At the outset of my PhD, my aim was to investigate the mechanisms responsible for the directed migration of primitive myeloid cells (PMCs) to wounds in Xenopus embryos. PMCs are the first blood cells to differentiate and become functional in Xenopus embryos, and have a notable migratory ability to be recruited by embryonic wounds before a functional vasculature is established. To find the mechanism underlying PMCs migration toward embryonic wounds, I first performed a screen to identify candidate cytoskeleton related genes, which might be responsible for facilitating the inflammatory response to injury in embryos. In situ hybridization and RT-PCR showed that coronin1a and l-plastin were specifically expressed in PMCs. I carried out loss-of-function experiments for coronin 1a and l-plastin in Xenopus embryos. Unfortunately neither knockdown affected the ability for PMCs to migrate during embryonic development or during the wound healing process. Loss-of-function experiments on coronin 1a and l-plastin also did not affect epidermal wound closure speed. Thus, although coronin 1a and l- plastin are expressed specifically in PMCs, they do not appear to be necessary for the migration of PMCs during development and during wound healing in Xenopuos embryos. Since my initial aim failed to provide insight into the mechanisms that mediate 9the inflammatory response to embryonic wounds, I decided to investigate the function of a previously identified monomeric actin protein during embryonic wound healing and appendage regeneration: namely Thymosin beta4 (Tβ4). In situ hybridization experiments showed that Tβ4 is expressed exclusively in the epidermis of developing frog embryos. Tβ4 knockdown embryos resulted in a significantly delay in the speed of wound closure during the early phase of wound healing. This delay correlated with a decrease in the actin contractile ring at the wound margin. Furthermore I found that the cell shapes of epidermal cells in the Tβ4 knockdown embryos were different from epidermal cells in control embryos. I hypothesize that this reduction caused the actin filaments changes in the epidermal cells, and were responsible for the failure of the cells to form an actin contractile ring, thus delaying the initial speed of wound closure. I tried to confirm that most of these defects specific to Tβ4, by performing rescue experiments with Tβ4 mRNA injections. Furthermore, I discovered that Tβ4 knockdown embryos displayed defects in tail development, including the absence of blood vessel branching within the fin of the tail. Finally, I found that the tails in Tβ4 knocked-down tadpoles failed to regenerate, while tails in control embryos regenerated completely following amputation. Both in situ hybridization and real-time PCR showed that Tβ4 was up regulated in the regenerated part of the tail in Xenopus tadpoles. Together with the tail amputation results, Tβ4 might be important for tail development and regeneration. These findings suggest that Tβ4 might play an important roles in the modulation of the actin cytoskeleton, which are essential for the proper behavior of epidermal cells during wound healing and appendage regeneration.

Les canaux calciques dépendants du voltage (CCVD) sont impliqués dans de nombreux processus cellulaires tels que la libération de neurotransmetteurs, la contraction musculaire ou encore la régulation de l'expression génique. Les CCVD sont constitués d'une sous-unité canalaire (alpha1 ou Cav) par laquelle les ions Ca2+ entrent dans le milieu intracellulaire, associée à différentes sous-unités auxiliaires, alpha2delta, beta et gammaqui régulent leur fonction. Ma thèse a contribué à la mise en évidence d'une nouvelle voie de régulation du couplage excitation-transcription impliquant la sous-unité beta4 des CCVD. Dans ce cadre, nous nous sommes intéressés à la compréhension des déterminants de l'entrée de beta4 dans le noyau et aux mécanismes de régulation de l'expression génique par cette sous-unité des CCVD. Un modèle animal nous a été particulièrement utile, la souris léthargique (lh), déficiente pour la sous-unité beta4 et considérée comme un modèle d'étude de l'épilepsie-absences. Une translocation de beta4 du cytoplasme vers le noyau est observée au cours de la différenciation neuronale. Cette translocation est dépendante de l'intégrité structurale de beta4 et plus précisément de l'interaction de ses domaines SH3 (Src Homology 3) et GK (Guanylate Kinase). La translocation de beta4 au noyau nécessite son association avec un partenaire : la sous-unité régulatrice de la protéine phosphatase 2A (PP2A), B56delta. La dépolarisation membranaire permet un décrochage de beta4 du canal et son association à B56delta. beta4 migre donc vers le noyau sous forme de complexe avec B56delta/PP2A. Une étude transcriptomique