Upon fertilization the paternal and maternal genomes unite, giving rise to the embryo, with its unique genetic code. All cells in the human body are derived from the fertilized ovum: hence they all contain (with a few exceptions) the same genetic composition. However, by selective processes, genes are turned on and off in an adaptable, and cell type-specific, manner. The aim of this thesis is to investigate how signals coming from outside the cell and epigenetic factors residing in the cell nucleus, cooperate to control gene expression. The transforming growth factor-β (TGF-β) superfamily consists of around 30 cytokines, which are essential for accurate gene regulation during embryonic development and adult life. Among these are the ligands TGF-β1 and bone morphogenetic (BMP) -7, which interact with diverse plasma membrane receptors, but signal via partly the same Smad proteins. Smad4 is essential to achieve TGF-β-dependent responses. We observed that by regulating transcription factors such as Id2 and Id3 in a specific manner, TGF-β1 and BMP-7 achieve distinct physiological responses. Moreover, we demonstrate that CTCF, an insulator protein regulating higher order chromatin conformation, is able to direct transcription by recruiting RNA polymerase II to its target sites. This is the first mechanistic explanation of how an insulator protein can direct transcription, and reveals a link between epigenetic modifications and classical regulators of transcription. We also detected that DNA loci occupied by CTCF replicate late. The timing of replication is a crucial determinant of gene activity. Genes replicating early tend to be active, whereas genes replicating late often are silenced. Thus, CTCF can regulate transcription at several levels. Finally, we detected a substantial cross-talk between CTCF and TGF-β signaling. This is the first time that a direct interplay between a signal transduction pathway and the chromatin insulator CTCF is demonstrated.

Chronic graft dysfunction (CGD) is an important post-transplant complication. CGD can be considered as an impaired repair process, which ultimately leads to the loss of graft function.To study non-immunological factors contributing to the development of CGD in kidney grafts we used in vitro and in vivo models, and clinical studies. We studied the actions of hyperlipidemia in vitro. LDL induced increased expression of TGF-β1 and TGF-β receptors type I and type II. Smad2 phosphorylation could be induced by conditioned medium from mesangial cells incubated with LDL. The effects of Fluvastatin and AT1 receptor blocker Candesartan cilexetil on aortic graft arteriosclerosis in the rat were evaluated. Fluvastatin neither alone nor in combination with Cyclosporine A affected allograft remodelling, but reduced neointima formation in isografts. Candesartan cilexetil treatment reduced graft arteriosclerosis. The effect is explained by the reduction of TGF-β1 expression. We investigated the effects of Carvedilol in patients with CGD. Carvedilol failed to alter the CGD progression despite the efficient control of blood pressure, and a beneficial effect on lipid pattern and oxidation. Close control of CyA blood levels is recommended due to interaction between CyA and Carvedilol. Measurement of Ab-oxLDL in kidney graft recipients demonstrated that these patients had lower Ab-oxLDL levels as compared with the control group. Decreased Ab-oxLDL levels were associated with graft loss due to acute rejection and with ischemic heart disease. In this thesis we have addressed several important complex issues, which are interconnected: (1) development of chronic graft dysfunction (2) lipoproteins and their role in inducing pathological conditions like atherosclerosis and graft damage, (3) oxidation, (4) TGF-β and its' role in different pathological conditions, including renal and vascular damage.

Transforming growth factor-β (TGF-β) signaling is initiated when the ligand binds to type II and type I serine/threonine kinase receptors at the cell surface. Activated TGF-β type I receptors phosphorylate R-Smads which relocate, together with co-Smads, to the cell nucleus and regulate transcription. Enhancement or repression of Smad-specific gene targets leads to intracellular protein compositions which organize functional complexes and thus govern cellular processes such as proliferation, migration and differentiation. TGF-β/Smad signaling relays are regulated by various post-translational modifications. From receptors to gene promoters, intricate interplays between phosphorylation, acetylation, ubiquitination and numerous other modifications, control Smad signaling initiation and duration. However, many steps in the cascade, including receptor internalization, Smad nuclear shuttling and transcriptional termination, still remain elusive. The open gaps in our understanding of these mechanisms most likely involve additional post-translational regulations. Thus, the aim of the present investigation was to identify novel modulators of TGF-β/Smad signaling. In the first part of this thesis, we show the importance of ADP-ribosylation in Smad-mediated transcription. We identified poly(ADP-ribose) polymerase 1 (PARP-1) as a Smad interacting protein. Our work revealed that PARP-1 forms direct interactions with Smad3/4, and PARylates residues in their MH1 domains. This modification restricts Smads from binding to DNA and attenuates Smad-activated transcription. PARylation is reversed by the glycohydrolase PARG. We provide evidence that PARG can de-ADP-ribosylate Smads, which enhances Smad-promoted gene regulation. In the second part, we examine a Smad-dependent gene target of TGF-β signaling, salt inducible kinase 1 (SIK). After induction, SIK cooperates with Smad7 and Smurf2 to downregulate the TGF-β type I receptor. The mechanism relies on both the kinase and UBA domain of SIK as well as the E3-ligase activity of Smurf2. In summary, we have unveiled two enzyme-dependent TGF-β/Smad modulatory mechanisms; SIK promoted receptor turnover and PARP-1/PARG-regulated Smad signaling.

