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Chinestra, Patrick. "Conception de biosenseurs des protéines RhoA, RhoB, RhoC". Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1715/.
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Notre équipe s'intéresse à la compréhension des mécanismes de dérégulation des voies de signalisation cellulaire dans la survenue et la maintenance des processus tumoraux, ainsi que leurs conséquences dans la réponse aux thérapies antitumorales. Nous nous intéressons particulièrement aux protéines Rho et à leurs régulateurs. Ils interviennent dans les voies de signalisation des récepteurs cellulaires conduisant à des modifications de l'adhérence, de la prolifération, de la motilité et de la balance survie/mort cellulaire. Les protéines RhoA, RhoB et RhoC sont des petites GTPases passant d'un état actif (liées au GTP) à un état inactif (liées au GDP) et dont l'homologie est proche de 85%. La surexpression des protéines RhoA et RhoC a été décrite dans un grand nombre de tumeurs; à l'inverse, on observe une diminution de l'expression de RhoB dans le mélanome et dans le cancer du poumon. La conception de fragments d'anticorps sélectifs de la conformation active des Rho, appelés biosenseurs, permettant d'évaluer l'activation de ces protéines in situ dans des coupes de tissus sains et cancéreux, pourrait aboutir à un usage pronostique de ces outils voire à la définition de nouveaux marqueurs thérapeutiques et permettrait de répondre à des questions plus fondamentales comme leurs localisations cellulaire, leur rôle dans la migration cellulaire, leur activation spatio-temporelle. A partir du scFvC1 sélectif de la conformation active des trois protéines RhoA, RhoB et RhoC et isolé précédemment dans l'équipe, nous avons créé une banque secondaire par mutagénèse aléatoire et opéré une sélection par phage display avec un objectif double : 1°) augmenter l'affinité du scFvC1 pour améliorer ses capacités d'interaction avec les protéines Rho actives natives en vue d'améliorer ses performances en immunohistologie ainsi que pour une utilisation intracellulaire. 2°) sélectionner des variants spécifiques de chaque Rho malgré leur très forte homologie. Nous montrons que la stratégie mise en œuvre permet d'augmenter l'affinité des scFv et de modifier leur sélectivité puisqu'un variant se lie préférentiellement à RhoA et RhoC. De plus, contrairement au svFvC1, ces scFv sont immunoprécipitants pour les Rho actives produites dans des cellules eucaryotes. En parallèle, nous avons mis au point une méthodologie permettant le marquage d'un scFv anti-RhoB obtenu au laboratoire, par l'action d'un dérivé fluorescent de la biotine sur une intéine exprimée en fusion C-terminale du scFv. Ceci permettra d'améliorer les techniques immunohistologiques avec des biosenseurs fluorescentsOur team is interested in understanding the mechanisms of deregulation of cell signaling pathways in the development and maintenance of tumor processes and their consequences in response to anti-tumor therapies. We focuse on Rho proteins as well as on their regulators. They are involved in signaling pathways of cell receptors leading to changes in adhesion, proliferation, motility and balance survival / cellular death. RhoA, RhoB and RhoC are small GTPases switching from an active state (GTP-bound) to an inactive state (GDP-bound) and the homology of which is close to 85%. Overexpression of RhoA and RhoC protein has been described in many tumors; in contrast, there was a decreased expression of RhoB in melanoma and lung cancer. Engineering antibody fragment specific of Rho active conformations, namely biosensors, would allow in situ assessment of these proteins activation in healthy or tumour samples. These tools could be further developed towards diagnosis or prognosis usage, or could even define novel therapeutics markers and be used to answer more fundamental question as their cellular localization, their role in cell migration, or their spatio-temporal activation. Starting from the scFvC1 previously isolated in the group and which is selective for RhoA, RhoB and RhoC active conformations, we have created by random mutagenesis a second library and performed a phage display selection with two aims: 1°) increase the C1 scFv affinity to enhance its interaction potential with active native Rho proteins in order to improve its performance in immuno-histology or to use it as an intracellular antibody. 2°) select variants with a selectivity towards strictly only one of the three Rho excluding the others despite their strong homology. We show that our strategy allowed an affinity increase of scFv and also a selectivity modulation as one variant preferentially binds RhoA and C. Moreover, in contrast to scFvC1 these scFvs immuno-precipitate active endogenous Rho Proteins in eukaryotic cells. In parallel, we have established a method allowing the labelling of an anti-RhoB scFv from the lab, by fusing it to an intein that induce covalent binding of a biotin fluorescent analogue. This approach will improve immuno-histological techniques using fluorescent biosensors
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Huppertz, Tilman [Verfasser]. "Charakterisierung der Rekrutierungsdomäne der kleinen Rho-GTPasen RhoA und RhoC / Tilman Huppertz". Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2009. http://d-nb.info/1023697491/34.
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Zandvakili, Inuk. "RhoA as a Potential Target in Lung Cancer". University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1445342196.
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Will, Laura Christine [Verfasser]. "Charakterisierung der Interaktion von p120ctn mit den Rho-GTPasen RhoA und RhoC / Laura Christine Will". Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1178320073/34.
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Will, Laura [Verfasser]. "Charakterisierung der Interaktion von p120ctn mit den Rho-GTPasen RhoA und RhoC / Laura Christine Will". Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1178320073/34.
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Soliman, Hesham. "The RhoA/Rho kinase pathway in diabetic cardiomyopathy". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43439.
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Diabetes mellitus leads to a unique pathological entity termed diabetic cardiomyopathy, the mechanisms of which have not been fully defined. In diabetic rat hearts, RhoA expression is increased and the RhoA/ROCK pathway is activated, while ROCK inhibition acutely improves contractile function of diabetic hearts. The mechanisms underlying this improvement and those responsible for the detrimental activation of RhoA/ROCK were investigated here.
Nitric oxide (NO) has been reported to upregulate RhoA expression in smooth muscle, and previous reports showed that iNOS expression is increased in diabetic rat hearts. In the first part of this thesis, the hypothesis that in diabetic cardiomyopathy, iNOS induction is responsible for increased RhoA expression was investigated. The results demonstrate that increased NO production from iNOS induction leads to RhoA upregulation in the diabetic heart and in isolated cardiomyocytes, contributing to the RhoA/ROCK mediated contractile dysfunction by increasing the total pool of RhoA available for activation.
In diabetic rat hearts, PKCβ₂ activation induces iNOS expression, leading to increased nitrosative/oxidative stress. This suggests that PKCβ2 might positively regulate RhoA expression, although in preliminary experiments inhibition of ROCK itself reduced RhoA expression. Therefore, in the second part, the hypothesis that PKCβ₂/iNOS and RhoA/ROCK interact together to form a positive feedback loop was tested. The results show that RhoA/ROCK overactivation is sustained by a positive feedback loop that involves PKCβ₂ activation and iNOS induction. This feedback loop requires an intact actin cytoskeleton and plays a key role in elevating superoxide production in diabetic rat hearts.
In the third part, the hypothesis that ROCK inhibition augments contraction by improving Ca²⁺ signaling was tested. Inhibition of ROCK improved contractile function and abolished the diabetes-induced delayed aftercontractions in isolated cardiomyocytes, in association with an improvement in Ca²⁺ transients.
Overall, the results show that in diabetic cardiomyopathy, overactivation of RhoA/ROCK contributes to contractile dysfunction by sustaining PKCβ2 activation, iNOS induction and superoxide production via a positive feedback loop that leads to impaired intracellular Ca²⁺ homeostasis. Inhibition of ROCK disrupts the loop, resulting in decreased oxidative stress, and improved Ca²⁺ handling and cardiomyocyte contraction, suggesting that ROCK inhibition might be a novel approach in treating diabetic cardiomyopathy.
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Srivastava, Kirtiman. "Pathophysiological role of RhoA/Rho-kinase under oxygen-glucose deprivation/reperfusion and hyperglycaemia". Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13533/.
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Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations.
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Ard, Ryan. "Regulation of RhoA Activation and Actin Reorganization by Diacylglycerol Kinase". Thesis, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/22669.
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Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.
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Bendris, Nawal. "Nouvelles fonctions de la Cycline A2 : régulation de l’invasion cellulaire et de la transition épithéliomésenchymateuse". Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20079.