réalisée pour comparer le profil d'expression dans le cervelet de souris lh par rapport aux souris wild-type a montré l'implication de beta4 dans la répression et l'activation d'un certain nombre de gènes. Particulièrement beta4 réprime fortement l'expression du gène qui code pour la tyrosine hydroxylase (TH). Dans le noyau, beta4 interagit avec un facteur de transcription, le récepteur aux hormones thyroïdiennes alpha(TRalpha). Cette association permet au complexe beta4/B56delta/PP2A de cibler la région promotrice du gène TH comme cela a été montré par des expériences d'immunoprécipitation de la chromatine (ChIP). Le complexe beta4/B56delta/PP2A est capable de s'associer aux histones et de déphosphoryler spécifiquement les histones H3 en Ser10 au niveau de la région promotrice du gène TH. Cette modification de la chromatine est corrélée avec le recrutement de Heterochromatin Protein 1 gamma (HP1gamma au niveau du promoteur du gène TH. HP1gamma est impliquée dans la formation d'hétérochromatine et pourrait expliquer la répression de l'expression du gène TH. Ainsi dans le cervelet de souris lh, l'absence de beta4 déclenche un dérèglement de cette voie de signalisation qui entraîne la sur-expression du géne TH. La mutation humaine R482X à l'origine de la délétion d'une partie du domaine C-terminal de beta4 et responsable d'une forme d'épilepsie juvénile myoclonique, perturbe la localisation nucléaire de beta4. En effet, le mutant beta1-481 incapable de s'associer à B56delta/PP2A et de migrer au noyau n'interagit pas avec les histones. La voie de signalisation permettant la régulation de l'expression génique par beta4 n'est donc plus assurée par le mutant. Ainsi, la fonction debeta4 ne se limite pas à son action cytoplasmique en tant que sous-unité auxiliaire des CCVD. En effet, ce travail montre combien dans le noyau,beta4, joue un rôle important dans la régulation de l'expression génique. / Voltage dependent calcium channels (VDCC) participate to various cellular processes such as neurotransmitters release, muscular contraction or gene expression regulation. VDCC are composed of a pore-forming subunit (alpha1 or Cav), that allows Ca2+ to enter the cells associated with auxiliary subunits, alpha2delta, beta and gamma. My thesis studies on a new signaling pathway in which the beta4 subunit of VDCC couples neuronal excitability to transcription. In particular it focuses on the understanding of the beta4 nuclear translocation determinants and the mechanisms of gene expression regulation by this VDCC subunit. beta4 translocation from the cytoplasm to the nucleus is observed during neuronal differentiation. This translocation depends on beta4 structural integrity and more precisely on interaction between the beta4 SH3 (Src Homology 3) and GK (Guanylate Kinase) domains. beta4 nuclear translocation is conditioned by its association with a partner: the regulatory subunit of phosphatase protein 2A (PP2A), B56delta. Membrane depolarization induces beta4 channel uncoupling and association with B56delta. Thus, beta4 migrates to the nucleus in complex with B56delta/PP2A. A study on gene expression generated by microarray was carried on to compare profiles of gene expression in lethargic (lh) mice, considered as spontaneous beta4 KO with wild-type (WT) mice cerebellum. This study proved the influence ofbeta4 on the repression and the activation of certain genes. Particularly, beta4 strongly represses tyrosine hydroxylase (TH) gene expression. In the nucleus, beta4 interacts with a transcription factor: the thyroid hormone receptor alpha (TR alpha). This association allows beta4/B56delta/PP2A complex to target the TH gene promoter as shown by chromatin immunoprecipitation (ChIP) experiments. This complex is also able to associate itself with histones and dephosphorylate Ser10 histone H3 in the TH gene promoter. This chromatin modification is correlated with HP1 gamma (Heterochromatin Protein 1 gamma) recruitment in the TH gene promoter. HP1 gamma is known to promote heterochromatin formation that could explain the TH gene repression by beta4. Thus, in lh mice cerebellum, the absence of beta4 triggers a complete disorganization of this signaling pathway that results in the up-regulation of the TH gene. R482X, the human mutation inducing the C-terminus domain deletion of beta4 and responsible for a form of juvenile myoclonic epilepsy prevents beta4 nuclear localization. In fact, the mutant beta1-481 unable to associate with B56delta/PP2A and to migrate to the nucleus does not interact with HP1gamma and histones. The signaling pathway allowing gene regulation by beta4 is prevented by the mutant. Thus, beta4 does not play a confined role in the cytoplasm as CCVD auxiliary subunit but also functions in the nucleus as a gene expression regulator.

