La niche des cellules souches hématopoïétiques représente un compartiment complexe et radiosensible. Sa protection est nécessaire pour la restauration de l'hématopoïèse faisant suite à la myélosuppression due à l'exposition aux rayonnements ionisants. Nous avons dans un premier temps étudié l'effet des RI sur les progéniteurs endothéliaux et mésenchymateux de la niche par une étude de radiosensiblilité et une étude d'évaluation de la mort cellulaire. Nous avons proposé par la suite une stratégie innovante de thérapie génique basée sur la sécrétion locale et à court terme du morphogène Sonic hedgehog visant à favoriser la réparation de niche vasculaire et de stimuler les cellules souches hématopoïétiques et les cellules progénitrices résiduelles. Nous avons étudié la réponse hématopoïétique des singes irradiés à 8-Gy gamma après une seule injection intra-osseuse de cellules souches mésenchymateuses xénogéniques, multipotentes et d'origine adipocytaire transfectées avec un plasmide pIRES2-eGFP codant la protéine Shh. La durée de thrombocytopénie et celle de neutropénie ont été significativement réduites chez les animaux greffés et les clonogènes sont normalisés à partir du 42e jour. Les aires sous la courbe des numération des plaquettes et des neutrophiles entre 0 et 30 jours ont été significativement plus élevée chez les animaux traités que chez les témoins. La greffe d'explants de MatrigelTM colonisés ou non avec des ASC chez des souris immunodéprimées a démontré une activité pro-angiogénique notable des ASC transfectées avec le plasmide Shh . Le suivi à long terme (180 à 300 jours) a confirmé une reconstitution durable dans les quatre singes greffés. Globalement cette étude suggère que la greffe de cellules souches multipotentes Shh-peut représenter une nouvelle stratégie pour la prise en charge des dommages radio-induits de la niche. / The hematopoietic stem cell niche represents a complex radiosensitive compartment whose protection is required for recovery from radiation-induced myelosuppression. We initially studied RI effects on endothelial and mesenchymal progenitors by an evaluating radiosensitivity and cell death. Then, we have proposed a new gene therapy strategy based on local and short term secretion of Sonic hedgehog morphogene to favour vascular niche repair and to stimulate residual hematopoietic stem and progenitor cells. We investigated the hematopoietic response of 8-Gy gamma irradiated monkeys to a single intra-osseous injection of xenogeneic multipotent mesenchymal stem cells transduced with a Shh pIRES2 plasmid. Thrombocytopenia and neutropenia duration were significantly reduced in grafted animals and clonogenics normalized from day 42. Areas under the curve of PLTs and ANCs between day 0 and day 30 were significantly higher in treated animals than in controls. Grafting MatrigelTM colonized or not with ASC in immunocompromized mice demonstrated a notable pro-angiogenic activity for Shh-ASC. Long term follow up (180-300 days) confirmed a durable recovery in the four grafted monkeys. Globally this study suggests that grafting Shh-multipotent stem cells may represent a new strategy to cure radiation-induced niche damage.