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L'agressivité des cancers est souvent liée au pouvoir métastatique des cellules tumorales et la dissémination de ces dernières peut survenir suite à un phénomène appelé la transition épithéliomésenchymateuse. Une analyse de l'expression de la Cycline A2 conduite sur des échantillons humains de tumeurs primaires colorectales et de leurs métastases correspondantes révèle que cette protéine est moins abondante dans ces dernières. Le travail décrit dans cette thèse a permis de relier la Cycline A2 au remodelage du cytosquelette d'Actine dans les fibroblastes. Cette régulation requiert la localisation cytoplasmique de la molécule ainsi que son domaine N-terminal qui ne lie pas les CDKs. Nos expériences suggèrent que cette nouvelle activité est la conséquence d'une liaison directe entre la GTPase RhoA et la Cycline A2. La présence de cette dernière augmente l'activation de RhoA par sa GEF in vitro. L'utilisation de cellules épithéliales mammaires normales a permis l'identification d'un autre partenaire, RhoC. Dans ce contexte cellulaire, l'invalidation de la Cycline A2 diminue l'activation de RhoA et, renforce celle de RhoC ce qui conduit à une augmentation de l'invasion cellulaire en matrice de collagène. Ces cellules acquièrent aussi des propriétés mésenchymateuses caractéristiques de l'EMT, et ce phénotype est exacerbé par la présence de RasV12. Ce travail établit donc l'existence de nouvelles fonctions pour la Cycline A2 qui viennent compléter le tableau de régulation de la motilité par les protéines du cycle cellulaire et contribuent à une meilleure compréhension de son rôle dans le cancerCancer aggressiveness is often associated with metastases occurrence and their dissemination can arise following an epithelial to mesenchymal transition (EMT). Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma of matched human tumors. This manuscript describes new links between Cyclin A2 and Actin cytoskeleton remodeling in fibroblasts. This regulation requires a cytoplasmic localization of the protein and its N-terminal domain, which is unable to bind CDKs. This new Cyclin A2 activity appears to be mediated by its binding to RhoA. Accordingly, the activity of its GEF is potentiated when Cyclin A2 is present, in vitro. Furthermore, we used a normal mammary epithelial cell line and identified another Cyclin A2 partner, RhoC. Cyclin A2 depletion in this context leads to a reciprocal RhoGTPase activation where RhoA activation is impaired and that of RhoC is increased. Moreover, cell invasiveness is increased in a collagen matrix following Cyclin A2 knockdown in these cells. In addition, the epithelial cells acquire mesenchymal properties, which are exarcerbated by the expression of RasV12 and are characteristic of an EMT. Our work completes the network involving cell cycle proteins in motility. These novel functions of Cyclin A2 will hopefully help to understand the impact of its deregulation in cancer
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Keller, Laura. "Conception de nano-anticorps conformationnels comme nouveaux outils d'étude de l'activité des GTPases de la sous-famille RHOA". Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30005/document.
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Les GTPases de la sous famille RHOA participent à la régulation de nombreuses voies de signalisation qui contrôlent la dynamique du cytosquelette cellulaire et une grande diversité de fonctions telles que la prolifération, la division, la migration et la polarité cellulaires. Ce sont de véritables interrupteurs moléculaires qui, en réponse à un stimulus, changent de conformation tridimensionnelle pour activer leurs protéines effectrices cibles. Elles existent donc sous deux formes, une forme inactive liant le GDP et une forme active, liant le GTP. La proportion de forme active est extrêmement régulée au niveau spatial et temporel dans une cellule et représente moins de 10% de sa totalité. Depuis près de 20 ans, le seul outil disponible pour étudier leur activation est constitué par le domaine de liaison d'un effecteur, le RBD. Peu stable, faiblement soluble et peu adaptable, de nouveaux outils sont nécessaires afin de mieux comprendre la fine régulation de ces protéines. Les anticorps à simple domaine, VHH ou nanobodies, sont caractérisés par leur stabilité, solubilité, haut rendement de production et versatilité de fonctionnalisation. A partir d'une nouvelle banque d'anticorps à simple domaine optimisée pour la production d'intracorps, nous avons isolés différents clones capables de reconnaître in vitro et de bloquer in cellulo la forme active de ces protéines. L'un de ces clones permettra le développement d'un nouvel outil de mesure de l'activité de ces protéines in vitro tandis qu'un autre, in cellulo, permettra de mieux comprendre la régulation spatiale et temporelle des protéines endogènesRHOA small GTPase belongs to a subfamily acting as a molecular switch activating major signaling pathways that regulate cytoskeletal dynamics and a variety of cellular responses such as cell cycle progression, cytokinesis, migration and polarity. RHOA activity resides in a few percent of GTP loaded protein, which is finely tuned by a crosstalk between regulators of the GTPase cycle. Manipulating a single RHO at the expression level often induces imbalance in the activity of other RHO GTPases, suggesting that more specific tools targeting these active pools are needed to decipher RHOA functions in time and space. We decided to use single domain antibodies, also known as VHH or nanobodies, as a new tool for studying RHOA activation. We produced and screened a novel fully synthetic phage display library of humanized nanobodies (NaLi-H1) to develop conformational sensors of the GTP loaded active conformation of RHO subfamily. We obtained several high affinity nanobodies against RHOA's active form which we characterized as RHO active antibodies in vitro and RHO signaling blocking intrabodies in cellulo. These new tools will facilitate and improve our current knowledge of this peculiar protein subfamily and will be a paradigm for the study of other RHO related small GTPases
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Böhringer, Christian. "The role of RhoA in corticogenesis". Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-148975.
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Agard, Christian. "Nouvelles approches pharmacologiques expérimentales ciblant la voie RhoA/Rho kinase dans l'hypertension artérielle pulmonaire". Nantes, 2009. http://www.theses.fr/2009NANT2091.
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L’hypertension artérielle pulmonaire (HTAP) est une maladie rare mais grave. Idiopathique, ou associée à certaines conditions, l’HTAP se caractérise par une obstruction progressive des artères pulmonaires distales. La physiopathologie de l’HTAP est complexe : dysfonction endothéliale, vasoconstriction artérielle, prolifération des cellules musculaires lisses (CML) de la média, thromboses in situ, fibrose et inflammation. Les mécanismes moléculaires de ces processus sont multiples et encore mal compris. La petite protéine G RhoA et son effecteur Rho kinase sont impliqués dans la pathogénie de l’HTAP car elles régulent les propriétés de contraction et de prolifération de la CML vasculaire. La sérotonine (5-HT), agent mitogénique et constricteur de la CML d’artère pulmonaire, et son transporteur (5-HTT) sont également impliqués dans l’HTAP. Cette thèse décrit l’intéraction entre la voie 5-HT/5-HTT et la voie RhoA/Rho kinase dans l’HTAP chez l’homme. Dans les poumons de patients avec HTAP, l’activation de RhoA/Rho kinase ainsi que la sérotonylation de RhoA, par transamination, ont été observées. In vitro, la prolifération, induite par la 5-HT, de CML d’artère pulmonaire met en jeu le 5-HTT et la voie RhoA/Rho kinase. Par ailleurs, nous avons testé la metformine, antidiabétique oral de la famille des biguanides, dans l’hypertension pulmonaire expérimentale chez le rat. En traitement préventif et curatif, la metformine a amélioré les paramètres hémodynamiques et les lésions de remodelage de l’hypertension pulmonaire. L’origine de l’effet thérapeutique est multiple : amélioration de la fonction endothéliale, inhibition de la prolifération des CML d’artère pulmonaire, inhibition de la vasoconstriction. Les effets s’expliquent au moins en partie par une inhibition de la voie RhoA/Rho kinase, possiblement médiée par l’activation de l’AMPK. Nos travaux ont contribué à l’analyse du rôle et de la régulation de la voie de signalisation RhoA/Rho kinase dans l’HTAP et ont permis d’identifier une nouvelle approche pharmacologique dans cette maladie
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Bei, Yihua. "Implications de la voie RhoA/Rho-kinases dans la physiopathologie des atteintes vasculaires et interstitielles pulmonaires des maladies respiratoires chroniques : études humaines et expérimentales chez la souris". Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05T011/document.
