La cytoarchitecture du néocortex repose sur la coordination spatiotemporelle des taux de production des neurones ‐via la régulation du cycle cellulaire de leurs progéniteurs‐ et de leur migration radiale vers la surface corticale. Chez le primate, les couches supragranulaires de l'aire 17 sont plus développées que celles de l'aire 18, conséquence d'une prolifération et d'une production neuronale accrues dans l'aire 17 à E77‐80. Des observations en vidéomicroscopie bi‐photonique, sur tranches organotypiques de cortex, révèlent que la migration radiale est plus rapide dans l'aire 17. Les variations aire‐spécifiques des taux de prolifération et de migration neuronale sont donc congruentes. L'étude des mécanismes moléculaires qui sous‐tendent la régulation coordonnée de la prolifération et de la migration est centrée sur le régulateur du cycle cellulaire p27kip1, qui via son domaine C‐terminal promeut la migration en inhibant la GTPase RhoA. Ce rôle pléiotrope de p27 a été exploré dans la migration nucléaire intercinétique (INM) qu'effectuent les précurseurs de la zone ventriculaire corticale, en synchronie avec les phases du cycle cellulaire. Des formes mutantes de p27 ou des shRNA ont été électroporés spécifiquement dans les neuroblastes d'embryons murins à E14‐15. Des observations en vidéomicroscopie bi‐photonique sur tranches organotypiques révèlent que le domaine C-terminal de p27 affecte l'INM, promeut les décisions différenciatives et la migration radiale. P27 se place donc au sein d'un réseau moléculaire contrôlant conjointement, et de façon aire-spécifique, les divisions successives des précurseurs corticaux, ainsi que la migration des neurones qui en sont issus / Cortical cytoarchitecture relies on the spatiotemporal coordination of neuronal production rate, precursors cell-cycle control and neuronal radial migration towards the cortical plate. In the primate, area 17 supragranular layers are more developed than in area 18, due to higher proliferation and neuronal production rates in area 17 between E77-80. Two-photon videomicroscopy observations on cortical organotypic slices revealed that radial migration is faster in area 17 than 18. This indicates that area-specific variations of proliferation and migration rates are congruent during corticogenesis. The study of molecular mechanisms underlying the coordinated regulation of proliferation and migration focused on the cell-cycle regulator p27kip1, which promotes migration, via inhibition of the Rhoa GTPase by its C-terminal domain. This p27 dual function could play a major role during the Interkinetic Nuclear Migration (INM) performed by cortical precursor cells from the ventricular zone, in synchrony with the cell-cycle phases. Mutant forms of p27 or shRNA were electroporated into neuroblasts of E14-15 mice embryos. Two-photon videomicroscopy observations on organotypic slices revealed that p27 affects INM, promotes differentiative divisions and neuronal radial migration, though its C-terminal domain. P27 is thus part of a molecular network which finely tunes, in an area-specific manner, the successive rounds of divisions of precursor, as well as the migratory behavior of the newborn neurons

O cristalino de vertebrados se origina a partir da invaginação do ectoderme que recobre a vesícula óptica. A invaginação epitelial em diversos modelos é causada pela constrição celular apical, mediada pela contração apical de actina e miosina II e regulada pela GTPase RhoA. Neste trabalho nós investigamos se a invaginação do cristalino em embriões de galinha ocorre devido à constrição celular apical e se este evento é controlado por RhoA. Actina filamentosa e miosina II são expressas na porção apical do cristalino durante a invaginação. Quando a polimerização de actina é inibida por Citocalasina D, o cristalino não invagina, sugerindo que a constrição celular apical poderia contribuir para a invaginação do cristalino. RhoA também é expressa durante o desenvolvimento do cristalino, mas a inibição de RhoA, por eletroporação da forma dominante-negativo, não impediu a invaginação do placóide do cristalino, não alterou a distribuição de miosina II na porção apical do cristalino nem sua ativação, indicando que a invaginação do cristalino independe de RhoA. / Vertebrate lens derives from invagination of the ectoderm that overlies optic vesicles. Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase RhoA. Here we investigate the possibility that chick lens placode invagination could also be driven by apical cell constriction and controlled by RhoA. We show that actin and myosin II are expressed at lens apical side during lens invagination. Actin polymerization inhibition by in ovo Cytochalasin D treatment prevents lens placode invagination, suggesting that lens placode invagination could be driven by apical cell constriction. RhoA GTPase is also expressed at apical portion of lens placode and during lens invagination. However, when we overexpressed by electroporation the dominant-negative RhoA in the pre-lens ectoderm invagination was not affected. Furthermore, dominant-negative RhoA didnt affect myosin II apical localization nor myosin II phosphorilation, indicating that in lens invagination this process is not regulated by GTPase RhoA.