Les récepteurs à dépendance (RDs) ont la particularité d’induire deux types de signalisation selon la disponibilité de leur ligand. En présence de leur ligand, ils induisent une signalisation positive favorisant la survie, la prolifération ou encore la différentiation cellulaire, tandis qu’en son absence, ils induisent la mort cellulaire par apoptose. Ptc et CDON, tous deux récepteurs du morphogène Shh, appartiennent à la famille des RDs. Ces deux récepteurs sont impliqués dans le développement embryonnaire, mais aussi dans le contrôle de la tumorigenèse et de l’homéostasie. La plupart des mécanismes moléculaires de la signalisation proapoptotique de ces récepteurs sont encore inconnus. Nous avons donc étudié les voies de signalisations proapoptotiques de ces récepteurs grâce à un crible d’ARN interférents. Parmi les partenaires potentiels identifiés, nous nous sommes intéressés à l’IGFBP7, caractérisé comme gène suppresseur de tumeurs dans de nombreux cancers. Nous avons mis en évidence que la mort induite par Ptc implique un contrôle transcriptionnel d’IGFBP7 de façon p53 dépendante. Dans un second temps, nous nous sommes intéressés au rôle des voies apoptotiques des récepteurs Ptc et CDON dans des cancers colorectaux associés à des maladies inflammatoires chroniques de l’intestin, pour lesquels il a été montré que le ligand Shh est surexprimé. La surexpression du ligand est l’un des mécanismes d’échappement au contrôle apoptotique exercé par les RDs acquit par les cellules tumorales. Notre étude vise à démontrer que le ciblage de Shh dans ces cancers pourrait induire une réactivation des voies apoptotiques des RDs Ptc et CDON, et ainsi constituer une nouvelle stratégie anti-tumorale / Dependence receptors (DRs) are able to inducing two types of signaling according to their ligand availability. In the presence of their ligand, they induce a positive signaling promoting survival, proliferation or cell differentiation, whereas in its absence, they induce cell death by apoptosis. Ptc and CDON, both receptors of the morphogen Shh, belong to the DRs family. Both receptors are involved in embryonic development, but also in the control of tumorigenesis and homeostasis. Most of the molecular mechanisms of the pro-apoptotic signaling of these receptors remain unknown. We therefore studied both receptors pro-apoptotic pathways through a shRNA screen. Among the identified potential partners, we have been interested in IGFBP7 shown as a tumor suppressor gene in many cancers. Secondly, we studied the role of Ptc and CDON pro-apoptotic pathways in colorectal cancers associated with chronic inflammatory bowel diseases, in which it has been shown that Shh is overexpressed. Overexpression of the ligand is one mechanism of pro-apoptotic control escapement by DRs acquired by tumor cells. Our study aims to demonstrate that targeting Shh in these cancers could induce reactivation of DRs pro-apoptotic pathways and thus constitute a new anti-tumor strategy

Recruitment of mural cells, i.e. pericytes and smooth muscle cells (SMC), is essential to improve the maturation of newly formed vessels. One of the major factors involved in this process is the endothelial cell-secreted Platelet-Derived Growth Factor BB (PDGF BB). Sonic hedgehog (Shh) has also been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms involved have not yet been elucidated. We first identified Shh as a target of PDGF BB and found that SMC respond to Shh not only by upregulating the Gli1-dependent canonical pathway, but also by activating ERK1/2 and PI3K-dependent non-canonical pathways. Moreover, we found that PDGF BB-induced SMC migration, involves Shh-dependent PI3K, ERK1/2 and Gli1 activation. In the mouse model of corneal angiogenesis, PDGF BB and Shh were expressed by endothelial cells and mural cells of VEGF-induced newly formed blood vessels, respectively. PDGF BB inhibition reduced Shh expression, confirming that Shh is a target of PDGF BB, as demonstrated by in vitro experiments. Finally, we found that inhibition of either PDGF BB or Shh signaling reduced NG2+ mural cell recruitment into neovessels and subsequently reduced the neo-vessel lifespan. In this work, we demonstrate, for the first time, that Shh is a key mediator of PDGF BB-induced mural cell migration and recruitment into neo-vessels and elucidates the molecular signaling pathway involved in this process. / Recruitment of mural cells, i.e. pericytes and smooth muscle cells (SMC), is essential to improve the maturation of newly formed vessels. One of the major factors involved in this process is the endothelial cell-secreted Platelet-Derived Growth Factor BB (PDGF BB). Sonic hedgehog (Shh) has also been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms involved have not yet been elucidated. We first identified Shh as a target of PDGF BB and found that SMC respond to Shh not only by upregulating the Gli1-dependent canonical pathway, but also by activating ERK1/2 and PI3K-dependent non-canonical pathways. Moreover, we found that PDGF BB-induced SMC migration, involves Shh-dependent PI3K, ERK1/2 and Gli1 activation. In the mouse model of corneal angiogenesis, PDGF BB and Shh were expressed by endothelial cells and mural cells of VEGF-induced newly formed blood vessels, respectively. PDGF BB inhibition reduced Shh expression, confirming that Shh is a target of PDGF BB, as demonstrated by in vitro experiments. Finally, we found that inhibition of either PDGF BB or Shh signaling reduced NG2+ mural cell recruitment into neovessels and subsequently reduced the neo-vessel lifespan. In this work, we demonstrate, for the first time, that Shh is a key mediator of PDGF BB-induced mural cell migration and recruitment into neo-vessels and elucidates the molecular signaling pathway involved in this process.

