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Chez les mammifères, l'environnement est un puissant régulateur de l'expression des gènes. Par exemple, en fonction de l'alimentation, la disponibilité en nutriments, en particulier en acides aminés, est responsable de l'induction de l'expression d'un certain nombre de gènes. Ainsi, des mécanismes moléculaires sont mis en jeu de façon à permettre la détection de ces variations et d'y répondre de façon appropriée. Lorsque l'un des neuf acides aminés essentiels vient à manquer, on observe une augmentation de la transcription de certains gènes. Cette activation de la transcription requière d'une part l'accumulation du facteur de transcription ATF4 et d'autre part la phosphorylation du facteur de transcription ATF2. La voie ATF4 a été identifiée et relativement bien décrite. En revanche les éléments régulateurs de la voie de signalisation en amonts du facteur ATF2 restent inconnus. Le but de ma thèse était donc de déterminer les différents intermédiaires responsables de la phosphorylation d'ATF2 en réponse à une carence en acides aminés. J'ai ainsi montré qu'une carence en acides aminés provoque la mise en jeu d'un module MAPK composé de MEKK1>MKK7>JNK2 contrôlant la phosphorylation d'ATF2 sur les résidus thréonine 69 et 71. J'ai montré que ce module est régulé en amont par deux GTPases Cdc42+Rac1 et par la protéine Gα12. Enfin, j'ai démontré l'impact de cette nouvelle voie de signalisation sur la transcription AARE-dépendante du gène ATF3 en réponse à une carence en acides aminés.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

PhD (Microbiology) / Department of Microbiology / Background Conversion of Cholesterol to 25-HydroxyCholesterol (25HC) by Cholesterol 25-hydroxylase (CH25H) has been shown to exert broad antiviral properties. Given its antiviral activities, CH25H is part of an increasingly appreciated connection between type I interferon (IFN-I) and lipid metabolism. Moreover, the details of this connection appear to differ in mouse and human cells. Nevertheless, the molecular basis for the induction of CH25H in humans is not known. Objective Elucidation of signaling and transcriptional events for induction of CH25H expression is critical to design therapeutic antiviral agents. Materials and methods: Wildtype THP-1 monocytic cell-line or THP-1 MyD88 Knockout cell-line were treated with PMA for 72 hours for differentiation into macrophages. Differentiated macrophages or Microglial cells were stimulated with either TLR-agonists, pro-inflammatory cytokine, or interferons, and CH25H mRNAs expression levels were measured by qPCR. Results In this study, we show that CH25H is induced by Zika virus infection or TLR stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines including 1L- 1, TNF-, and IL-6, and this induction depends on STAT-1 transcription factor. Additionally, we have observed that ATF3 weakly binds to the CH25H promoter, suggesting co-operation with STAT-1. However, ZIKV induced CH25H was independent of type I interferon. Conclusion This study has demonstrated for the first time that pro-inflammatory cytokines such as 1L-1, TNF-, and IL-6 induce CH25H expression. Moreover, this provides further understanding to the connection between innate immunity and sterol metabolism and encourages the exploration of cytokines in antiviral immunity. / NRF

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Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In obesity their production is dysregulated, which is one of the major factors contributing to the onset of a chronic low-grade systemic inflammation representing a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis was to analyze the secretion of selected adipocytokines (adiponectin, IL6 and MCP1) by in-vitro differentiated adipocytes, isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. In case of adiponectin, the secretion of its isoforms was analyzed too. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. At the same time the development of the endoplasmic reticulum (ER) in the course of adipogenesis was monitored by indirect...

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