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Les Hedgehogs (Hh) sont des morphogènes multifonctionnels qui ont des rôles importants dans l'embryogenèse et la morphogenèse de plusieurs organes. Leur rôle dans le développement de la glande gastrique humaine n'est pas connu. Peu est connu de la signalisation Hh, plus précisément de Sonic Hedgehog (Shh), dans la glande gastrique humaine adulte, autant dans sa localisation et son association aux différents types cellulaires gastriques que dans son rôle fonctionnel dans cet organe.Les modèles murins de délétion classique des molécules de la signalisation Hh subissent, dans la grande majorité, une mort néonatale ou au stade somites rendant ces modèles inutiles pour l'étude du maintien de l'homéostasie du tube digestif adulte, autant au niveau de l'intestin que de l'estomac. Dans un premier temps, les patrons d'expression des différents gènes clés impliqués dans la voie de signalisation Hh dans le développement foetal humain de la glande gastrique ont été déterminés à l'aide de tissus gastriques foetaux âgés entre 14 et 20 semaines de gestation. Dans un deuxième temps, les rôles fonctionnels et physiologiques de Shh, dans l'homéostasie de l'intestin adulte et dans la morphogenèse et homéostasie de l'estomac ont été élucidés par la génération de souris possédant une délétion conditionnelle de Shh à l'épithélium de l'intestin ou de l'estomac à l'aide du système Cre/loxP. L'analyse des patrons d'expression des ligands SHH et Indian Hedgehog (IHH), des récepteurs PATCHED et SMOOTENED, des effecteurs GLI et de gènes cibles de la voie de signalisation Hh dans la glande gastrique humaine en développement, a confirmé la présence de la majorité de ces protéines dès 14 semaines de gestation. Ces effecteurs n'ont été détectés qu'au niveau des cellules à mucus de surface, des cellules du foveolae et des cellules zymogéniques mais pas au niveau des cellules pariétales. L'expression strictement épithéliale de presque toutes ces protéines suggère fortement l'activation exclusivement autocrine de cette voie de signalisation dans la glande gastrique humaine foetale. Ces patrons d'expression diffèrent de ceux retrouvés dans la glande gastrique murine foetale ou adulte. L'étude du modèle murin Villine -Cre; ShhloxP/loxP , qui possède une délétion de Shh à l'épithélium intestinal, a permis d'élucider les rôles fonctionnels de Shh dans l'homéostasie de l'intestin. Nous avons constaté que Shh a un rôle spécifique dans l'homéostasie intestinale puisque les souris expérimentales présentent un allongement villositaire. Shh est également impliqué dans la maturation des cellules caliciformes et dans l'inhibition de la prolifération épithéliale intestinale.Les résultats montrent aussi que Ihh semble, quant à lui, avoir un rôle compensateur puisqu'il se relocalise dans les régions qui expriment normalement Shh. L'étude du modèle murin Foxa3 -Cre; ShhloxP/loxP , qui possède une délétion de Shh dans l'épithélium gastrique, a permis d'élucider les rôles de Shh dans le développement et le maintien de la glande gastrique. Nous avons déterminé que Shh a un rôle dans la morphogenèse de la glande gastrique et dans le maintien de ce dernier. En effet, l'architecture foveolae-glande est fortement affectée par la perte épithéliale de Shh.Les résultats démontrent également que Shh a un rôle déterminant dans la différenciation des cellules pariétales puisque ces dernières sont absentes chez les souris expérimentales.

Doctor of Philosophy / This thesis offers the first English translation of Eduardo De Filippo’s last play, Gli esami non finiscono mai (1973). It analyzes the play in the context of the dramatist’s career and describes the philosophical shift that took place in Eduardo’s dialectic as he progressed from a post-war, neorealist drama like Napoli milionaria! toward the existential reflections present in his last play. Unlike previous studies, this work concentrates on Eduardo’s philosophical journey from neorealism to existential query and identifies the factors that influenced his thinking process. To this end, I have evaluated the plays most relevant to the development of his philosophy and the socio-political context in which they were written. The influence of the Neapolitan traditional dialect theater, along with that of Luigi Pirandello, his American contemporaries, Arthur Miller and Eugene O’Neill, and William Shakespeare, is also examined. Important social issues that directly affected the author, such as the struggle in Italy for the legalization of divorce and the plight of children born out of wedlock, are highlighted to illustrate how they contributed to the disillusionment and pessimism present in Eduardo’s last play. From the rather hopeful ending of Napoli milionaria! Eduardo was reduced at the end of his life to sheer desperation in Gli esami non finiscono mai. Italy had changed but it had not moved on. By focusing on the playwright’s final play, this thesis offers a new perspective on a twentieth century dramatist who is much more complex than is commonly acknowledged. De Filippo is revealed as a dramatist who transcended the Neapolitan comic theatrical traditions he sprang from and created a theater of political and social engagement that endures today.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