O Tumor Odontogênico Queratocístico (TOQC) é considerado uma entidade com alta taxa de recidiva e agressividade local, apresentando também frequente associação com a Síndrome do Carcinoma Basocelular Neviode (SCBCN), por isso a patogêneses de este tumor tem sido intensamente estudada. A transformação e proliferação de células neoplásicas normalmente envolvem a desregulação de vias de sinalização que participam do desenvolvimento embrionário normal, principalmente a via Sonic Hedgehog (Shh). A expressão de certas proteínas presentes nesta via foi detectada em vários tumores odontogênicos, sugerindo que desempenha um papel importante nas interações epiteliais e na proliferação de células tumorais. Embora o papel da via de sinalização Shh não esteja bem estabelecida no desenvolvimento de TOQCs, sugere-se que sua ativação pode ser correlacionada com o comportamento clínico agressivo destas lesões. O objetivo deste estudo foi avaliar a expressão imunoistoquímica das proteínas da via de sinalização Shh em TOQCs esporádicos e associados a SCBCN, além de comparar a sua expressão entre lesões recorrentes e não recorrentes. Para isso foi realizado um estudo retrospectivo onde as características clinicopatológicas de 62 pacientes foram avaliadas, a expressão imunoistoquímica das proteínas Shh, Ptch1, Ptch2, Smo, Gli1, Gli2 e Gli3 foi analisada em todas as amostras, comparando TOQCs sindrômicos (29 lesões) e TOQCs esporádicos (57 lesões), assim como sua respectiva recorrência. Com este estudo foi possível observar que as proteínas Shh, Smo e Gli1 revelaram aumento da expressão em TOQCs associados a SCBCN, em comparação com tumores esporádicos. Shh mostrou expressão citoplasmática intermédia dentro da camada basal em tumores sindrômicos, Smo por sua vez revelou forte expressão nuclear e citoplasmática nas camada basal e suprabasal de tumores sindrômicos, enquanto que a expressão de Gli1 foi mais elevada apenas no citoplasma de TOQCs associados a síndrome em comparação com tumores esporádicos. No que diz respeito a sua recorrência, as proteínas Ptch1 e Gli2 mostraram maior expressão em TOQCs esporádicos e recorrentes, enquanto que a expressão Gli1 foi mais relevante nos tumores recorrentes e associados a SCBCN. Os nossos resultados sugerem que a maior expressão das proteínas Shh, Smo e Gli1 em TOQCs pode contribuir para o diagnóstico precoce de lesões associadas com a SCBCN. Da mesma forma, as proteínas Ptch1 e Gli2 pode predizer o risco de recorrência de TOQCs esporádicos, enquanto Gli1 sugere uma potencial associação de recorrência em tumores sindrômicos. / Keratocystic Odontogenic Tumor (KCOT) is considered an entity of high recurrence rates and local aggressiveness, also often presenting association with Nevoid basal cell carcinoma syndrome (NBCCS), and the pathogenesis of this tumor has been therefore intensively studied. The transformation and proliferation of neoplastic cells usually involve deregulation of signaling pathways participating in normal embryonic development, mainly via Sonic Hedgehog (Shh). The expression of certain proteins of this pathway has been detected in several odontogenic tumors, suggesting that plays an important role in epithelial interactions and proliferation of tumor cells. Although the role of Shh signaling pathway is not well established in the development of KCOTs it has been suggested that its activation can be correlated to the aggressive clinical behavior of these lesions. The aim of this study is therefore to evaluate the immunohistochemical expression of proteins of Shh signaling pathway in sporadic KCOTs and associated with NBCCS and to compare their expression between recurring and non-recurring lesions.thus, a retrospective study was performed where the clinicopathological features of 62 patients were evaluated, the immunohistochemical expression of the Shh, Ptch1, Ptch2, Smo, Gli1, Gli2 and Gli3 protein, was analyzed in all samples, comparing syndromic KCOTs ( 29 lesions) and sporadic KCOTs (57 lesions), and also their respective recurrence. The results showed that the expression of Shh, Smo and Gli1 proteins was increased in KCOTs associated with NBCCS compared to sporadic tumors. Shh showed intermediate cytoplasmic expression within the basal layer syndromic tumors, Smo in turn showed strong nuclear expression and cytoplasmic in basal and suprabasal layers of syndromic tumors, while Gli1 cytoplasm expression was higher only in KCOTs associated syndrome compared with sporadic tumors. Regarding recurrent tumors, Ptch1 and Gli2 proteins showed higher expression in sporadic and recurrent KCOTs, while Gli1 expression was more significant in recurrent tumors and associated NBCCS. Our results suggest that increased expression of Shh, Smo and Gli1 proteins in KCOTs can contribute to early diagnosis of KCOTs associated with the NBCCS. Likewise, Ptch1 and Gli2 proteins can predict the risk of recurrence of sporadic KCOTs, while Gli1 suggest a potential association to recurrence into syndromic tumors.