Introdução: Caderinas e integrinas são importantes para a manutenção da integridade tecidual e transdução de sinal durante o desenvolvimento da pele. A distribuição destas moléculas no desenvolvimento da pele humana foi investigada e associada com os marcadores de diferenciação, Citoqueratinas (CK) e involucrina (INV). Método: Usando a técnica de imunoistoquímica foram investigadas as proteínas E- e P- caderinas, integrinas beta- 1 e -4, CK 10, CK 14 e INV em fragmentos de pele de várias regiões corpóreas de 7 fetos humanos (semana gestacional de 4 a 24, todos pesando até 500 g). Resultados: Na fase inicial do desenvolvimento, integrinas beta-1 e -4 and E- and P- caderinas estavam presentes na membrana plasmática das células epiteliais em todos as camadas do epitélio. CK14 e CK10 foram observadas em todas as camadas epiteliais e a INV fracamente detectada em células da camada mais superficial. Em estágios mais avançados, integrinas foram detectadas em todas as camadas epiteliais, com expressão polarizada principalmente na camada basal. E- caderina foi detctada em todas as camadas, menos no estrato cornificado e a P- caderina foi observada em camadas mais profundas do epitélio. CK14 estava presente na camada basal, CK 10 no estrato suprabasal e a INV foi observada no estrato cornificado. Conclusão: Caderinas e integrinas são essenciais para o desenvolvimento da pele, sendo espacialmente e temporalmente regulados. Suas expressões são relatas com a expressão da maturação de marcadores da epiderme. / Introduction: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P- cadherins, integrins beta-1 and -4, CK10, CK 14 and INV was assessed in skin fragments of 7 human fetuses (gestacional weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P- cadherins were present on epithelial cell membranes in all layers. CK 14 and CK 10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E- cadherin was detected in all layers, but the cornified stratum and P- cadherin were observed in the lower layers. CK 14 was expressed in layer, CK 10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Introdução: Caderinas e integrinas são importantes para a manutenção da integridade tecidual e transdução de sinal durante o desenvolvimento da pele. A distribuição destas moléculas no desenvolvimento da pele humana foi investigada e associada com os marcadores de diferenciação, Citoqueratinas (CK) e involucrina (INV). Método: Usando a técnica de imunoistoquímica foram investigadas as proteínas E- e P- caderinas, integrinas beta- 1 e -4, CK 10, CK 14 e INV em fragmentos de pele de várias regiões corpóreas de 7 fetos humanos (semana gestacional de 4 a 24, todos pesando até 500 g). Resultados: Na fase inicial do desenvolvimento, integrinas beta-1 e -4 and E- and P- caderinas estavam presentes na membrana plasmática das células epiteliais em todos as camadas do epitélio. CK14 e CK10 foram observadas em todas as camadas epiteliais e a INV fracamente detectada em células da camada mais superficial. Em estágios mais avançados, integrinas foram detectadas em todas as camadas epiteliais, com expressão polarizada principalmente na camada basal. E- caderina foi detctada em todas as camadas, menos no estrato cornificado e a P- caderina foi observada em camadas mais profundas do epitélio. CK14 estava presente na camada basal, CK 10 no estrato suprabasal e a INV foi observada no estrato cornificado. Conclusão: Caderinas e integrinas são essenciais para o desenvolvimento da pele, sendo espacialmente e temporalmente regulados. Suas expressões são relatas com a expressão da maturação de marcadores da epiderme. / Introduction: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P- cadherins, integrins beta-1 and -4, CK10, CK 14 and INV was assessed in skin fragments of 7 human fetuses (gestacional weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P- cadherins were present on epithelial cell membranes in all layers. CK 14 and CK 10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E- cadherin was detected in all layers, but the cornified stratum and P- cadherin were observed in the lower layers. CK 14 was expressed in layer, CK 10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

<p dir="ltr">Seizure involves a sudden, uncontrolled electrical disturbance of the brain due to many different causes apart from epilepsy, for example, high fever, low level of blood sugar, alcohol withdrawal, and many more, including the infections in the brain. In fact, epilepsy is a group of chronic neurological disorders characterized by recurrent unprovoked sudden-onset seizures. It stands as one of the prevalent brain disorders globally, impacting over 70 million individuals. The origins of epilepsy are multifaceted, coming from a mix of genetic and environmental factors including genetic predispositions, brain-related conditions (like tumors or strokes), infectious diseases, and traumatic brain injuries. Seizures can be partly referred to the dysregulation of ion channels, including voltage-gated sodium channels which will impact the action potential (electrical impulses that are responsible for the communication that takes place between neurons in the brain). These voltage-gated sodium channels mediate the depolarization responsible for the generation and conduction of action potentials. They are