RhoB est une petite GTPase rapidement activée par les facteurs de croissance et les stress cellulaires, qui régule des processus biologiques fondamentaux comme la migration, l'angiogenèse, la réparation de l'ADN, l'apoptose ainsi que la réponse à des thérapeutiques anticancéreuses. L'activité des petites GTPases est finement régulée par leur localisation subcellulaire. Cependant, l'activation de RhoB en cellules vivantes n'avait jamais été investiguée. Ce travail a permis d'adapter et de valider une méthode innovante d'analyse des interactions protéine-protéine par complémentation split-GFP tripartite, pour la détection sensible et spécifique de l'activation des petites GTPases en cellules vivantes. Nous avons ensuite développé un modèle cellulaire optimisé par la combinaison de la technologie split-GFP tripartite et d'un intracorps anti-GFP amplificateur de fluorescence, pour détecter la régulation de l'activation de RhoB avec une haute résolution spatiale. Ce biosenseur a mis en évidence la translocation de la forme active de RhoB en réponse au sérum à partir des endosomes pour s'accumuler au niveau de la membrane plasmique, révélant ainsi une nouvelle plateforme de signalisation membranaire de RhoB. Ce biosenseur permettra d'analyser le profil d'activation de RhoB et d'autres petites GTPases, sous d'autres stimulations ou dans différents contextes cellulaires, et d'identifier leurs partenaires et les modulateurs de leur activation. / RhoB is a small GTPase that is rapidly activated in response to growth factors and cellular stress. It regulates fundamental biological processes such as cell migration, angiogenesis, DNA repair, apoptosis and response to anticancer therapies. Small GTPases activity is tightly regulated by their subcellular localization. However, RhoB activation had never been investigated in living cells. In this work, we have adapted and validated an innovative method of protein-protein interactions analysis using tripartite split-GFP complementation, for the sensitive and specific detection of small GTPases activation in living cells. Then, we developed an optimized cellular model by combining the tripartite split-GFP technology with an anti-GFP intrabody fluorescence-enhancer to detect the regulation of RhoB activation with high spatial resolution. This biosensor highlighted the translocation of active RhoB from endosomes to accumulate at the plasma membrane upon serum stimulation, revealing a novel membrane signaling platform of RhoB. Future studies based on this biosensor will enable the analysis of RhoB activation profile and other small GTPases upon various stimuli or in different cellular contexts, as well as the identification of the GTPases partners and activation modulators.

In order to effectively treat disorders whose pathology is marked by neovascularization, a better understanding of the pathways that mediate the processes involved in angiogenesis is needed. To this end we have identified two important pathways that regulate endothelial cell capillary morphogenesis, a key process in angiogenesis. We have identified the small GTPase RhoB as being induced by vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Depletion of RhoB inhibited endothelial cell VEGF - mediated migration, sprouting, and cord formation. Cells depleted of RhoB showed a marked increase in RhoA activation in response to VEGF. Defects in cord formation in RhoB - depleted cells could be partially restored through treatment with the Rho inhibitor C3 transferase or ROCK I/II inhibitors, indicating increased RhoA activity and enhanced downstream signaling from RhoA contribute to the phenotype of decreased cord formation observed in cells depleted of RhoB. Interestingly, we did not observe a significant change in RhoC activity in RhoB - depleted cells suggesting differential regulation of RhoA and RhoC by RhoB in HUVECs. We have also identified microRNA - 30b (miR - 30b) as being negatively regulated by VEGF and as being a negative regulator of HUVEC capillary morphogenesis. Overexpression of miR - 30b significantly reduced HUVEC cord formation in vitro, while inhibition of miR - 30b enhanced cord formation. Neither overexpression nor inhibition of miR - 30b affected migration or viability of endothelial cells. Interestingly, miR - 30b regulated the expression of TGFβ2 but not TGFβ1, with overexpression of miR - 30b inducing expression of TGFβ2 mRNA and protein, and inducing phosphorylaton of Smad2 , suggesting TGFβ2 produced in response to miR - 30b overexpression functions in an iii autocrine manner to stimulate HUVECs . MiR - 30b effects on TGFβ2 expression were found to be regulated to an extent by ATF2, as miR - 30b overexpressing cells exhibited increased levels of phosphorylated ATF2 , with depletion of ATF2 via siRNA resulting in inhibition of miR - 30b - induced TGFβ2 expression. Treatment of HUVECs with TGFβ2 inhibited cord formation, while TGFβ1 had no effect, indicating a major difference in how endothelial cells respond to these two related growth factors. Inhibition of TGFβ2 with a neutralizing antibody restored cord formation in miR - 30b overexpressing cells to levels similar to control cells, thus identifying TGFβ2 expression as contributing to the inhibitory effects of miR - 30b overexpression on capillary morphogenesis. Thus, we have identified two signaling pathways regulated by VEGF in HUVECs that further our understanding of the process of angiogenesis and may provide novel targets for therapeutic intervention into diseases involving angiogenesis.