No description available.

<p>Cerebral cavernous malformations (CCMs) are multicavernous vascular lesions affecting the central nervous system. Affected individuals have a lifetime risk of recurrent headaches, focal neurological deficits, seizures, and intracerebral hemorrhage leading to stroke. Patients tend to fall into two classes: familial cases with a known family history and multiple lesions, and; sporadic cases with no family history and single lesions. This epidemiological pattern suggests a two-hit mutational mechanism for CCM. While somatic mutations have been identified in lesions from familial patients, it is unknown if sporadic cases follow the same genetic mechanism. Using a next-generation sequencing strategy, I have identified somatic mutations from sporadic CCM lesions in the three known CCM genes, including one lesion bearing two independent mutations in CCM1. These data support a two-hit mutation mechanism in CCM for sporadic patients.</p><p>The mechanism of CCM pathogenesis (how mutations in one of the three CCM genes causes lesions to form and develop) is currently unknown. We developed mouse models that recapitulate the human disease. We have further shown that inhibition of Rho Kinase decreases the number of late-stage, multicavernous lesions. This is the first potential therapeutic strategy to specifically treat CCM, and suggests that the RhoA pathway is a central player in CCM pathogenesis.</p> / Dissertation

In the present study, we have examined the expression of protease-activated receptors (PARS) and characterized their signaling pathways in rabbit gastric muscle cells. Immunoblot analysis revealed expression of PARl and PAR2 but not PAR3 or PAR4 in smooth muscle. The PARl agonist TFLLR activated Gq, G12, and Gi3, but not Gil, Gi2, G13, Gs or Gz, whereas the PAR2 agonist SLIGRL activated Gq, G13, Gil, and Gi2, but not Gi3, G12, Gs, or Gz. Both PARl and PAR2 agonists stimulated PI hydrolysis and Rho kinase activity and inhibited cAMP formation. PAR1-stimulated PI hydrolysis was abolished in cells expressing Gαq minigene, but was not affected in cells expressing Gαi minigene or in cells treated with pertussis toxin (PTx). PAR2-stimulated PI hydrolysis was partially inhibited in cells expressing Gαq or Gαi minigene and in cells treated with PTx. PAR1- and PAR2-stimulated Rho kinase activity was abolished in cells expressing Gα12 or Gα13 minigene, respectively. Both PARl and PAR2 agonists induced a transient initial contraction that was selectively blocked by the inhibition of PI hydrolysis with U73122 and MLC kinase activity with ML-9. PAR1-induced sustained contraction was preferentially inhibited by the PKC inhibitor bisindolylmaleimide and to a minor extent by the Rho kinase inhibitor Y27632, whereas PAR2-induced sustained contraction was preferentially inhibited by Y27632. Activation of both PARl and PAR2 induced MLC20 phosphorylation, whereas phosphorylation of MYPTl and CPI-17 are receptor-specific: only PARl induced CPI-17 phosphorylation and only PAR2 induced MYPTl phosphorylation.Activation of PARl and PAR2 also induced IκBα degradation and NF-κB activation; the effects were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting NF-κB is downstream of RhoA. PAR1- and PAR2-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (PKI), IKK2 (IKKIV), or NF-κB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IκBα (IκBα (S32A/S36A)), or phosphorylation-deficient RhoA (RhoA(S188A)). In addition, activation of PARl induced Gα12 phosphorylation, which was abolished by bisindolylmaleimide, suggests that phosphorylation was mediated by PKC derived from the activation of RhoA. Only PAR1-stimulated Rho kinase activity was significantly augmented by the PKC inhibitor. The effect of PKC inhibitor was additive to that of the PKA inhibitor.