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La voie RhoA/Rho-kinases (ROCK) joue un rôle important dans la physiopathologie de l’hypertension pulmonaire (HTP) par son implication dans le dysfonctionnement endothélial, la constriction et le remodelage des vaisseaux pulmonaires. Selon les classifications internationales, la bronchopneumopathie chronique obstructive (BPCO) et la pneumopathie infiltrante diffuse (PID) sont deux causes fréquentes d’HTP ayant en commun plusieurs mécanismes physiopathologiques dont le dysfonctionnement endothélial, le remodelage vasculaire et la fibrose parenchymateuse. Les objectifs de ce travail étaient d’étudier le rôle de la voie RhoA/ROCK dans la physiopathologie de la BPCO et de la PID avec ou sans HTP et de préciser les anomalies moléculaires liées à la perturbation de la signalisation de cette voie dans chacune de ces situations.Le dysfonctionnement endothélial est un événement essentiel dans l’initiation et la progression de la BPCO. L’activation de la voie RhoA/ROCK dans le dysfonctionnement endothélial systémique et pulmonaire a été mise en évidence chez les tabagiques avec ou sans BPCO. Les résultats de notre première étude montrent l’existence d’une activation de la voie RhoA/ROCK au niveau des artères pulmonaires chez les patients BPCO ayant un dysfonctionnement endothélial, et une corrélation entre l’activité de la RhoA et l’expression génique et l’activité de la NO synthase endothéliale (NOS-3).L’HTP est une complication grave des PID. Nous avons montré dans notre deuxième étude l’implication de la voie RhoA/ROCK dans la réponse inflammatoire et la fibrose pulmonaire (FP) dans un modèle murin de PID induite par injection intratrachéale de bléomycine (BLM). Nous avons ensuite testé l’effet préventif du fasudil, un inhibiteur des ROCK, sur l’apparition de la FP et l’HTP expérimentales induites par la BLM. Les résultats de cette deuxième étude montrent que la FP et l’HTP sont associées à une activation de la voie RhoA/ROCK dans ce modèle murin et que le fasudil inhibe la réponse inflammatoire, la FP et l’HTP, via l’inhibition de la phosphorylation de Smad2/3 de la voie de signalisation par le TGF-β1.La FP et l’HTP représentent deux causes principales de mortalité liée à la sclérodermie systémique (ScS). Nous avons étudié le rôle de la voie RhoA/ROCK dans la physiopathologie de la fibrose cutanée et l’atteinte pulmonaire dans un modèle murin de ScS induite par injection intradermique d’acide hypochloreux (HOCl). Les résultats de cette troisième étude montrent l’association entre la fibrose cutanée induite par l’HOCl et l’activation de la voie RhoA/ROCK au niveau de la peau, et l’effet préventif du fasudil sur la fibrose cutanée et pulmonaire, en partie via l’inhibition de la phosphorylation de Smad2/3 et de l’activation des protéines ERK1/2. Ces résultats suggèrent l’implication de la voie RhoA/ROCK dans la physiopathologie de la BPCO et de la PID avec ou sans HTP. La voie RhoA/ROCK pourrait de ce fait représenter une nouvelle cible thérapeutique dans la BPCO et la PID avec ou sans HTP.Mots-clés : RhoA, Rho-kinases, fasudil, BPCO, fibrose pulmonaire, hypertension pulmonaireThe RhoA/Rho-kinases (ROCK) pathway plays a pivotal role in the pathophysiology of pulmonary hypertension (PH) as its abnormal activation leads to endothelial dysfunction, sustained vasoconstriction and pulmonary vascular remodeling. According to the international classification of PH, chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) represent two main causes of PH associated with chronic respiratory diseases. These two causes have in common major pathophysiological mechanisms such as endothelial dysfunction, vascular remodeling and interstitial fibrosis. The aims of the present study were to investigate the role of the RhoA/ROCK pathway in the pathophysiology of lung vascular and interstitial injuries in COPD and ILD with or without development of PH, and to study the molecular mechanisms associated with regulation of the RhoA/ROCK pathway in each of these situations.The pulmonary endothelial dysfunction is an essential event in the initiation and progression of COPD. Although the role of the RhoA/Rho-kinase pathway in pulmonary endothelial dysfunction has been demonstrated in smokers with normal lung function, little is known about its role in patients with COPD. The results of our first study demonstrated an increase in RhoA and ROCK activity in pulmonary arteries of patients with COPD, simultaneously with an altered pulmonary endothelial-dependent vasodilation. The increased RhoA activity in patients with COPD was correlated with an impairment of the gene expression and activity of endothelial NO synthase (eNOS).PH associated with pulmonary fibrosis (PF) considerably worsens prognosis of ILD. The results of our second study showed an activation of the RhoA/ROCK pathway in lung tissues of mice intoxicated by intratracheal instillation of bleomycin (BLM). BLM induced severe PF and PH in mice, associated with an increased RhoA and ROCK activity in the lung. We further demonstrated that long-term treatment with fasudil, a selective ROCK inhibitor, reduced BLM-induced lung inflammation, lung fibrosis and PH in mice, at least in part, via inhibition of Smad2/3 phosphorylation in TGF-β1 signaling.PF and PH represent two leading causes of death in patients with systemic sclerosis (SSc). In our third study, we investigated the role of the RhoA/ROCK pathway in the pathophysiology of skin fibrosis and lung injuries in a murine model of SSc induced by intradermal injection of hypochlorous acid (HOCl). We demonstrated that HOCl-induced skin fibrosis was associated with an activation of the RhoA/ROCK pathway in the fibrotic skin, and that long-term treatment with fasudil reduced both skin and lung fibrosis through inhibition of the phosphorylation of Smad2/3 and ERK1/2 in the fibrotic skin.These results suggest the implications of the RhoA/ROCK pathway in the pathophysiology of lung vascular and interstitial injuries in COPD and ILD with and without development of PH. The RhoA/ROCK pathway might be a promising therapeutic target for patients with COPD or ILD with and without PH
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Gluth, Markus. "Untersuchungen zum Einfluss von RhoA und der RhoA Effektorkinase PKN auf die TNF-induzierte Barrieredysfunktion in humanen intestinalen Epithelzellen". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16529.
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Chronisch entzündliche Darmerkrankungen stellen eine Gruppe von chronischen, häufig in Schüben verlaufenden Erkrankungen mit rezidivierenden Entzündungen des Gastrointestinaltraktes dar. Es konnte gezeigt werden, dass eine gestörte Barrierefunktion einen wichtigen Schritt für die Pathogenese darstellt und dass das Zytokin Tumornekrosefaktor alpha (TNF) eine entscheidende Rolle dabei spielt. Die Rolle der kleinen GTPase RhoA bei der TNF-induzierten Barrieredysfunktion ist aufgrund der Komplexität der Signalwege nicht vollständig verstanden. Daher sollte der Einfluss von RhoA und der RhoA Effektorkinase PKN auf diese Prozesse in vitro mit Hilfe eines induzierbaren Expressionssystems untersucht werden, welches die kontrollierte Expression einer konstitutiv aktiven (KA) RhoA- und PKN-Mutante sowie einer dominant negativen (DN) PKN-Mutante ermöglichte. Die Induktion der KA RhoA Expression führte zu einer Störung der epithelialen Barriere. Eine simultane Interferon-gamma und TNF-Behandlung resultierte ebenfalls in einer gestörten Barrierefunktion, welche in KA RhoA Zellen weniger stark ausgeprägt war. Die TNF-Behandlung führte zu einer Aktivierung von PKN, weshalb dieses Protein ein Kandidat für die Vermittlung dieser Effekte darstellte. Inhibition von PKN mit Inhibitoren oder der Expression der DN Mutante führten zu einer Aggravierung der TNF-induzierten Barrieredysfunktion, welche durch eine Verringerung des transepithelialen elektrischen Widerstandes und eine erhöhte Ionenpermeabilität charakterisiert war. Diese Veränderungen wurden von einer Erhöhung des Myosin Leichtketten und NF-kappaB p65-Phosphorylierungsniveaus sowie von morphologischen Veränderungen begleitet. Im Gegensatz dazu konnten diese Veränderungen durch die Expression der KA PKN Variante abgeschwächt bzw. verhindert werden. Diese Ergebnisse liefern Hinweise auf eine potenzielle Rolle der RhoA Effektorkinase PKN bei der Modulation der TNF-induzierten Barrieredysfunktion in intestinalen Epithelzellen.Inflammatory bowel diseases are relapsing systemic inflammatory diseases of the gastrointestinal tract associated with high morbidity and costs. A plethora of studies demonstrated that impaired intestinal barrier function is a key step in the pathogenesis of inflammatory bowel diseases and that the cytokine tumor necrosis factor alphpa (TNF) is of pivotal importance for this effect. Although the small GTPase RhoA has been implicated in the control of tight junction function, its role in TNF induced barrier dysfunction is not entirely understood due to the complexity of its downstream signaling pathways. Therefore, the contribution of RhoA and its effector kinase PKN on TNF induced barrier dysfunction was investigated in vitro. An inducible expression system that allowed the doxycyline controlled expression of a constitutively active (CA) RhoA and PKN mutant as well as a dominant negative (DN) PKN mutant was generated. Induction of CA RhoA expression led to an impaired epithelial barrier. Simultaneous Interferon-gamma and TNF treatment also resulted in barrier perturbation, but this defect was attenuated when CA RhoA was expressed. As treatment with TNF resulted in activation of the RhoA effector kinase PKN, this protein constitutes a candidate molecule for the mediation of these effects. Inhibition of PKN by inhibitory compounds as well as expression of a dominant negative PKN mutant aggravated TNF-induced barrier dysfunction, characterized by a decline in transepithelial electrical resistance and increased ion permeability. These alterations were accompanied by an increase in myosin light-chain and NF-kappaB p65 subunit phosphorylation level as well as morphological changes of the tight junctions. Conversely, expression of a CA PKN mutant attenuated or prevented these changes. These results provide support for a potential role of the RhoA effector kinase PKN in modulating the barrier disrupting effects of TNF in the intestinal epithelium.
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Stirling, Lee. "Dual Roles for Rhoa/Rho-Kinase in the Regulated Trafficking of a Voltage-Sensitive Potassium Channel". ScholarWorks @ UVM, 2009. http://scholarworks.uvm.edu/graddis/223.
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Kv1.2 is a member of the Shaker family of voltage-sensitive potassium channels and contributes to regulation of membrane excitability. The electrophysiological activity of Kv1.2 undergoes tyrosine kinase-dependent suppression in a process involving RhoA. We report that RhoA elicits suppression of Kv1.2 ionic current by modulating channel endocytosis. This occurs through two distinct pathways, one clathrin-dependent and the other cholesterol-dependent. Activation of RhoA downstream effectors Rho-kinase (ROCK) or protein kinase N (PKN) via the lysophosphatidic acid (LPA) receptor elicits clathrin-dependent Kv1.2 endocytosis and consequent attenuation of its ionic current. LPA-induced channel endocytosis is blocked by ROCK inhibition , dominant negative PKN, or by clathrin RNAi. In contrast, steady-state endocytosis of Kv1.2 in un-stimulated cells is cholesterol-dependent. Inhibition of basal ROCK with Y27632 or basal PKN with HA1077 increases steady-state surface Kv1.2. The Y27632-induced increase persists in the presence of clathrin RNAi and, in the presence of the sterol-binding agent filipin, does not elicit an additive effect. Temperature block experiments in conjunction with studies that perturb trafficking of newly synthesized proteins from the Golgi demonstrate that basal ROCK affects cholesterol-dependent trafficking by modulating the recycling of constitutively endocytosed Kv1.2 back to the plasma membrane. Both receptor-stimulated and steady-state Kv1.2 trafficking modulated by RhoA/ROCK require the activation of dynamin as well as the ROCK effector LIM kinase, indicating a key role for actin remodeling in RhoA-dependent Kv1.2 regulation.
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Kaarbo, Mari, i n/a. "The Role of RhoA in Early Heart Development". Griffith University. School of Biomolecular and Biomedical Science, 2005. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060105.091005.