O Tumor Odontogênico Queratocístico (TOQC) é considerado uma entidade com alta taxa de recidiva e agressividade local, apresentando também frequente associação com a Síndrome do Carcinoma Basocelular Neviode (SCBCN), por isso a patogêneses de este tumor tem sido intensamente estudada. A transformação e proliferação de células neoplásicas normalmente envolvem a desregulação de vias de sinalização que participam do desenvolvimento embrionário normal, principalmente a via Sonic Hedgehog (Shh). A expressão de certas proteínas presentes nesta via foi detectada em vários tumores odontogênicos, sugerindo que desempenha um papel importante nas interações epiteliais e na proliferação de células tumorais. Embora o papel da via de sinalização Shh não esteja bem estabelecida no desenvolvimento de TOQCs, sugere-se que sua ativação pode ser correlacionada com o comportamento clínico agressivo destas lesões. O objetivo deste estudo foi avaliar a expressão imunoistoquímica das proteínas da via de sinalização Shh em TOQCs esporádicos e associados a SCBCN, além de comparar a sua expressão entre lesões recorrentes e não recorrentes. Para isso foi realizado um estudo retrospectivo onde as características clinicopatológicas de 62 pacientes foram avaliadas, a expressão imunoistoquímica das proteínas Shh, Ptch1, Ptch2, Smo, Gli1, Gli2 e Gli3 foi analisada em todas as amostras, comparando TOQCs sindrômicos (29 lesões) e TOQCs esporádicos (57 lesões), assim como sua respectiva recorrência. Com este estudo foi possível observar que as proteínas Shh, Smo e Gli1 revelaram aumento da expressão em TOQCs associados a SCBCN, em comparação com tumores esporádicos. Shh mostrou expressão citoplasmática intermédia dentro da camada basal em tumores sindrômicos, Smo por sua vez revelou forte expressão nuclear e citoplasmática nas camada basal e suprabasal de tumores sindrômicos, enquanto que a expressão de Gli1 foi mais elevada apenas no citoplasma de TOQCs associados a síndrome em comparação com tumores esporádicos. No que diz respeito a sua recorrência, as proteínas Ptch1 e Gli2 mostraram maior expressão em TOQCs esporádicos e recorrentes, enquanto que a expressão Gli1 foi mais relevante nos tumores recorrentes e associados a SCBCN. Os nossos resultados sugerem que a maior expressão das proteínas Shh, Smo e Gli1 em TOQCs pode contribuir para o diagnóstico precoce de lesões associadas com a SCBCN. Da mesma forma, as proteínas Ptch1 e Gli2 pode predizer o risco de recorrência de TOQCs esporádicos, enquanto Gli1 sugere uma potencial associação de recorrência em tumores sindrômicos. / Keratocystic Odontogenic Tumor (KCOT) is considered an entity of high recurrence rates and local aggressiveness, also often presenting association with Nevoid basal cell carcinoma syndrome (NBCCS), and the pathogenesis of this tumor has been therefore intensively studied. The transformation and proliferation of neoplastic cells usually involve deregulation of signaling pathways participating in normal embryonic development, mainly via Sonic Hedgehog (Shh). The expression of certain proteins of this pathway has been detected in several odontogenic tumors, suggesting that plays an important role in epithelial interactions and proliferation of tumor cells. Although the role of Shh signaling pathway is not well established in the development of KCOTs it has been suggested that its activation can be correlated to the aggressive clinical behavior of these