The Tibetan Book of the Dead and the Jewish Tractate Mourning are important texts about death in their respective traditions. The Tibetan Book of the Dead is a manual read by the living to the deceased as the deceased journeys through the many realms of the after-life. It is an abstract, philosophical text. The Tractate Mourning on the other hand, is a highly empirical and pragmatic text that guides the living through their loss. It is concerned only with the living left behind and offers no guidance to the deceased. Despite this profound difference however, this thesis has as its objective to show that both traditions, as evidenced through these texts, share an underlying emotion: compassion. Through the concern shown to the deceased as he or she stumbles through the often terrifying realms of the after-life in the Tibetan tradition, and through the precise and detailed instructions given to the living in the Jewish tradition as the mourners are guided through their grief, both texts exhibit profound compassion.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Doctor of Philosophy / This thesis offers the first English translation of Eduardo De Filippo’s last play, Gli esami non finiscono mai (1973). It analyzes the play in the context of the dramatist’s career and describes the philosophical shift that took place in Eduardo’s dialectic as he progressed from a post-war, neorealist drama like Napoli milionaria! toward the existential reflections present in his last play. Unlike previous studies, this work concentrates on Eduardo’s philosophical journey from neorealism to existential query and identifies the factors that influenced his thinking process. To this end, I have evaluated the plays most relevant to the development of his philosophy and the socio-political context in which they were written. The influence of the Neapolitan traditional dialect theater, along with that of Luigi Pirandello, his American contemporaries, Arthur Miller and Eugene O’Neill, and William Shakespeare, is also examined. Important social issues that directly affected the author, such as the struggle in Italy for the legalization of divorce and the plight of children born out of wedlock, are highlighted to illustrate how they contributed to the disillusionment and pessimism present in Eduardo’s last play. From the rather hopeful ending of Napoli milionaria! Eduardo was reduced at the end of his life to sheer desperation in Gli esami non finiscono mai. Italy had changed but it had not moved on. By focusing on the playwright’s final play, this thesis offers a new perspective on a twentieth century dramatist who is much more complex than is commonly acknowledged. De Filippo is revealed as a dramatist who transcended the Neapolitan comic theatrical traditions he sprang from and created a theater of political and social engagement that endures today.

Pour maintenir l'organisation et la polarité du tissu épithélial, de nouvelles jonctions cellulaires ont besoin de se former lors de la division cellulaire. Pour comprendre les mécanismes de formation de la jonction durant la cytokinèse, nous avons exploré dans les tissus épithéliaux de la Drosophile, la formation des jonctions septées tricellulaires (TCJs), critique à la fois dans la fonction de barrière tissulaire, dans l'homéostasie des cellules souches, ainsi que dans l'orientation du fuseau mitotique. Durant les dernières étapes de la constriction de l'anneau contractile, les membranes des deux cellules filles et des cellules voisines localisées sous la jonction adhérente (JA) restent enchevêtrées dans une structure à 4 cellules apposée au corps intermédiaire. Les constituants protéiques de la jonction septée, Discs-large (Dlg) et Neuroglian (Nrg), ainsi que les composants de la TCJ, Gliotactin (Gli) et Anakonda (Aka), s'accumulent dans cette structure à 4 cellules. Par la suite, la descente basale du corps intermédiaire est corrélée au détachement des membranes des cellules voisines, au désengagement des cellules filles de leurs voisines, et à la formation de TCJs matures. Le détachement des cellules voisines du corps intermédiaire est indépendant de l'abscision. Au contraire, la perte de la fonction Gli ou Aka empêche le détachement entre les cellules filles-voisines et le mouvement du corps intermédiaire. Ainsi, nous proposons que les protéines de la TCJ contrôlent une étape additionnelle de la cytokinèse, nécessaire au désengagement des cellules filles et de leurs voisines durant la cytokinèse épithéliale. / To maintain epithelial tissue organisation and polarity, new cell-cell junctions need to be formed upon cell division. To understand the mechanisms of junction formation during cytokinesis, we explored in Drosophila epithelial tissues, the de novo formation of tricellular septate junctions (TCJs), which are critical to tissue barrier function, stem cell homeostasis and mitotic spindle orientation. During the final stages of cytokinetic ring constriction, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. Protein constituents of the septate junction Discs-large (Dlg) and Neuroglian (Nrg) and the components of the TCJ Gliotactin (Gli) and Anakonda (Aka) accumulate in this 4-cell structure. Subsequently, a basal descent of the midbody correlates with the detachment of the neighbouring cell membranes, disengagement of the daughter cells from their neighbours and the formation of mature TCJs. The detachment of the neighbouring cells from the midbody is independent of abscission. On the contrary, the loss of Gli or Aka function prevents the resolution of the connection between the daughter-neighbour cells and the midbody movement. Altogether, we propose that TCJ proteins control an additional step of cytokinesis necessary for the disentanglement of the daughter cells and their neighbours during epithelial cytokinesis.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Proper central nervous system development is critical for survival and depends on complex intracellular and extracellular signaling to regulate neural progenitor cell growth and differentiation; however, the mechanisms that mediate molecular crosstalk between pathways during neurogenesis are not fully understood. Here, we explored the integration of the Hedgehog (Hh) signaling pathway with the two critical developmental pathways, Receptor Tyrosine Kinase (RTK) and Notch signaling, in the growth and maintenance of neural progenitors in the developing neuroretina. We found combined and sustained RTK and Hh signaling was sufficient to establish long-term retinal progenitor cell (RPC) cultures and these cells maintained neurogenic and gliogenic, but not retinogenic, competence in vitro and in vivo. In addition, we identified crosstalk between Notch and Hh signaling, where Notch is required for Hh-mediated proliferation and Gli protein accumulation, and gain-of-function of Notch is sufficient to extend the window of Hh responsiveness in a subset of Müller glia. Both Hh-RPC monolayer establishment and Notch mediated Hh-responsiveness required Gli2. Taken together, we identified molecular cross-communication between the Hh pathway and two major pathways, Notch and RTK, during retinogenesis, advancing our understanding of mechanisms that influence Hh to control neural progenitor growth.