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RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.
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Sahai, Erik Anand. "Functional analysis of RhoA and its effector molecules". Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300839.
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Kaarbo, Mari. "The Role of RhoA in Early Heart Development". Thesis, Griffith University, 2005. http://hdl.handle.net/10072/366791.
Pełny tekst źródłaStreszczenie:
RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text
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Krause, Sven Matthias. "Small Rho-GTPase function in myelinating cells of the vertebrate peripheral nervous system with focus on RhoA /". Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17732.
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Turner, Stephanie J. "RhoA, B, and C in cancer study of statin-induced changes in Rho signaling, and identification of isoform-specific Rho effectors /". Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3359512.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed July 7, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 77-106).
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Silva, Gisele Espinha Teixeira da. "Sinalização da GTPase RhoA nas respostas celulares após estresse genotóxico promovido por radiação ultravioleta". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-19102016-165552/.
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A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida.The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.
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Yusuf, Muhammad Zuhair. "cAMP signaling reverses platelet spreading via inhibition of RhoA". Thesis, University of Hull, 2016. http://hydra.hull.ac.uk/resources/hull:14381.
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Prostacyclin (PGI₂) is a key regulator of platelet function. There is a significant field of research which outlines the role of PGI₂ in inhibiting platelets in circulation, and therefore playing a major role in the prevention of excessive thrombus formation. PGI₂ signalling is countered by strong activatory stimuli leading to platelet spreading and thrombus formation. However little is known if PGI₂ plays a role after platelet activation. Therefore, the aim of this study was to identify the effect of PGI₂ on already activated platelets. Therefore, thrombi were formed on fibrinogen and/or collagen, and then PGI₂ was flowed over the top. This revealed a significant reduction in thrombus surface area on fibrinogen, and both surface area coverage, and thrombus height on collagen, demonstrating the ability of PGI₂ to modulate a pre-formed thrombus. To understand the mechanism behind this event it was postulated that PGI₂ was modulating the actin cytoskeleton of an activated platelet, leading to thrombus instability. To identify if cytoskeletal changes were induced by PGI₂, platelet spreading was monitored. Platelet spreading follows a defined sequence of events, with the formation of filopodia, actin nodules, lamellipodia and finally stress fibres. By understanding the percentage of platelets containing each structure, it would be possible to identify the role of PGI₂ within platelet spreading. Therefore, platelets were allowed to spread, before stimulation with PGI₂. This identified that PGI₂ induced a significant reduction in stress fibre formation whilst inducing actin nodule formation. Alongside this there was a reduction in the surface area of the platelet. Further to this PGI₂ was shown to induce a strong elevation of cAMP within fully spread platelets. This reversal of stress fibre formation occurred in a PKA dependent manner as inhibition of PKA induced an inhibition of the stress fibre reversal induced by PGI₂. Stress fibre formation is linked to RhoA activity. Inorder to understand if PKA was modulating RhoA, the phosphorylation and activity status of RhoA was monitored. We showed clearly that PKA both induced the phosphorylation, but also significantly reduced the activity of RhoA in spread platelets. Further to this, there was a reduction in the phosphorylation of the light chains of Myosin II. Thus, PGI₂ modulates the actin cytoskeleton via a PKA dependent inactivation of RhoA, leading to stress fibre reversal and thrombus instability.
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Lawder, John J. "The Role of RhoA in GPR116 Mediated Alveolar Homeostasis". University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1563526829841322.
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Del, Re Dominic Pasquale. "RhoA as a mediator of cardiomyocyte survival and apoptosis". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3316411.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed Sept. 9, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 121-141).
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Jatho, Aline. "The role of the monomeric GTPase RhoA in cardiac fibroblasts". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://hdl.handle.net/11858/00-1735-0000-0023-98EA-5.
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Der spezifische Knockdown von RhoA in neonatalen kardialen Rattenfibroblasten führte auf molekularem Level zu einer Reduktion des Myofibroblastenmarkers α-Glattmuskelaktin und zu einem Anstieg im modifizierten acetylierten Tubulin. Auf subzellulärer Ebene konnte ein Verlust von Stressfasern, Aktinstrukturen höherer Ordnung und eine erhöhte Dichte des Golgi-Apparats beobachtet werden. Außerdem waren die Fokaladhäsionen kürzer und zufällig verteilt, was auf einen Verlust der Zellpolarität hinweist. Auf dem zellulären Level erhöhte der Knockdown von RhoA die Zellfläche aber nicht das Volumen. Diese Veränderungen führten zu einer schnelleren Adhäsion unabhängig vom Substrat, eine Reduktion der Migration in 2D und im Gegensatz dazu eine verbesserte Migration durch eine poröse Membran. Außerdem war die mitogene Antwort der Zellen auf einen Serumstimulus stark reduziert. Eine Veränderung in Zellviabilität konnte zudem nicht beobachtet werden. Die Expression und Sekretion des Fibrose-assoziierten Faktors CTGF war in gehungerten Zellen mit einer Reduktion in RhoA Expression signifikant vermindert, was jedoch in der Anwesenheit eines Serumstimulus aufgehoben werden konnte. Auf einer heterogenen multizellulären Ebene verringerte der Knockdown von RhoA die kontraktile Funktion von generierten künstlichen Herzgeweben unter Kalziumstimulation. Dies ging einher mit einer Reduktion der Expression von α-Glattmuskelaktin und Calsequestrin. Durch die Verwendung
spezifischer Inhibitoren der Rho-assoziierten Kinase (ROCK) und HDAC6 konnten einige dieser zellulären Veränderungen imitiert und demensprechend einem Effektorprotein zugeordnet werden. Der ROCK Inhibitor Fasudil konnte die morphologischen Veränderungen und die reduzierte Migrationskapazität in Wildtyp-Fibroblasten abbilden, wobei eine
Reduktion der Proliferation nach der Verwendung des HDAC6 Inhibitors Tubastatin A beobachtet wurde.
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Lima, Victor Vitorino. "Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17133/tde-15082013-114532/.
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LIMA, V.V. Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1. 2012. 106 f. Tese (Doutorado) - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. A O-Glicosilação com N-acetilglucosamina (O-GlcNAc) é uma modificação pós-traducional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: UDP-NAc transferase (OGT) e O-GlcNAcase (OGA). A enzima OGT catalisa a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina ou treonina das proteínas alvo. Por outro lado, a OGA catalisa a remoção hidrolítica de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvos da O-GlcNAc, e recentemente demonstramos que a expressão de proteínas modificadas com O-GlcNAc está aumentada em artérias de ratos com hipertensão DOCA-sal. Considerando que a produção de endotelina-1 (ET-1) encontra-se aumentada na vasculatura de diferentes modelos de hipertensão sensível ao sal, nós investigamos a hipótese de que o aumento da resposta vascular contrátil induzida pela ET-1 é decorrente da hiperativação da via RhoA/Rho cinase, mediada pelo aumento dos níveis de proteínas O-GlcNAc. Durante a realização de nossos experimentos, demonstramos que a exposição de aortas ou células do músculo liso vascular (CMLV) à ET-1 (0,1 mol/L) aumenta a vasoconstrição para fenilefrina (PE) e serotonina, bem como os níveis de proteínas O-GlcNAc, além de modular a expressão das enzimas OGT e OGA. A infusão de ET-1 (2 pmol/Kg/min) por 14 dias também promoveu aumento dos níveis vasculares de proteínas O-GlcNAc e da resposta contrátil da aorta à PE. O tratamento de aortas ou CMLV com ST045849 (inibidor da OGT, 100 µMol/L) ou atrasentan (antagonista do receptor ETA, 1 mol/L), preveniu o aumento dos níveis de proteínas O-GlcNAc induzido pela ET-1. Além disso, o tratamento com atrasentan por cinco semanas (atrasentan - 5 mg/kg/dia, por via oral) normalizou os níveis vasculares de proteínas O-GlcNAc em ratos DOCA-sal e também diminuiu a resposta contrátil da aorta à PE. A transfecção de CMLV com siRNA para OGT aboliu o efeito da ET-1 sobre os níveis de proteínas O-GlcNAc. Considerando que o aumento nas contrações da aorta à PE, após o tratamento com PUGNAc (inibidor seletivo da OGA) ou ET-1, foi abolido pelo inibidor de Rho cinase (Y-27632, 1 mol/L) e que a ET-1 ativa a via de sinalização da RhoA/Rho cinase, decidimos investigar se aumento dos níveis de proteínas O-GlcNAc ativa/modula a via RhoA/Rho cinase. A incubação de CMLV com ET-1 não mudou a expressão protéica das formas totais de ROCK-, ROCK-, CPI-17, MYPT-1 ou MLC, porém aumentou a expressão das formas fosforiladas da MYPT-1 (Tre853), CPI-17 (Tre38) e MLC (Tre18/Ser19). Estes efeitos não foram observados quando CMLV foram tratadas com ST045849, atrasentan ou previamente transfectadas com o siRNA para OGT. Também observamos que a ET-1 aumentou a atividade e a expressão protéica da RhoA, assim como a expressão da PDZ-Rho GEF e p115-Rho GEF. Este efeito foi abolido, quando CMLV foram previamente transfectadas com siRNA para OGT, incubadas com o inibidor da OGT ou tratadas com o antagonista de receptores ETA. Em conclusão, nossos dados fornecem evidências de que a ET-1 aumenta os níveis vasculares de proteínas O-GlcNAc, resultando na ativação da via RhoA/Rho cinase e no aumento da reatividade vascular. É possível que o aumento de proteínas O-GlcNAc, induzido pela ET-1, possa representar um novo mecanismo para a disfunção vascular induzida por este potente peptídeo.LIMA, V.V. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of RhoA/Rho-kinase pathway. 2012. 106 f. Ph.D. Thesis - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. Glycosylation with O-linked -N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). Whereas OGT catalyses the addition of O-GlcNAc to the hydroxyl group of serine and threonine residues of a target protein, OGA catalyses the hydrolytic cleavage of O-GlcNAc from post-translationally-modified target. Proteins with an important role in vascular function are targets for O-GlcNAcylation and we have recently shown that the vascular content of O-GlcNAc-proteins is augmented in arteries from DOCA-salt rats. Since endothelin-1 (ET-1) production is increased in the vasculature of salt-sensitive forms of hypertension, we tested the hypothesis that O-GlcNAc contributes to the vascular effects of ET-1, via activation of the RhoA/Rho-kinase pathway. Incubation of rat aortas or vascular smooth muscle cells (VSMCs) with ET-1 (0,1 mol/L) produced a time-dependent increase in O-GlcNAc levels, decreased expression of O-GlcNAc transferase (OGT) and -N-acetylglucosaminidase (OGA), key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine (PE) vasoconstriction. ET-1 effects were not observed when vessels were previously instilled with anti-OGT antibody or after incubation with an OGT inhibitor (ST045849, 100 mol/L). Aortas from DOCA-salt rats, which exhibit increased pre-pro-ET-1 expression, displayed increased contractions to PE and augmented levels of O-GlcNAc proteins. Treatment of DOCA-salt rats with atrasentan (ETA antagonist) abrogated augmented vascular levels of O-GlcNAc and prevented increased PE vasoconstriction. Aortas from rats chronically infused with low rate of ET-1 (2 pmol/Kg/min, 14days) exhibited increased O-GlcNAc-proteins and enhanced PE responses. These changes are similar to those induced by PUGNAc (OGA inhibitor which increases O-GlcNAc levels). ET-1 as well as PUGNAc augmented contractions to PE in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632 (1 mol/L). Incubation of VSMCs with ET-1 did not change expression of ROCK-, ROCK-, CPI-17, MYPT-1 or MLC, but increased phosphorylation levels of MYPT-1 (Thr853), CPI-17 (Thr38) and MLC (Thr18/Ser19). The effects of ET-1 on MYPT-1, CPI-17 and MLC phosphorylation were prevented by the OGT inhibitor and OGT siRNA transfection, as well as by atrasentan. ET-1 increased RhoA expression and activity in VSMCs, and this effect was abolished by OGT siRNA transfection and OGT inhibition. ET-1 also augmented expression of PDZ-Rho GEF and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, OGT inhibition (ST045849) and ETA receptor blockade (atrasentan, 1 mol/L). In conclusion, our data strongly suggest that ET-1 augments O-GlcNAc levels and this modification contributes to increase vascular contractile responses, via activation of the RhoA/Rho-kinase pathway. We speculate that the modulatory effect of ET-1 on O-GlcNAcylation may represent a novel mechanism underlying the vascular effects of the peptide.
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Assis, Welton Ferreira de [UNESP]. "Efeitos da manutenção em ambiente enriquecido em aspectos cognitivos e nas proteínas AKT, RhoA e RhoE musculares de ratos diabéticos". Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/150223.
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O diabetes mellitus é um quadro patológico que traz diversas complicações como prejuízos metabólicos, endócrinos, cognitivos, sarcopenia, emagrecimento, hiperfagia e polidispia. Estudos prévios têm demonstrado que a manutenção de animais em ambiente enriquecido traz um conjunto de benefícios através dos estímulos que oferece, sendo a atividade física um desses estímulos. Apesar disso, poucos estudos investigaram os efeitos da manutenção de animais diabéticos em ambiente enriquecido. Desta forma, a presente dissertação teve por objetivos investigar os efeitos do diabetes na atividade física realizada no ambiente enriquecido e investigar os efeitos do ambiente enriquecido em parâmetros bioquímicos, morfométricos, cognitivos e nas proteínas AKT, RhoA e RhoE musculares. Para isso, na primeira etapa do desenho experimental, ratos wistar foram distribuídos em dois grupos: controle e diabetes. O diabetes foi induzido por estreptozotocina via intraperitonial (50 mg/kg) e os animais foram inseridos em gaiolas amplas contendo objetos coloridos e rodas de atividade com contador de giros. O rastreamento foi realizado pelo sistema para análises cinemáticas, Digital Video For Biomechanics - Windows 32 bits (DVIDEOW® ) e software Matlab® . Na segunda etapa do desenho experimental, os animais foram distribuídos nos seguintes grupos: controle, diabetes e diabetes gaiola enriquecida. O diabetes foi induzido por estreptozotocina via intraperitonial (50 mg/kg) e os animais foram mantidos em gaiolas padrão ou ambiente enriquecido. A massa corporal, ingestão hídrica e alimentar foram coletadas duas vezes por semana e a glicemia no início e final do experimento. Ao final do período experimental foi registrado o comprimento corporal e coletadas amostras do músculo gastrocnêmio para as análises da fosforilação da AKT e expressão de RhoA e RhoE. Para estas análises, foram utlizados o teste t ou Mann-Whitney para as análises entre dois grupos e análise de variância (ANOVA) two way, com posthoc de Bonferroni (significância de 5%) para as demais análises. O software SPSS® foi utilizado para as análises. O diabetes reduziu o acúmulo de atividade física no ambiente enriquecido. A manutenção no ambiente enriquecido amenizou o aumento da hiperglicemia e melhorou, parcialmente, o desempenho no labirinto aquático de Morris dos animais diabéticos. A redução na massa corporal, índice de Lee e o aumento na ingestão hídrica e alimentar nesses animais diabéticos não foram influenciados pelo ambiente enriquecido. Ainda, neste período de 6 semanas, não houve efeitos do diabetes ou do ambiente enriquecido na fosforilação da AKT e expressão das proteínas RhoA e RhoE. Pode ser concluído que a manutenção no ambiente enriquecido traz benefícios aos animais diabéticos, apesar deste realizar menos atividade física que animais controles.
Diabetes mellitus is a pathological condition that brings several complications such as metabolic, endocrine, cognitive loss, sarcopenia, weight loss, hyperphagia and polydipsia. Previous studies have shown that keeping animals in enriched environment brings a set of benefits through the stimuli it provides, and the physical activity is one of these stimuli. Despite this, few studies have investigated the effects of the maintenance of animals in diabetic enriched environment. The present dissertation aimed to investigate the effects of diabetes on physical activity performed in the enriched environment and to investigate the effects of enriched environment on biochemical parameters, morphometric, cognitive, and AKT, RhoA and RhoE proteins in the muscle. For this, in the first step of the experimental design, wistar rats were distributed into two groups: control and diabetes. Diabetes was induced streptozotocin via intraperitoneal (50 mg/kg) and the animals were placed in large cages, containing colored objects and the wheels of activity and a counter of revolutions. The tracking was carried out by the system for analysis, kinematics, Digital Video For Biomechanics - 32-bit Windows (DVIDEOW®) and Matlab® software. In the second step of the experimental design, the animals were distributed into the following groups: control, diabetic and diabetic - enriched cage. Diabetes was induced streptozotocin via intraperitoneal (50 mg/kg) and the animals were kept in cages of standard or enriched environment. The body mass, the intake of water and food were collected twice per week and the blood glucose at the beginning and end of the experiment. At the end of the experimental period was recorded and the length of the body and collected samples of the gastrocnemius muscle for the analysis of the phosphorylation of AKT and expression of RhoA and RhoE. For these analyses, were used the t test or the Mann-Whitney test for analyses between two groups and analysis of variance (ANOVA) two way, with posthoc Bonferroni (significance of 5%) for all other analyses. The software SPSS® was used for the analyses. Diabetes reduced the accumulation of physical activity in the enriched environment. The maintenance in the enriched environment slowdown the increase of hyperglycemia and improved, in part, the performance of the diabetic animals in the water maze of Morris. The reduction in the body mass, index of Lee and the increase in the intake of water and food of these diabetic animals were not influenced by the enriched environment. Still, in this period of 6 weeks, there were no effects of the diabetes or from the enriched environment in the phosphorylation of AKT and expression of the RhoA and RhoE proteins. It can be concluded that maintenance in enriched environment brings benefits to the animals and diabetic patients, although this perform less physical activity than control animals.
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Assis, Welton Ferreira de. "Efeitos da manutenção em ambiente enriquecido em aspectos cognitivos e nas proteínas AKT, RhoA e RhoE musculares de ratos diabéticos /". Rio Claro, 2016. http://hdl.handle.net/11449/150223.