crucial in the generation and continuous electrical signals of the tissues that respond rapidly, like the neurons, and thus forming part of their function. In epilepsy, therefore, it is relevant to that domain in which abnormal functions of these sodium channels come up. Any change or dysfunction of these channels affect the excitability of the neurons themselves, with the consequence that an increased probability occurs in which abnormal electrical activity can be generated, hence the convulsions. Voltage-gated sodium channels are made up of large transmembrane proteins, having a single alpha subunit and related beta subunits. The beta subunit is an auxiliary protein that modulates channel gating, kinetics, surface expression, and the unique resurgent current, thereby influencing neuronal excitability and signaling. Resurgent currents represent a kind of current that can develop during action potential repolarization. They are characterized by a resurgent sodium current, the current which follows the initial sodium inflow in depolarization. Resurgent sodium currents are characterized by a rebound increase in sodium current during the repolarization phase of the action potential. Unlike the classic transient sodium current that inactivates rapidly upon membrane depolarization, the resurgent current is facilitated by the partial block and unblock of the sodium 17 channel pore by the β subunit or other intracellular molecules during the repolarization phase. This allows sodium ions to flow into the cell when this blockage removed before it goes to closed state. It is believed widely to be of keen importance in neuronal excitability. The role of resurgent currents in epilepsy is likely genetically influenced with some environmental influence. Genetic mutations and dysregulation of the gene code for voltage-gated sodium channels, especially those related to beta subunits, can be linked to some atypical resurgent current. This increases the chance of having a seizure, which could develop into epilepsy. Four beta subunits have been identified up to now. As such, my investigation will focus on the beta 4 subunit and its possible involvement in increased susceptibility to seizures. My study will involve a genetically modified mouse β4 knockout (K.O) of the voltage-gated sodium channel, which will be compared with a wild type (WT) mouse model. To facilitate this comparison, I will prepare cortical brain slices from both the genetically modified and WT mice using a (Leica VT1200s vibratome). These slices will then be analyzed with multi-electrode arrays to detect electrical activity and measure the neurons' electrical responses. Additionally, I use 4- Aminopyridine, a potassium channel blocker, to stimulate electrical activity in the neurons and brain slices. Using the methodology outlined above, I aimed to investigate the ability to induce and measure neuronal activity in the β4 K.O mouse model. This involved comparing the neuronal activity between the β4 K.O and WT mice in terms of frequency and amplitude. The analysis of the recorded data was performed using Spike2 software, in conjunction with the multi-electrode array recordings. Furthermore, I explored whether variations in temperature (body temp vs 40℃) affect neuronal activity differently in β4 K.O compared to WT mice. In conclusion, my observations revealed that neuronal activity could indeed be induced in the β4 K.O mice, with a noted decrease in the frequency of this activity compared to WT mice, but an increase in amplitude. These outcomes were consistent at both normal body temperature and at an elevated temperature of 40°C, as analyzed using Spike2 software. However, when conducting a statistical analysis using a two-way ANOVA to compare between the β4 K.O and WT mice, and between body temperature and 40°C conditions, no significant differences were observed. Despite this, it is a general observation and conclusion that β4 K.O mice exhibit altered neuronal activity 18 compared to WT mice. To gain a deeper understanding of the role of the β4 subunit on the alpha subunit of the voltage-gated sodium channel, adopting alternative methods such as patch clamp techniques or in vivo studies with intracranial electrodes may be beneficial. This suggestion comes considering various challenges and limitations encountered during my study, such as maintaining the viability of the slices for extended periods and minimizing noise in multi-electrode array (MEA) recordings. Mutations of β-subunit-encoding genes have been associated with such a wide array of debilitating diseases that include epilepsy, cancer, neuropathic pain, and febrile seizures, to some of the most prevalent conditions in neurodegeneration. Further study will be needed to better understand the biology of these important proteins and their potential for use as new targets for several disease states. Even so, the role of β4 remains somewhat controversial.</p>