No description available.

L’efficacité de la réponse immunitaire adaptative repose tout particulièrement sur la motilité des lymphocytes T naïfs entre la circulation sanguine et les organes lymphoïdes secondaires, leur permettant ainsi de rencontrer un antigène spécifique. De nombreuses voies de signalisation sont impliquées dans ce phénomène. En particulier, les Rho GTPases y jouent un rôle central, par leur capacité à moduler le cytosquelette d’actine. Nous avons identifié la protéine Fam65b comme nouveau régulateur de la circulation des lymphocytes T. En effet, nous avons montré que la diminution de l’expression de Fam65b dans des LT primaires humains induit une augmentation de leur polarisation, leur adhésion et leur migration in vitro. Afin d’étudier son rôle dans un contexte plus physiologique, nous avons développé au laboratoire une souris Fam65b-/-, dans laquelle l’expression de Fam65b est supprimée dans le lignage T. Les lymphocytes T issus de ces souris présentent un contenu global en F-actine réduit, une plus grande quantité de L-sélectine et d’intégrines actives à leur surface, et une migration moins rapide et moins rectiligne que leurs équivalents WT. Nous n’avons pu observer, avec nos méthodes, aucune différence significative de polarisation, de migration in vitro ou d’entrée dans les organes lymphoïdes secondaires pour les LT Fam65b-/-. Nous avons identifié les Rho GTPases comme médiateurs de ces effets de Fam65b. Nous avons observé, en cytométrie de flux, que les niveaux de RhoA-GTP et de Rac-GTP sont plus élevés dans les LT murins Fam65b-/-, et que cela est aussi vrai pour RhoA-GTP dans les LT humains exprimant de faibles niveaux de Fam65b. Nous avons identifié, dans des expériences in vitro, le mécanisme par lequel Fam65b inhibe l’activité de RhoA, puisqu’il ralentit sa charge en GTP par les protéines GEF. Nous avons montré, par des techniques de biochimie, que l’activation de RhoA en aval d’une stimulation chimiokine est permise par la dissociation de RhoA et de Fam65b, probable conséquence de la phosphorylation de Fam65b. Cette dissociation a aussi été observée pour Fam65b et Rac1, mais les mécanismes mis en jeu restent à déterminer. D’autre part, l’expression de Fam65b est sous le contrôle du facteur de transcription FOXO1, connu pour son rôle dans le contrôle de l’écotaxie (homing) via la régulation de l’expression de molécules permettant l’entrée dans les ganglions lymphatiques. Fam65b, régulateur atypique de l’activité des Rho GTPases, représente donc un lien inédit entre la voie PI3K/FOXO1 et les Rho GTPases. / The motility of naive T lymphocytes between the blood and secondary lymphoid organs is essential to the efficiency of the adaptative immune response, and allows those cells to meet their cognate antigen. Numerous signaling pathways are involved in this phenomenon, such as Rho GTPases, modulators of the actin cytoskeleton. We have identified Fam65b as a new regulator of T lymphocytes recirculation. We have shown that a decrease of Fam65b expression in human primary T cells increases the morphological polarization, the adhesion and the in vitro migration of those cells. Looking for a more physiological model, we developed, in the lab, a Fam65b KO (Knock-Out) mouse, specific to the T lineage. In those animals, T cells showed decreased levels of F-actin, an increase in the display of L-selectin and integrins, and a slower and less straight migration, compared to WT (Wild-Type) T cells. On the other hand, we weren't able to see any significant differences in the morphological polarisation, the in vitro migration or the homing capacity of the Fam65b KO T cells. We have identified Rho GTPases as mediators of the effects of Fam65b. We showed, in flow cytometry, that the amount of RhoA-GTP and Rac-GTP are increased in the Fam65b KO cells. The RhoA-GTP levels are also increased in human primary T cells expressing low levels of Fam65b. We have identified, in in vitro experiments, that Fam65b slows down RhoA loading with GTP by its GEF proteins, thus inhibiting RhoA activity. Moreover, we showed that Fam65b dissociates from RhoA after chemokine stimulation of T cells, thus allowing RhoA activation. The phosphorylation of Fam65b is a probable cause to this phenomenon. Fam65b also dissociates from Rac1 in these conditions, although no mechanism is yet known. Furthermore, the transcription factor FOXO1 controls the expression of Fam65b. FOXO1 is also known to control the homing capacity of T cells, since it controls the expression of molecules involved in the entry of lymphocytes in the lymph nodes. Fam65b, an atypical regulator of Rho GTPases activity, thus represents a new connection between the PI3K/FOXO1 and the Rho GTPases pathways.