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Orientador: José Alexandre Curiacos de Almeida LemeBanca: Julio Wilson dos Santos
Banca: Andressa Coope dos Santos
Resumo: O diabetes mellitus é um quadro patológico que traz diversas complicações como prejuízos metabólicos, endócrinos, cognitivos, sarcopenia, emagrecimento, hiperfagia e polidispia. Estudos prévios têm demonstrado que a manutenção de animais em ambiente enriquecido traz um conjunto de benefícios através dos estímulos que oferece, sendo a atividade física um desses estímulos. Apesar disso, poucos estudos investigaram os efeitos da manutenção de animais diabéticos em ambiente enriquecido. Desta forma, a presente dissertação teve por objetivos investigar os efeitos do diabetes na atividade física realizada no ambiente enriquecido e investigar os efeitos do ambiente enriquecido em parâmetros bioquímicos, morfométricos, cognitivos e nas proteínas AKT, RhoA e RhoE musculares. Para isso, na primeira etapa do desenho experimental, ratos wistar foram distribuídos em dois grupos: controle e diabetes. O diabetes foi induzido por estreptozotocina via intraperitonial (50 mg/kg) e os animais foram inseridos em gaiolas amplas contendo objetos coloridos e rodas de atividade com contador de giros. O rastreamento foi realizado pelo sistema para análises cinemáticas, Digital Video For Biomechanics - Windows 32 bits (DVIDEOW® ) e software Matlab® . Na segunda etapa do desenho experimental, os animais foram distribuídos nos seguintes grupos: controle, diabetes e diabetes gaiola enriquecida. O diabetes foi induzido por estreptozotocina via intraperitonial (50 mg/kg) e os animais foram mantidos em gaiolas padrão ou ambiente enriquecido. A massa corporal, ingestão hídrica e alimentar foram coletadas duas vezes por semana e a glicemia no início e final do experimento. Ao final do período experimental foi registrado o comprimento corporal e coletadas amostras do músculo gastrocnêmio para as análises da fosforilação da AKT e expressão de RhoA e ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Diabetes mellitus is a pathological condition that brings several complications such as metabolic, endocrine, cognitive loss, sarcopenia, weight loss, hyperphagia and polydipsia. Previous studies have shown that keeping animals in enriched environment brings a set of benefits through the stimuli it provides, and the physical activity is one of these stimuli. Despite this, few studies have investigated the effects of the maintenance of animals in diabetic enriched environment. The present dissertation aimed to investigate the effects of diabetes on physical activity performed in the enriched environment and to investigate the effects of enriched environment on biochemical parameters, morphometric, cognitive, and AKT, RhoA and RhoE proteins in the muscle. For this, in the first step of the experimental design, wistar rats were distributed into two groups: control and diabetes. Diabetes was induced streptozotocin via intraperitoneal (50 mg/kg) and the animals were placed in large cages, containing colored objects and the wheels of activity and a counter of revolutions. The tracking was carried out by the system for analysis, kinematics, Digital Video For Biomechanics - 32-bit Windows (DVIDEOW®) and Matlab® software. In the second step of the experimental design, the animals were distributed into the following groups: control, diabetic and diabetic - enriched cage. Diabetes was induced streptozotocin via intraperitoneal (50 mg/kg) and the animals were kept in cages of standard or enriched environment. The body mass, the intake of water and food were collected twice per week and the blood glucose at the beginning and end of the experiment. At the end of the experimental period was recorded and the length of the body and collected samples of the gastrocnemius muscle for the analysis of the phosphorylation of AKT and expression of RhoA and RhoE. For these analyses, ... (Complete abstract click electronic access below)
Mestre
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Leppert, Amanda Fitch. "GENETIC ANALYSIS OF RHOA SIGNALING DURING EPITHELIAL MORPHOGENESIS IN DROSOPHILA". Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4430.
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Epithelial morphogenesis is contingent upon cell shape changes. Cell shape changes are the driving force for the metamorphosis of the adult Drosophila leg from the leg imaginal disc precursor. Genetic analysis has identified several Drosophila genes involved in regulating cell shape changes during leg disc morphogenesis. These include members of the RhoA signaling pathway and the product of the Stubble-stubbloid (Sb-sbd) locus, a transmembrane serine protease. Mutations in the Sb-sbd gene interact genetically with the members of the RhoA signaling pathway, however the nature of the relationship between Sb-sbd serine protease activity and RhoA signaling is not understood. To identify additional components of the RhoA signaling pathway that may help us to understand the role of the Sb-sbd protease in RhoA signaling the Drosophila genome was systematically scanned for genes that interact with Sb-sbd and RhoA mutations using deletions/deficiencies of specified regions of each chromosome. A total of 201 deficiencies uncovering approximately 84.9-91% of the euchromatic genome and spanning the X, second, and third chromosoms were tested. Of the 201 deficiencies tested, five putative interacting genetic regions and one gene within these deficiencies were identified. The candidate gene Eip78C encodes a nuclear steroid hormone receptor previously identified as having an important role in metamorphosis.M.S.
Department of Biology
Arts and Sciences
Biology
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Zhou, Xuan. "RhoA GTPase Controls Cytokinesis and Programmed Necrosis of Hematopoietic Progenitors". University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378197381.
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Schömel, Franziska [Verfasser]. "Einfluss von RhoA auf die Invasivität von Mammakarzinomzellen / Franziska Schömel". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1234847086/34.
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Moose, Devon Lyle. "Rhoa-myosin II pathway confers resistance to fluid shear stress". Thesis, University of Iowa, 2018. https://ir.uiowa.edu/etd/6221.
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The second leading cause of death in the United States is cancer, and approximately 90% of cancer related deaths are due to metastasis. When cancer metastasizes, cell from the tumor enter the circulation where they are exposed to hemodynamic forces. One of the main mechanical forces of the circulation is fluid shear stress (FSS), which was thought to be the main reason for metastatic inefficacy. However, recent studies have shown that in vitro cancer cells are more resistant to FSS than non-transformed epithelial cells. Additionally, that loss of viability cancer cells experience is biphasic in nature. Investigations into this adaptive response have shown that the Young’s Modulus of cancer cells is increased.
Further investigating the adaptive phenomena, RhoA activity is shown to be increased in cancer cells and not non-transformed cells after exposure to two brief pulses of FSS. Also, extracellular calcium is also essential to maintain resistance upon exposure to FSS, although, through unknown mechanisms. Additionally, inhibiting myosin II sensitizes cell to FSS both in vivo and in vitro.
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MARCHESI, STEFANO. "DEPDC-1B MODULATES RHOA-DEPENDENT CELL ADHESION DURING MITOTIC ENTRY". Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/214609.
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One of the most frequent alterations occurring in cancer is the inactivation of the mechanisms controlling cell cycle progression: the cellular checkpoints. Among the different existing checkpoints, the one controlling the G2/M transition holds great interest because of its implications for genome stability and tumorigenesis.
Previous work in our lab identified DEPDC-1B as an E1A-induced gene during cell-cycle re-entry of differentiated cells (Nicassio et al. 2005). In this study, DEPDC-1B was found a cell cycle regulated-gene, with transcriptional expression peaking at the G2 and M phases (Nicassio et al. 2005). The DEPDC-1B protein possesses the structural features of a signaling protein, with two conserved domains (a DEP and a Rho-GAP domain) putatively involved in RhoGTPase signaling. Therefore, we hypothesized the existence of a previously un-described signaling pathway involving both DEPDC-1B and Rho-GTPases in the control of G2/M transition.
Our results identify DEPDC-1B as a key player in the regulation of mitotic entry in vertebrates. DEPDC-1B ablation causes a delay in mitotic entry in human cell lines and impairs mitotic progression in vivo during the early stages of Zebrafish development. By the use of biochemical and genetic approaches we provide evidence for a DEPDC- 1B/RhoA complex, possibly with the participation of the focal adhesion-specific receptor phosphatase PTPRF, able to regulate focal adhesion and cytoskeleton dynamics at the G2/M transition. Our model suggests that DEPDC-1B acts as a negative modulator of RhoA activity, and requires both the DEP and Rho-GAP domains, although the latter appears to be an atypical Rho-GAP that does not possess any GAP activity per se. We observed that, even in Zebrafish development, DEPDC-1B control on cell cycle progression is RhoA-dependent and involves both domains of DEPDC1B.
Our results also indicate that alterations of adhesion during the G2 phase occur as a
consequence of either DEPDC-1B/PTPRF silencing or RhoA hyper-activation. Such modulation of expression of the three genes is able to induce a delay in the G2/M transition. We, therefore, suggest the existence of an “Adhesion Checkpoint”, centered on the DEPDC-1B/RhoA axis, which controls cell detachment at the G2/M transition by the coordinated disassembly of focal adhesive structures. This previously uncharacterized mechanism appears to be essential for the proper progression to mitosis in vitro and in vivo and might potentially impact also on the mechanisms controlling DNA damage response at the G2/M phase and cellular transformation.
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Howe, Grant Alexander. "Identification of Mechanisms Regulating Endothelial Cell Capillary Morphogenesis". Thesis, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26196.
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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.
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Salo, Paul David. "Protein prenylation inhibitors reveal a novel role for rhoa and rhoc in trafficking of g protein-coupled receptors through recycling endosomes". Diss., Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/26711.
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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008.Committee Co-Chair: Hud, Nicholas; Committee Co-Chair: Radhakrishna, Harish; Committee Member: Doyle, Donald; Committee Member: Fahrni, Christoph; Committee Member: McCarty, Nael. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Loomis, Rebecca Jo Su Lishan. "The role of RhoA signaling pathways in regulating HIV-1 replication". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,517.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.
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Rao, Milly Yushu. "Role of the RhoA/ROCK pathway in hypertension associated with diabetes". Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/38344.
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Hypertension is the most common cardiovascular complication associated with diabetes mellitus. The RhoA/ROCK pathway has been implicated in the regulation of vascular smooth muscle contractile responses associated with hypertension and diabetes. Here we wished to determine the role of the RhoA/ROCK pathway in vascular smooth muscle contractile responses in different types of diabetic models and its contribution to diabetes-associated hypertension.