lesions. The aim of this study is therefore to evaluate the immunohistochemical expression of proteins of Shh signaling pathway in sporadic KCOTs and associated with NBCCS and to compare their expression between recurring and non-recurring lesions.thus, a retrospective study was performed where the clinicopathological features of 62 patients were evaluated, the immunohistochemical expression of the Shh, Ptch1, Ptch2, Smo, Gli1, Gli2 and Gli3 protein, was analyzed in all samples, comparing syndromic KCOTs ( 29 lesions) and sporadic KCOTs (57 lesions), and also their respective recurrence. The results showed that the expression of Shh, Smo and Gli1 proteins was increased in KCOTs associated with NBCCS compared to sporadic tumors. Shh showed intermediate cytoplasmic expression within the basal layer syndromic tumors, Smo in turn showed strong nuclear expression and cytoplasmic in basal and suprabasal layers of syndromic tumors, while Gli1 cytoplasm expression was higher only in KCOTs associated syndrome compared with sporadic tumors. Regarding recurrent tumors, Ptch1 and Gli2 proteins showed higher expression in sporadic and recurrent KCOTs, while Gli1 expression was more significant in recurrent tumors and associated NBCCS. Our results suggest that increased expression of Shh, Smo and Gli1 proteins in KCOTs can contribute to early diagnosis of KCOTs associated with the NBCCS. Likewise, Ptch1 and Gli2 proteins can predict the risk of recurrence of sporadic KCOTs, while Gli1 suggest a potential association to recurrence into syndromic tumors.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

Le syndrome de Gorlin est une maladie rare caractérisée par de nombreuses anomalies du développement. Ces manifestations cliniques, dues à des mutations d'un acteur essentiel de la voie de signalisation sonic hedgehog, incluent aussi une hyper-radiosensibilité et une forte prédisposition à développer des carcinomes basocellulaires. Etant donné l'implication de défaut de la réparation de l'ADN au niveau des affections liées à l'hyper-radiosensibilité, nous avons décidé d'étudier l'effet des mutations du gène PTCH1 sur la réponse aux dommages de l'ADN afin de mieux comprendre les mécanismes cellulaires et moléculaires conduisant au phénotype Gorlin.Cette étude permet de mettre en évidence une défaillance globale des systèmes de réparation des dommages de l'ADN dans les fibroblastes issus de patients Gorlin par rapport à des fibroblastes normaux. Elle met notamment en exergue un écroulement de la réparation par excision de bases (BER) responsable de la réparation des dommages oxydatifs. / The Gorlin syndrome is a rare genetic disorder characterized by several developmental abnormalities. Due to mutations in PTCH1, a key player of the sonic hedgehog signaling pathway, clinical manifestations also includes hyper-radiosensitivity and an increased predisposition to the development of basal cell carcinomas. Given the implication of DNA repair system defects in hyper-radiosensitivity pathologies, we decided to study the effect of PTCH1 mutations on the DNA damage response in order to better understand the cellular and molecular mechanisms leading to Gorlin's phenotype.This study demonstrate a global failure of the DNA damage repair systems in Gorlin fibroblasts with respect to controls. It highlights in particular the collapse of the base excision repair pathway (BER) responsible for the repair of oxidative DNA damage.

