2 activity in DC is necessary to induce a Th1 polarization by controlling the secretion of IFN- by LT, and maintains a pre-existing Th17 response. Moreover, CK2 in DC negatively controls a spontaneous Th2 response.Finally, CK2 controls the expression of Nrf2 target genes mRNA. Nrf2 is a protective transcription factor playing a major role in detoxification, oxidative stress and allergic inflammation generated by contact sensitizers. Nrf2 activation involves different kinases and we highlight that c-Jun could be bound to Nrf2 to generate an active transcriptional complex in response to chemicals.

Au cours de ma thèse, mes projets de recherche ont visé à mieux comprendre les mécanismes moléculaires contrôlant la prolifération et la spécification des cellules progénitrices dans la rétine du xénope à travers trois projets principaux. Le réseau de régulation qui contrôle la spécification des cellules progénitrices vers les sous-types neuronaux est à ce jour très peu connu. C’est dans ce contexte que j’ai étudié le rôle du facteur de transcription à domaine bHLH, Ascl1, dans la détermination des sous-types rétiniens au cours du développement. Par des approches in vivo de gain et perte de fonction d’Ascl1, des expériences d’épistasie et la recherche de ses cibles transcriptionnelles, j’ai pu mettre en évidence qu’Ascl1 (i) est impliqué dans la genèse des neurones GABAergiques rétiniens, (ii) qu’il est épistatique sur des facteurs glutamatergiques tels que Neurog2, NeuroD1 ou Atoh7, (iii) que son activité GABAergique est conférée par son domaine basique de liaison à l’ADN et (iv) que cette activité implique la régulation directe du facteur de transcription Ptf1a. Ces données ajoutent donc une nouvelle pièce au réseau transcriptionnel gouvernant la spécification des sous-types GABAergiques au cours du développement de la rétine. La mise en place correcte des types et sous-types cellulaires de la rétine nécessite une coordination avec le moment de sortie du cycle cellulaire des progéniteurs rétiniens. Dans ce contexte, j’ai contribué à l’avancée d’un projet visant à étudier le réseau de signalisation contrôlant la prolifération des précurseurs de la rétine. Par des approches in vivo, génétiques et pharmacologiques, cette étude a montré que les voies Wnt et Hedgehog s’antagonisent pour réguler l’activité proliférative des cellules souches et progénitrices rétiniennes. Nos données préliminaires suggèrent que ces voies agissent de façon opposée à la fois sur la sortie et sur la cinétique du cycle cellulaire. Ce travail nous a conduit à proposer un modèle selon lequel ces voies Wnt et Hedgehog réguleraient la balance entre prolifération et différenciation dans la rétine post-embryonnaire. Enfin, dans le but d’élargir nos connaissances sur les réseaux de signalisation et les réseaux transcriptionnels impliqués dans le contrôle de la prolifération et de la détermination cellulaire dans la rétine, j’ai également contribué à la recherche de nouveaux marqueurs spécifiques des différentes populations cellulaires rétiniennes au travers d’un crible à grande échelle par hybridation in situ. De nombreux gènes spécifiquement exprimés dans les cellules souches ou les cellules progénitrices constituent des gènes candidats pour de futures approches fonctionnelles. / My thesis research work aimed to better understand the molecular mechanisms underlying proliferation and specification of retinal progenitors in Xenopus through three main projects. As the mechanisms governing specification of retinal progenitors towards the different neuronal subtypes are still poorly understood, I focused my work on the role of Ascl1, a bHLH transcription factor, in cell-subtype determination during retinogenesis. Using in vivo gain- and loss-of-function experiments, I have investigated Ascl1’s epistatic relationships with other bHLH factors and identified its transcriptional targets. My results indicate that Ascl1 (i) is implicated in the genesis of retinal GABAergic neurons (ii) is epistatic to glutamatergic factors such as Neurog2, NeuroD1 and Atoh7 (iii) that its basic DNA-biding domain is sufficient for its GABAergic-inducing activity (iv) and that this activity involves a direct regulation of the Ptf1a transcription factor. The correct order of neural cell types and subtypes formation is tightly coordinated with the timing of cell-cycle exit of retinal progenitors. Ongoing work in the laboratory, to which I have contributed, was therefore investigating the role of signaling pathways controlling retinal precursor proliferation in this process. Using in vivo genetic and pharmacological tools, we have shown that an antagonistic cross-regulation between Wnt and Hedgehog signaling governs stem cell and progenitor proliferation in post-embryonic retina. Preliminary data shows that Wnt and Hedgehog have opposite effects on both cell cycle exit and kinetics and may therefore regulate the proliferation/differentiation balance in the post-embryonic retina. Lastly, in order to broaden our knowledge on the transcriptional and signaling networks which govern proliferation and cell fate determination in the retina, I have participated in a large scale screen by in situ hybridization aiming to identify new molecular markers of different retinal cell population. Many genes that are exclusively expressed in retinal stem cells or progenitors are promising candidates for future functional studies.