Our results suggest that the involvement of ROCK may be more important in U-46619- than in PE-induced contractile responses in endothelium denuded mesenteric resistance arteries from control rats. On the other hand, as found in our previous work and other reports, PE-induced contractile responses were attenuated by inhibition of ROCK in rat superior mesenteric arteries, suggesting that ROCK is likely to play an essential role in PE-induced contraction in superior mesenteric arteries.
Blood pressure in GK rats was normalized by one-week treatment with fasudil, a known ROCK inhibitor, suggesting that the elevated blood pressure in GK rats may be due to enhanced ROCK pathway. However, agonist-induced contractile responses in GK mesenteric resistance arteries were not augmented compared to control arteries, and moreover, expression of ROCK/RhoA as well as activity of ROCK were similar between control and GK arteries. The specific causes of the discrepancy remain unknown.
In the presence of L-NAME, ROCK inhibitor significantly attenuated contraction induced by PE or U-46619 in mesenteric resistance arteries from GK rats, suggesting that agonist-induced contractile responses in mesenteric resistance arteries from GK rats were likely to be ROCK-mediated. This was further supported by Western blotting results, in that phosphorylation of MYPT was significantly increased by U-46619. Interestingly, U-46619-induced contraction in mesenteric resistance arteries from control rats was no longer sensitive to inhibition of ROCK in the presence of L-NAME, although the reasons were unclear.
In conclusion, results from the GK rat study do not support the hypothesis that activation of the RhoA/ROCK pathway contributes to the development of diabetes-associated hypertension in GK rats by enhancing vascular smooth muscle contractile responses. To further investigate this study, a more ROCK selective ROCK inhibitor is needed.
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Böhringer, Christian [Verfasser], i Carsten [Akademischer Betreuer] Culmsee. "The role of RhoA in corticogenesis / Christian Böhringer. Betreuer: Carsten Culmsee". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1027066259/34.
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Dovas, Athanassios. "Regulation of RhoA by PKCa during syndecan-4-mediated cell adhesion". Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434916.
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Betuing, Sandrine. "Regulation 2-adrenergique du cytosquelette dans les preadipocuytes : implication de rhoa". Toulouse 3, 1998. http://www.theses.fr/1998TOU30202.
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Le tissu adipeux blanc est physiologiquement important comme lieu de stockage des triglycerides, source d'energie. Parmi les hormones et neurotransmetteurs regulant l'activite du tissu adipeux blanc, les catecholamines exercent un role non negligeable en agissant par les recepteurs 2-adrenergiques conduit (1) a la regulation negative de la lipolyse et (2) a la liberation d'acide lysophosphatidique dans le milieu extracellulaire. La mise en evidence recente au laboratoire d'une receptivite 2-adrenergique dans les preadipocytes a permis de s'interroger sur le role de ces recepteurs dans ces cellules. Les premieres etudes realisees ont mis en evidence un effet trophique des catecholamines sur les preadipocytes via l'activation des recepteurs 2-adrenergiques. La premiere partie de notre travail a mis en evidence une regulation du cytosquelette et de l'adhesion cellulaire suite a une stimulation 2-adrenergique. Dans les preadipocytes, une stimulation des recepteurs 2-adrenergiques entraine une augmentation de l'adhesion cellulaire, une reorganisation des filaments d'actine en fibres de tension, la formation de pseudopodes (lamellipodes) et d'adhesion focale. L'exploration de la transduction du signal nous a conduit a montrer que ces effets morphologiques sont dependants des proteines rho. L'etude des mecanismes moleculaires mis en jeu dans cette activation a permis de proposer que, contrairement a l'activation de la proteine p21ras, l'activation de la proteine rhoa est independante des sous-unites des proteines g heterotrimeriques. La seconde partie de notre travail nous a conduits a montrer, pour la premiere fois, que la stimulation des recepteurs 2-adrenergiques inhibe la differenciation des preadipocytes. En effet, la morphologie cellulaire et l'accumulation des triglycerides sont alterees a la suite d'une stimulation 2-adrenergique. Les mecanismes cellulaires impliques dans cette regulation reste a determiner. Ce travail renforce l'hypothese d'une implication des recepteurs 2-adrenergiques dans l'hyperplasie du tissu adipeux blanc. D'un point de vue plus fondamental, cette etude a aussi contribue a une meilleure comprehension des mecanismes intracellulaires impliques dans la regulation d'une voie de transduction susceptible d'etre couplee aux recepteurs a sept passages transmembranaires.
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Chen, Weihua. "Role of RhoA/ROCK pathway in angiogenesis and their potential values in prostate cancer treatment". Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05T047/document.
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Le cancer de la prostate est la principale cause de décès chez les hommes des pays occidentaux. Traitement du cancer de la prostate résistant à la castration (CRPC) métastatique est encore limité. RhoA/ROCK sont les régulateurs principaux du cytosquelette et sont impliqués dans l'angiogenèse et l'invasion tumorale. Dans la première étude (partie 1), nous avons étudié les effets anti-angiogéniques de fasudil ( inhibiteur de ROCK ) in vitro. Des cellules endothéliales de la veine ombilicale sont stimulé par les cellules du cancer de la prostate. La prolifération est détectée par la Bromodéoxyuridine (BrdU). La migration est détectée par la cicatrisation des plaies. Les conditions de l'angiogenèse in vitro sont évaluées par la formation des tubes et un ellipsoïde test de germination. Fasudil inhibe la prolifération et la migration des cellules endothéliales induite par le cancer de la prostate. Dans les tests in vitro de l'angiogenèse, les formations des tubes et des germes sphériques sont inhibées significativement par fasudil dans d'une manière dépendante de la dose. Selon les résultats de Western Blot, l'expression de MYPT-1 a été significativement réduite après le traitement de Fasudil, confirmé l'inhibition de l'activité de ROCK par fasudil. Dans la deuxière étude (partie 2 et 3), l'expression et l'activité de RhoA sont évaluées chez 34 tissues paraffinées et 20 échantillons congelés de la prostate, obteni de 45 malades du cancer de la prostate qui à été reçu une prostatectomie radicale. Les motifs de l'expression de RhoA sont detecté par la coloration immunomarquage et western blot. Les différence entre le centre, l'avant et plus loin autour de la tumeur sont évalué. L’activité de RhoA sont évalué par G-LISA. Le gradient d'augmentation de l'expression de RhoA a été trouvé du centre a la region autour de la tumeur. L'expression de RhoA est supérieure significativement dans les cellules de l’avant de la tumeur par rapport au centre de la tumeur par Immunohistochimie (p = 0001). Gleason score était significativement supérieur chez des patients avec l'expression de RhoA élevée dans l’avant et centre de la tumeur (p =0044 et 0039, respectivement). Après un suivi médian de 52 mois, le taux de récidive est plus élevé chez des patients avec l'expression de RhoA élevée dans l’avant de la tumeur (62,5% contre 35%), une tendance évidente a la différence significative (P = 0089). Il n'y a pas de corrélation entre l'expression de RhoA, PSA et la stadification pathologique. On a aussi découvert que l'expression de le ROCK2, mais pas l'expression de le ROCK1, est eleve significativement dans l’avant de la tumeur du cancer de la prostate. En conclusion, nous avons trouvé que fasudil inhiber la prolifération, la migration, la formation de capillaires et de la sphère de la germination des cellules endothéliales vasculaires d’'une manière dépendante de la dose. Ces effets peut-être grâce à l'inhibition de l'activité de la ROCK résultant de la sécrétion de cellules du cancer de la prostate. Nous avons aussi trouvé que l'expression de la RhoA et ROCK2 dans l’avant de la tumeur de la prostate sont plus élevés. La corrélation de l'expression de RhoA avec Gleason score et récidive est identifiée. Cela montre l’association entre la voie RhoA/ROCK et l’agressive du cancer de la prostate. L'étude décrite ici peut fournir de nouveaux traitements cible la voie RhoA/ROCK contre l’angiogenèse et agressive du cancer de la prostate. Fasudil peut être utile comme agent anti - angiogénique, doit être étudiée pour leur rôle potentiel dans le traitement du cancer de la prostateProstate cancer remains a major cause of mortality among males in western countries. Treatment options for metastatic castration-resistant disease remain limited. There is a continuing unmet need for new systemic interventions in patients with progressive prostate cancer. RhoA/Rho-associated protein kinases (ROCK) are key regulators of the cytoskeleton and have been implicated in PCa angiogenesis and tumour invasion. In the first study (Part I), we investigated the anti-angiogenic effects of fasudil, a ROCK inhibitor, on PCa-induced angiogenesis in vitro. Proliferation of PCa-conditioned human umbilical vein endothelial cells (HUVECs) was assessed using a bromodeoxyuridine (BrdU) assay, and migration was assessed with a wound healing assay. In vitro angiogenesis of PCa-conditioned HUVECs was evaluated by tube formation and a spheroid sprouting assay. Fasudil inhibited PCa-induced endothelial cell proliferation, and also decreased PCa-induced endothelial cell migration. In the in vitro angiogenesis assay, tube formation and spheroid sprouts were significantly inhibited at fasudil in a dose dependent manner. Western blotting results showed that expression of phosphorylated myosin phosphatase target subunit 1 (MYPT-1) was significantly lower after fasudil treatment, confirming that fasudil inhibited ROCK activity in these model systems. In the second study (Part II & III), we evaluated RhoA expression and activity in a total of 34 paraffin embedded and 20 frozen prostate specimens, respectively, obtained from 45 patients treated with radical prostatectomy for clinically localized cancer. The expression patterns of RhoA were tested by immunohistochemical staining and Western blotting, and further compared between the tumour centre, tumour front and distant peritumoral tissue. RhoA activity was assessed by G-LISA. Our results showed an increasing gradient of expression from the centre to the periphery of index tumour foci. RhoA expression was indeed significantly higher at the tumour front as compared to tumour centre, using immunohistochemistry (p=0.001). Gleason score was significantly higher in the patients with higher RhoA expression in both the tumour front and tumour centre (p=0.044 and 0.039, respectively). After a median follow-up of 52 months, the rate of PSA relapse was higher in patients with a higher RhoA expression at the tumour front (62.5% vs 35%), although the difference was not significant (p=0.089). There was no association between RhoA expression and PSA, pathological stage. We also found ROCK2 expression, but not ROCK1 expression, was significantly higher in the prostate cancer tumor front. In conclusion, we found fasudil significantly inhibits the key steps of endothelial cell angiogenesis, including proliferation, migration, capillary tube formation and spheroid sprouting, in a dose-dependent manner. These effects may due to inhibition of ROCK activity induced by PCa cell secretions. We also identified higher RhoA and ROCK2 expression in human prostate tumour front. The correlation of higher RhoA expression with higher Gleason score and higher rate of cancer relapse. This indicated the association of RhoA/ROCK2 pathway with aggressiveness of prostate cancer. The insights described here may provide the foundation for novel therapeutic approaches targeting RhoA/ROCK pathway to inhibit angiogenesis and clinically aggressiveness of PCa. Fasudil may be a useful anti-angiogenic agent and should be investigated further for its potential role in PCa treatment
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Rochelle, Tristan. "Signalisation des GTPases de la famille Rho dans les phénotypes migratoires induits par les différentes formes de Bcr-Abl". Thesis, Poitiers, 2012. http://www.theses.fr/2012POIT1401/document.