Le syndrome cutané radio-induit (SCR) est une lésion cutanée spécifique secondaire à une irradiation localisée de forte dose, qui se caractérise par des douleurs importantes, des vagues inflammatoires extensives, un défaut de revascularisation et de cicatrisation. De nouvelles stratégies thérapeutiques peuvent être envisagées pour la prise en charge de cette pathologie avec l'émergence récente des cellules souches mésenchymateuses. Ces cellules multipotentes ont des propriétés de transdifférenciation et de sécrétion de facteurs favorisant la régénération tissulaire. L'association de la chirurgie d'exérèse, des greffes de peau autologue et des injections de cellules souches autologues isolées à partir de la moelle osseuse (Bone Marrow Mesenchymal Stem Cells ou BM-MSC) a confirmé ces hypothèses mais leur utilisation reste limitée à des cas de patients isolés.L'objectif de ce travail était d'optimiser la prise en charge du SCR par thérapie cellulaire pour l'étendre aux cas d'accidents de masse ou d'irradiation hétérogène associée.Les cellules souches dérivées du tissu adipeux (ASC pour Adipose tissue derived Stem Cells), plus abondantes que les BM-MSC et présentant des propriétés pro-angiogéniques, anti-apoptotiques et immunomodulatrices équivalentes voire supérieures, ont été choisies pour développer cet outil de thérapie cellulaire. La faisabilité de différentes stratégies thérapeutiques a été évaluée dans ce travail : la constitution de banques cellulaires autologues et allogéniques, une potentialisation des effets des ASC par thérapie génique transitoire et l'utilisation du sécrétome des ASC.La constitution de banques autologues ou allogéniques de cellules souches permet de pallier le délai d'obtention de greffons de taille suffisante en termes de quantité de cellules souches. Cette stratégie évaluée sur un modèle in vivo de miniporc a démontré la pertinence des banques d'ASC autologues mais a également mis en évidence les difficultés liées aux injections d'ASC allogéniques en particulier de type immunologique.La stratégie d'optimisation des ASC par manipulation ex vivo a pour but de potentialiser leurs effets par la sécrétion d'une protéine ad hoc impliquée dans les processus de réparation tissulaire et ainsi, de diminuer la quantité de cellules injectées et donc le temps de culture. La protéine sélectionnée dans ce travail est la protéine Sonic Hedgehog (Shh) décrite comme présentant des propriétés pro-angiogéniques et anti-apoptotiques. L'évaluation d'un protocole de thérapie génique transitoire utilisant des ASC transfectées par une construction plasmidique codant pour Shh (ASC-Shh) a montré la faisabilité de cette stratégie sur un modèle in vivo de miniporc.Les effets pro-angiogéniques de même que les effets du sécrétome des ASC et ASC-Shh sur des fibroblastes irradiés ont confirmé le rôle des facteurs paracrines des ASC dans la survie cellulaire et mis en évidence la potentialisation de leurs effets pro-angiogéniques par Shh. Ainsi l'utilisation du sécrétome des ASC ou ASC-Shh apparaît également comme une stratégie pertinente permettant de s'affranchir de la compatibilité donneur-receveur lors des greffes de cellules souches.Ces différentes techniques basées sur les cellules souches dérivées du tissu adipocytaire représentent donc des stratégies prometteuses pour la prise en charge du syndrome cutané radio-induit. / Cutaneous radiation syndrome (CRS) is a specific skin lesion caused by high dose local irradiation. It is characterized by severe pain, extensive inflammatory waves, incomplete revascularization and wound healing. For the last few years, mesenchymal stem cells (MSC) have become a promising cell source for cell therapy. MSC are multipotent cells which can transdifferentiate and secrete many paracrine factors involved in tissue regeneration, and they may be considered as a new therapeutic strategy for the management of this pathology. This was confirmed by the results of works associating surgical resection, autologous skin graft and autologous bone marrow mesenchymal stem cells (BM-MSC) injections, although the use of the latter remains limited to isolated cases of patients.The aim of this work was to optimize the management of CRS using stem cell therapy in order to extend it to mass casualties or associated heterogeneous irradiation.Adipose tissue derived stem cells (ASC) were chosen to develop cell therapy as they are more abundant than BM-MSC and exhibit higher pro-angiogenic, anti-apoptotic and immunomodulation properties.The feasibility of different therapeutic strategies has been evaluated in this work: the establishment of autologous and allogeneic cell banks, potentiation of ASC effects by transient gene therapy and the use of ASC secretome.The constitution of autologous or allogeneic stem cell banks overcomes the cell expansion delay required to obtain stem cells of clinical quantity. This strategy was evaluated in an in vivo minipig model of CRS and the relevance of autologous ASC banks was shown but this study highlighted difficulties associated with allogeneic injections especially immunological ones.Here is also reported an ASC optimization by ex vivo manipulation that aims to decrease the amount of injected cells and culture time through the secretion of an ad hoc protein involved in tissue repair processes and thus to, decrease the amount of injected cells and therefore the culture time. The protein chosen was Sonic Hedgehog (Shh), known to be involved in pro-angiogenic and anti-apoptotic processes. The evaluation of a transient gene therapy protocol using ASC transfected with a plasmid construct encoding Shh (ASC-Shh) confirmed the feasibility of this strategy in the same minipig model of CRS.The pro-angiogenic effects as well as other effects of ASC and ASC-Shh secretome on irradiated fibroblasts have confirmed the role of ASC paracrine factors in cell survival and demonstrated potentiation of their pro-angiogenic effects by Shh. Thus the use of ASC or ASC-Shh secretome also appears as a relevant strategy to overcome the donor-recipient compatibility during stem cell transplants.These techniques based on adipose tissue derived stem cells represent promising strategies for the management of cutaneous radiation syndrome.