Les canaux calciques dépendants du voltage (CCVD) sont impliqués dans de nombreux processus cellulaires tels que la libération de neurotransmetteurs, la contraction musculaire ou encore la régulation de l'expression génique. Les CCVD sont constitués d'une sous-unité canalaire (alpha1 ou Cav) par laquelle les ions Ca2+ entrent dans le milieu intracellulaire, associée à différentes sous-unités auxiliaires, alpha2delta, beta et gammaqui régulent leur fonction. Ma thèse a contribué à la mise en évidence d'une nouvelle voie de régulation du couplage excitation-transcription impliquant la sous-unité beta4 des CCVD. Dans ce cadre, nous nous sommes intéressés à la compréhension des déterminants de l'entrée de beta4 dans le noyau et aux mécanismes de régulation de l'expression génique par cette sous-unité des CCVD. Un modèle animal nous a été particulièrement utile, la souris léthargique (lh), déficiente pour la sous-unité beta4 et considérée comme un modèle d'étude de l'épilepsie-absences. Une translocation de beta4 du cytoplasme vers le noyau est observée au cours de la différenciation neuronale. Cette translocation est dépendante de l'intégrité structurale de beta4 et plus précisément de l'interaction de ses domaines SH3 (Src Homology 3) et GK (Guanylate Kinase). La translocation de beta4 au noyau nécessite son association avec un partenaire : la sous-unité régulatrice de la protéine phosphatase 2A (PP2A), B56delta. La dépolarisation membranaire permet un décrochage de beta4 du canal et son association à B56delta. beta4 migre donc vers le noyau sous forme de complexe avec B56delta/PP2A. Une étude transcriptomique réalisée pour comparer le profil d'expression dans le cervelet de souris lh par rapport aux souris wild-type a montré l'implication de beta4 dans la répression et l'activation d'un certain nombre de gènes. Particulièrement beta4 réprime fortement l'expression du gène qui code pour la tyrosine hydroxylase (TH). Dans le noyau, beta4 interagit avec un facteur de transcription, le récepteur aux hormones thyroïdiennes alpha(TRalpha). Cette association permet au complexe beta4/B56delta/PP2A de cibler la région promotrice du gène TH comme cela a été montré par des expériences d'immunoprécipitation de la chromatine (ChIP). Le complexe beta4/B56delta/PP2A est capable de s'associer aux histones et de déphosphoryler spécifiquement les histones H3 en Ser10 au niveau de la région promotrice du gène TH. Cette modification de la chromatine est corrélée avec le recrutement de Heterochromatin Protein 1 gamma (HP1gamma au niveau du promoteur du gène TH. HP1gamma est impliquée dans la formation d'hétérochromatine et pourrait expliquer la répression de l'expression du gène TH. Ainsi dans le cervelet de souris lh, l'absence de beta4 déclenche un dérèglement de cette voie de signalisation qui entraîne la sur-expression du géne TH. La mutation humaine R482X à l'origine de la délétion d'une partie du domaine C-terminal de beta4 et responsable d'une forme d'épilepsie juvénile myoclonique, perturbe la localisation nucléaire de beta4. En effet, le mutant beta1-481 incapable de s'associer à B56delta/PP2A et de migrer au noyau n'interagit pas avec les histones. La voie de signalisation permettant la régulation de l'expression génique par beta4 n'est donc plus assurée par le mutant. Ainsi, la fonction debeta4 ne se limite pas à son action cytoplasmique en tant que sous-unité auxiliaire des CCVD. En effet, ce travail montre combien dans le noyau,beta4, joue un rôle important dans la régulation de l'expression génique.

Despite the importance of integrins in epithelial cell biology surprisingly little is known about their regulation. It is known that they form hemidesmosomes (HDs), are actively involved in cell contacts during cell migration/invasion, and are key signaling molecules for survival and growth. However, there has been a distinct lack of understanding about what controls the dynamic integrin localization during cell activation and movement. Growth factors, such as EGF, are elevated during wound healing and carcinoma invasion leading to phosphorylation of ITGβ4 and the disassembly of the HD and mobilization of ITGβ4 to actin-rich protrusions. More recently the phosphorylation of a novel site on ITGβ4 (S1424) was found to be distinctly enriched on the trailing edge of migrating cells, suggesting a possible mechanism for the dissociation of ITGβ4 from HDs. Arrestin family member proteins are involved in the regulation of cell surface proteins and vesicular trafficking. In this study, we find that over-expression of arrestin family member ARRDC3 causes internalization and proteosome-dependent degradation of ITGβ4, while decreased levels of ARRDC3 stabilizes ITGβ4 levels. These results lead us to a new mechanism of ITGβ4 internalization, trafficking and degradation. During migration, ARRDC3 co-localizes with ITGβ4 on the lagging edge of cells but has a distinct distribution on the leading edge of cells. Additional immuno co-precipitation experiments demonstrate that ARRDC3 preferentially binds to ITGβ4 when phosphorylated on S1424. Using confocal microscopy, we show that the expression pattern of ARRDC3 on the lagging edge of a migrating cell is identical to the expression pattern of ITGβ4-pS1424. We demonstrate that ARRDC3 expression represses cell proliferation, migration, invasion, growth in soft agar and tumorigenicity. Collectively, our data reveals that ARRDC3 is a negative regulator of β4 integrin and demonstrates how this new pathway impacts biologic processes in stem cell and cancer biology. Additionally, as ARRDC3 is highly expressed in several tissues and conserved across species, our results are likely to be translated to other models.