L'angiogenèse est impliquée dans différents processus tels que la cicatrisation, la croissance tumorale et les maladies ischémiques. RhoA, connu pour son implication dans la prolifération et la migration des cellules, est régulé par la voie NO/PKG. Le sildenafil, inhibiteur de la phosphodiestérase 5, permet d'activer cette voie, ce qui constitue une stratégie thérapeutique intéressante dans le traitement des maladies ischémiques. Nous avon montré que le sildenafil améliore la croissance collatérale dès 7 jours de traitement, sans modifier la densité capillaire, via un mécanisme PI3K/akt-NO-dépendant et HIF/VEGF-indépendant. Ces différentes voies sont down-régulées à 21 jours. Enfin cette revascularisation s'accompagne d'un remodelage des artères de résistance, suggérant que le sildenafil favorise non pas l'angiogenèse mais l'artiogenèse. Enfin, nous avons mis en évidence un effet dose-dépendant des polyphenols du vin rouge, un autre puissant vasodilattaeur, sur la revascularisation post-ischémique : à fortes doses les densités vasculaires sont diminuées, associées à une inhibition des voies PI3K/Akt-NO et des MMPs. De faibles doses entraînent une augmentation des densités vasculaires par stimulation des voies PI3K/akt-No, sans affecter les MMPs. Par conséquent, nous avons mis en évidence un effet anti-angiogénique (fortes doses) et un effet pro-angiogenique (faibles doses) des plyphenols. Ce travail nous permet donc de mieux comprendre les mécanismes d'action de deux puissants vasodilatateurs dans revascularisation post-ischémique. Nous pouvons ainsi envisager de nouvelles stratégies thérapeutiques dans le traitement des maladies ischémiques ou du cancer.

GTPases are integral components of virtually every known signal transduction pathway, and mutations in GTPases frequently cause disease. A genomic analysis identified and annotated 174 GTPases in the sea urchin genome (with 90% expressed in the embryo), covering five classes of GTP-binding proteins: the Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR GTPases, and the translational GTPases. The sea urchin genome was found to contain large lineage-specific expansions within the Ras superfamily. For the Rho, Rab, Arf and Ras subfamilies, the number of sea urchin genes relative to vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increased overall numbers, such that total sea urchin gene numbers of these GTPase families approximate vertebrate gene numbers. This suggests lineage-specific expansions as an important component of genomic evolution in signal transduction. A focused analysis on RhoA, a monomeric GTPase, shows it contributes to multiple signal transduction pathways during sea urchin development. The data reveal that RhoA inhibition in the sea urchin results in a failure to invaginate during gastrulation. Conversely, activated RhoA induces precocious archenteron invagination, complete with the associated actin rearrangements and extracellular matrix secretion. Although RhoA regulates convergent extension movements in vertebrates, our experiments show RhoA activity does not regulate convergent extension in the sea urchin. Instead, the results suggest RhoA serves as a trigger to initiate invagination, and once initiation occurs RhoA activity is no longer involved in subsequent gastrulation movements. RhoA signaling was also observed during endomesodermal specification in the sea urchin. Data show that LvRhoA activity is required, downstream of a partially characterized Early Signal, for SoxB1 clearance from endomesodermal nuclei (and subsequent expression of GataE and Endo16 genes). Investigations also suggest that within the endomesoderm, RhoA clears SoxB1 as part of Wnt8 signaling, as activated RhoA is sufficient to rescue Wnt8-inhibited embryos. These data provide evidence of the first molecular components involved in SoxB1 clearance, as well as highlight a previously unrecognized role for RhoA during endomesodermal specification. These analyses suggest RhoA signaling is integral to the proper specification and morphogenesis of the sea urchin endomesoderm. / dissertation

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 107-130).