Morphogens are secreted signalling molecules that are expressed in restricted groups of cells within the developing tissue. From there, they are secreted and travel throughout the target field and form concentration gradients. These concentration profiles endow receiving cells with positional information. A number of experiments in Drosophila demonstrated that the morphogen Decapentaplegic (Dpp) forms activity gradients by inducing the expression of several target genes above distinct concentration thresholds at different distances from the source. This way, Dpp contributes to developmental fates in the target field such as the Drosophila wing disc. Although the tissue distribution as well as the actual shape and size of the Dpp morphogen concentration gradient has been visualized, the cell biological mechanisms through which the morphogen forms and maintains a gradient are still a subject of debate. Two hypotheses as to the dominant mechanism of movement have been proposed that can account for Dpp spreading throughout the Drosophila wing imaginal target tissue: extracellular diffusion and planar transcytosis, i. e. endocytosis and resecretion of the ligand that is thereby transported through the cells. Here, I present data indicating that implications of a theoreticalanalysis of Dpp spreading, where Dpp transport through the target tissue is solely based on extracellular diffusion taking into account receptor binding and subsequent internalization, are inconsistent with experimental results. By performing Fluorescence Recovery After Photobleaching (FRAP) experiments, I demonstrate a key role of Dynamin-mediated endocytosis for Dpp gradient formation. In addition, I show that most of GFP-Dpp traffics through endocytic compartments at the receiving epithelial cells, probably recycled through apical recycling endosomes (ARE). Finally, a Dpp recycling assay based on subcellular photouncage of ligand is presented to address specifically the Dpp recycling event at the receiving cells.

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

Ce travail a été mené afin d'étudier l'effet du FGF 2 et du TGF-β1 sur les étapes précoces de la régénération dentinaire en utilisant la micro-encapsulation de ces facteurs dans une matrice pour les protéger et contrôler leur libération et ensuite l'application des microparticules obtenues en coiffage pulpaire direct dans un modèle de culture de dents entières. Ce travail consiste d'abord à l'optimisation des moyens techniques mis en oeuvre pour réaliser l'encapsulation du TGFβ1, FGF-2 à l'aide de l'acide poly (lactique-glycolique) PLGA. Les études de la caractérisation colloïdal et physico chimique des microparticules montre que les microparticules gardent leurs caractéristiques physicochimiques après séchage et resuspension dans l'eau. La procèdes optimisé a été ensuite utilisé pour encapsuler les facteurs de croissance. L'encapsulation de FGF-2 et TGF-β1 a été obtenue avec une taille, une efficacité d'encapsulation et une profile de libération adaptés au type d'application choisi. Les études biologiques ne montrent aucun effet toxique des particules sur les fibroblastes pulpaires. Les facteurs de croissance ont gardé leur activité biologique spécifique. Un modèle de culture de dent entier humain a été utilisé pour réaliser l'application de nos microparticules comme un matériau de coiffage dentaire pour confirmer leurs activités biologiques ex-vivo. Ces microparticules peuvent être utiles dans les études des étapes précoces de la régénération dentinaire, l'activation et la migration des cellules progénitrices de la pulpe dentaire

Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.

Wound healing and carcinogenesis are defined as complex, adaptive processes which are controlled by intricate communications between the host and the tissue microenvironment. A number of phenotypic similarities are shared by wounds and cancers in cellular signaling and gene expression. Radiotherapy is the second most effective modality of cancer treatment after surgery and can be used, either alone or in combination with chemotherapy. Recent findings suggest that radiotherapy apart from tumor cell death also rapidly and persistently modifies the tissue microenvironment. These modifications affect cell phenotype, tissue metabolism, bidirectional exchanges and signaling events between cells. The complex interactions between stromal cells and cancer cells are of immense interest and in The First Part of My Thesis, I tried to explore the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts, irrespective of their RhoB status, do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms respectively, TGF-β1 and MMP-mediated. We also found that co-irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response. Secondly, our in vivo experiments, tends to confirm the in vitro data showing that irradiated tumor bed does not stimulate tumor growth and escape. Our results also challenges the view that irradiated stroma would promote migration of carcinoma cells as we show that independently from their genotype co-irradiation of fibroblasts and carcinoma cells repressed carcinoma cell migration and confirmations studies are currently performed in vivo. The Third Part of My Project, was dedicated to investigate the effect on CTC release after radiotherapy. Consistently with the results reported after surgery , the number of CTC increases in the blood stream after radiotherapy probably due to radiation-induced vascular injury induced or/and by EMT induction in tumor cells but these cells seemed to be entrapped into the cardiac cavity. The significance of these CTC to metastatic development is still under investigation but there is evidence for a metastasis-promoting effect of RT from animal studies.Thus the microenvironment can exert antagonist stimulatory or inhibitory effects on malignant cells.

Les cellules souches neurales quiescentes (CSN) sont le réservoir de la neurogenèse adulte, permettant de produire des nouveaux neurones tout au long de la vie. Cependant, la neurogenèse décroit au cours du vieillissement, provoquant des déclins cognitifs incurables. Afin de mieux comprendre les mécanismes qui contrôlent la prolifération des CSN, nous avons mis en place une méthode de tri par cytométrie en flux qui permet pour la première fois d'isoler les CSN quiescentes et leurs cellules filles dans la ZSV adulte murine. Cette technique nous a permis de prouver que le blocage de la voie GABAAR in vivo provoque l'entrée en cycle des CSN quiescentes. Ainsi, les signaux GABA produits par les neuroblastes dans la ZSV permettent de maintenir les CSN dans leur état de quiescence. Au cours du vieillissement, nous montrons que la production progressive de TGFβ1 par les cellules endothéliales de la niche allonge la phase G1 des CSN activées, diminuant sensiblement la production de nouveaux neurones, sans toutefois diminuer le stock de CSN. Nous mettons ainsi en évidence deux voies majeures contrôlant la prolifération des CSN in vivo, la voie du GABAAR et la voie TGF-β/Smad-3. En vue d'une application thérapeutique, nous prouvons que leur blocage pharmacologique permet de stimuler efficacement la neurogenèse in vivo.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.