Les vésicules extracellulaires (VE), incluant exosomes et microparticules (MP), vecteurs d’information biologique, peuvent moduler la fonction de cellules cibles. Une élévation du taux de VE circulantes est observée dans les pathologies cardiovasculaires dont l’obésité est l’un des facteurs de risque majeur. Cependant, il existe peu de données concernant la production de VE adipocytaires et leur capacité à moduler le métabolisme des adipocytes. Tout d’abord, nous avons caractérisé de manière morphologique et biochimique les MP et les exosomes adipocytaires. Nous montrons une production accrue de ces VE dans un contexte d’obésité murine. L’analyse protéomique des VE adipocytaires révèle un enrichissement spécifique des MP et des exosomes en protéines clé du métabolisme énergétique et de l’inflammation, respectivement. Dans une seconde partie, nous avons étudié l’effet de MP lymphocytaires portant le morphogène Hedgehog (MPHh+) sur la différenciation adipocytaire. A l’instar d’une activation classique de la voie de signalisation Hh, les MPHh+ inhibent l’adipogenèse. Bien que dépendant du récepteur Smoothened (Smo), cet effet inhibiteur est indépendant des facteurs de transcription Gli. Nous montrons que les MPHh+ activent un axe anti-adipogénique Smo/Lkb1/Ampk pouvant être stimulé par un nouvel agoniste de Smo, le GSA-10. Nos résultats démontrent, d’une part, la capacité des adipocytes à sécréter des VE, et d’autre part, le potentiel fonctionnel des MPHh+ à inhiber l’adipogenèse par une voie de signalisation Hhnon-canonique. Les VE pourraient contribuer aux dysfonctions métaboliques associées à l’obésité en véhiculant des messages métaboliques à l’échelle de l’organisme. / Extracellular vesicles (EV), including microparticles (MP) and exosomes, are able to modulate target cell function through exchange or transfer of biological material. Although EV are present in the blood of healthy individuals, an elevated quantity of circulating EV is associated with cardiovascular diseases, which obesity is a major cardiovascular risk factor. Nevertheless, few studies have reported the ability of adipocytes to release EV and their implication in adipose tissue metabolism. First of all, we could determine morphological and biochemical features of adipocyte-derived exosomes and MP through a combination of methods. We were able to demonstrate an increase in adipocyte EV production in a murine model of obesity. Proteomic analysis of adipocyte EV further revealed a specific enrichment of proteins crucial for glucose and lipid metabolism and related to inflammation in MP and exosomes respectively. We then evaluated the ability of lymphocytes-derived MP harboring the Sonic Hedgehog morphogen to control adipocyte differentiation. Activation of the Hedgehog canonical pathway inhibited adipogenesis, as did MPHh+. Surprisingly, although Smo dependent, inhibitory potential of such MP did not involve the Gli transcription factors. We show that MPHh+ inhibit adipocyte differentiation through a Smo/Lkb1/Ampk axis as does a new agonist of Smo, GSA-10. Our results demonstrate, on one hand, the ability of adipocyte to release EV and on the other hand, the capacity of MPHh+ to control adipogenesis through a non-canonical Hh signaling pathway. In conclusion, EV might contribute to obesity related metabolic dysfunctions through systemic regulation of metabolic pathways.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.