Cerebral palsy (CP) as a neurodevelopmental disorder that affects individuals’ movement, posture, and balance and occurs in every 2-3 out of 1,000 live births. Symptoms of CP can include seizures, hydrocephalus, impairment of the limbs, and learning disabilities. External contributors to CP are well known, but there are 80% of CP cases are idiopathic and in which no brain injury is reported. Recently, several genetic studies have shown that deleterious de novo mutations in CP patients may be implicated in CP pathogenesis. One such potentially deleterious de novo mutation of RhoB was identified in two CP patients. RhoB encodes for RHOB protein, a Rho GTPase that regulates the actin cytoskeleton. Biochemical and structural analyses of RhoB (S73F) protein suggested that the RhoB mutation generates a hyperactive form of RhoB. However, how the RhoB (S73F) protein may interfere with brain development and can contribute to CP is unknown. To determine whether RhoB (S73F) expression affects cortical development, we used in utero electroporations in mice to study the effect of RhoB (S73F) expression on cellular morphology, polarity, migration, and Golgi localization in the embryonic mouse model at E15.5 and P0, comparing it to a RhoB overexpression model as well as control. To address changes in cell morphology, we examined the cell size, shape, and volume of RhoB expressing cells using Imaris software. We show that RhoB overexpression and RhoB (S73F) expression cause detrimental changes in cell shape, polarity, and neuritogenesis. Furthermore, RhoB (S73F) expressing cells migrate less compared to RhoB overexpressing cells and control. Interestingly, we found that RhoB (S73F) expressing cells that did not migrate away from the ventricular surface still became neurons. To determine the effect of RhoB (S73F) expression on the subcellular environment, we examined the localization of the Golgi apparatus, and found the Golgi to be mislocalized and fragmented when RhoB (S73F) was expressed. Overall, this study shows that overexpression of RhoB is sufficient to cause changes in cell morphology, polarity, migration, and subcellular localization of the Golgi. Importantly, expression of RhoB (S73F) is distinct and unique from RhoB overexpression, causing more severe changes in cell size, shape, polarity, cell process number, and Golgi localization that result in failed neuronal migration. This data suggests the potential for genetic mutations to enact changes within the structure and function of cortical cells, which may contribute to the pathogenesis of CP. / Cell Biology

Made available in DSpace on 2014-12-17T14:08:35Z (GMT). No. of bitstreams: 1 KleberCOCpdf.pdf: 2178209 bytes, checksum: 588b533d30c060af9cf941e7001d3372 (MD5) Previous issue date: 2009-04-22 / In recent years, the DFA introduced by Peng, was established as an important tool capable of detecting long-range autocorrelation in time series with non-stationary. This technique has been successfully applied to various areas such as: Econophysics, Biophysics, Medicine, Physics and Climatology. In this study, we used the DFA technique to obtain the Hurst exponent (H) of the profile of electric density profile (RHOB) of 53 wells resulting from the Field School of Namorados. In this work we want to know if we can or not use H to spatially characterize the spatial data field. Two cases arise: In the first a set of H reflects the local geology, with wells that are geographically closer showing similar H, and then one can use H in geostatistical procedures. In the second case each well has its proper H and the information of the well are uncorrelated, the profiles show only random fluctuations in H that do not show any spatial structure. Cluster analysis is a method widely used in carrying out statistical analysis. In this work we use the non-hierarchy method of k-means. In order to verify whether a set of data generated by the k-means method shows spatial patterns, we create the parameter ? (index of neighborhood). High ? shows more aggregated data, low ? indicates dispersed or data without spatial correlation. With help of this index and the method of Monte Carlo. Using ? index we verify that random cluster data shows a distribution of ? that is lower than actual cluster ?. Thus we conclude that the data of H obtained in 53 wells are grouped and can be used to characterize space patterns. The analysis of curves level confirmed the results of the k-means / Nos ?ltimos anos, o DFA introduzido por Peng, foi estabelecido como uma importante ferramenta capaz de detectar autocorrela??o de longo alcance em s?ries temporais com n?o-estacionaridade. Esta t?cnica vem sendo aplicado com sucesso a diversas ?reas tais como: Econofis?ca, Biof?sica, Medicina, F?sica e Climatologia. No presente trabalho, utilizamos a t?cnica do DFA para obter o expoente de Hurst (H) do perfil el?trico de densidade (RHOB) de 53 po?os provindos do Campo Escola de Namorado. Neste trabalho queremos saber se podemos, ou n?o, utilizar este expoente para caracterizar espacialmente o campo. Duas hip?teses surgem: Na primeira o conjunto dos H reflete a geologia local, po?os com mesmo H se encontram pertos, e ent?o se pode pensar em utilizar H em procedimentos geoestat?sticos espaciais. Na segunda hip?tese cada po?o tem seu H, a informa??o dos H de cada po?o est? descorrelacionada e o conjunto dos perfis mostra apenas flutua??es aleat?rias em H que n?o revelam qualquer estrutura espacial. A an?lise de agrupamentos ? um m?todo bastante utilizado na realiza??o de an?lises estat?sticas. Nesta disserta??o utilizamos o m?todo de agrupamento n?o hier?rquico chamado m?todo do k-m?dia. Com o objetivo de verificar se um conjunto de dados gerados pelo m?todo do k-m?dia, ou de forma aleat?ria, forma padr?es espaciais, criamos o par?metro ? (?ndice de vizinhan?a). Altos ? implicam em dados mais agregados, baixos ? em dados dispersos ou sem correla??o espacial. Com aux?lio deste ?ndice e do m?todo de Monte Carlo verificamos que os dados agrupados aleatoriamente apresentam uma distribui??o mais baixa de ? do que os obtidos dos dados concretos e agrupados pelo k-m?dia. Desta forma conclu?mos que os dados de H obtidos nos 53 po?os est?o agrupados e podem ser usados na caracteriza??o espacial de campos. A an?lise de curvas de n?vel confirmou o resultado do k-m?dia

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 201 pages. Includes vita. Includes bibliographical references.