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Les oncogènes Bcr-Abl (p190bcr-abl et p210bcr-abl) sont issus d'une translocation chromosomique t(9,22) qui fusionne en phase les gènes bcr et c-abl. p210bcr-abl est généralement responsable de la Leucémie Myéloïde Chronique (LMC) alors que p190bcr-abl induit un sous type de Leucémie Aigue Lymphoblastique (LAL). La seule différence structurale entre ces deux protéines est la présence d'un domaine DH/PH au sein de p210bcr-abl activateur spécifique de RhoA. L'expression de Bcr-Abl dans la lignée Ba/F3 est associée au déclenchement d'une migration spontanée, dépourvue de directionnalité, sous la dépendance de la GTPase Rac1.L'activation de RhoA, spécifique des cellules Ba/F3p210, est associée à un phénotype migratoire amœboïde dans une matrice de Matrigel™ en 3D où les cellules Ba/F3p190 dépourvues de RhoA activé, présentent une mobilité de type roulement. Dans ce travail, nous avons mis en évidence que l'activation spécifique de ROCK1 par RhoA détermine deux voies parallèles et mutuellement indispensables pour le mouvement amœboïde : 1) la voie de la Chaine Légère de Myosine (CLM) 2) celle des protéines de la famille ADF (Actin Depolymerizing Factor), et plus particulièrement l'isoforme ADF/destrine. Nous démontrons également l'existence d'invadopodes spécifiquement dans les cellules Ba/F3p190, dont la formation est sous la dépendance de l'absence d'activation de RhoA corrélée à une augmentation de l'activation de Cdc42. Enfin nous démontrons que la voie de signalisation RhoA/ROCK est spécifiquement activée dans les progéniteurs hématopoïétiques CD34+ issus de patients atteints de LMC et ce, indépendamment de l'activité tyrosine kinase de Bcr-AblBcr-Abl chimeric oncogenes (p190bcr-abl and p210bcr-abl) result from the t(9,22) chromosomal translocation that fuse the bcr and the c-abl genes. p210bcr-abl and p190bcr-abl are associated with Chronic Myelogenous Leukemia (CML) and a subset of Acute Lymphoblastic Leukemia (ALL) respectively. The only difference between these two chimeras is the presence of a specific RhoA-GEF domain in the p210bcr-abl oncogene. Bcr-Abl expression in Ba/F3 lymphoblasts induces spontaneous migration of these cells without apparent directionality. Motility triggering of Bcr-Abl-expressing Ba/F3 depends on the RhoGTPase Rac1.RhoA activity is associated with a typical amoeboid movement of Ba/F3p210 cells embedded in Matrigel™ 3D matrix, whereas the Ba/F3p190 cells, devoid of RhoA activity, display a rolling-type motility. In this work we showed that activation of the RhoA effector ROCK1 triggers two parallel pathways which are both necessary for amoeboid movement: 1) the Myosin Light chain (MLC) pathway 2) ADF family proteins (Actin Depolymerizing Factor) pathway, specifically the ADF/destrin isoform. Besides, we showed that Ba/F3p190 cells could assemble invadopodia-like structures. The formation of these structures is driven by the reduction of RhoA activity associated with the absence of the DH/PH domain in p190bcr-abl and correlates with an increase in Cdc42 activity. We finally demonstrated that the RhoA/ROCK pathway is constitutively activated in CD34+ cells isolated from CML patients while not in their normal counterparts. We also demonstrated that this activation is independent of the tyrosine Kinase activity of Bcr-Abl
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Nyamandi, Vongai. "The role of RhoA/ROCK signaling in the development of diabetic cardiomyopathy". Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/64116.
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Craig, Graham Peter. "INOS mediates increased RhoA expression and altered cell signaling in diabetic cardiomyopathy". Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/32344.
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The RhoA/ROCK pathway is a well established signaling pathway which
regulates cellular contractility. Previous studies, from this lab have shown that acute
inhibition of ROCK improves heart function in 12 week STZ-diabetic rats. Here we
wished to determine, 1) whether the RhoA/ROCK pathway was upregulated in the
diabetic rat heart, 2) what factors may. be contributing to altered activity of this pathway
during diabetes, and 3) determine the consequences of the altered RhoA/ROCK
signaling might be.
RhoA expression was significantly elevated in hearts and cardiomyocytes from
rats with STZ-induced diabetes. Our preliminary studies also showed elevated iNOS
expression in hearts from rats with STZ-induced diabetes. We hypothesized that NO
from iNOS mediates elevated RhoA expression. This hypothesis is supported by a
number of our observations. Elevated iNOS expression and activity was observed to be
concomitant with increased RhoA expression in diabetic rat hearts. In cultured
cardiomyocytes exposure to the NO donor, SNP, and the iNOS inducer, LPS , caused
elevated RhoA expression. The iNOS inhibitor, L-NIL, blocked increased RhoA
expression in LPS treated cardiomyocytes and in hearts from diabetic animals.
Increases in RhoA expression correlated strongly with increased levels of active
RhoA in diabetic cardiomyocytes. We also observed increased phosphorylated LIMK, a
marker for activation of the RhoA/ROCK pathway. LIMK phosphorylation was reduced to
levels similar to control after chronic treatment of diabetic rats with L-NIL, suggesting
that iNOS also contributes to increased activity of this pathway in the STZ-diabetic rat
heart.
The actin cytoskeleton is a well established downstream target of the
RhoA/ROCK pathway. The level of polymerized actin in diabetic rat cardiomyocytes was
found to be significantly elevated, but was normalized after acute ROCK inhibition.
Given this observation, we suggest that normalization of actin polymerization may
contribute to ROCK inhibitor-mediated improvement of contractile function of hearts from
diabetic rats.
The findings presented in this thesis indicate a central role for iNOS in the
upregulation of the RhoA/ROCK pathway, which is believed to contribute to impaired
contractility in the diabetic heart. Our findings also support the suggestion that ROCK is
an excellent therapeutic target in the treatment of diabetic cardiomyopathy.Pharmaceutical Sciences, Faculty of
Graduate
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Tirrell, Lee Sean. "The role of RhoA GTPase activating protein DLC2 in painful diabetic neuropathy". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/195956.
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Neuropathy is a major complication that affects nearly half of all patients with diabetes, greatly decreasing their quality of life. Patients experience a wide range of symptoms including pain, numbness, weakness and other morbidities. While its pathogenesis has been the focus of extensive research, there are still few effective treatment options available for this disease. The discovery of novel molecular targets underlying this diabetic neuropathy may lead to the development of new, more effective therapeutics.
DLC2, a Rho GTPase-activating protein with specific activity for RhoA, was shown to be involved in pain signaling. Mice deficient for this protein (DLC2-/-) have increased RhoA activity in their peripheral nerves, and have heightened pain responses compared to wild type (DLC2+/+) in acute pain tests, displaying increased sensitivity to noxious thermal and inflammatory stimuli. DLC2-/- mice also show elevated blood glucose levels, lower body weight and increased sensitivity to blood glucose compared to wild type. Because of the hyperalgesia to acute pain displayed by DLC2-/- mice compared to wild type, and since the RhoA pathway is known to be involved in the pathogeneses and maintenance of diabetes and its complications, these mice were used to investigate more clinically relevant, chronic pain in a model of diabetic neuropathy.
Streptozotocin (STZ), given in multiple low doses over five days (MLDS treatment), was used to induce diabetes in DLC2+/+ and DLC2-/- mice, and their pain responses were tested 8 weeks later. Diabetic DLC2-/- mice (DLC2-/--STZ) were hyperalgesic to thermal stimuli from the hot plate test compared to diabetic DLC2 wild type mice (DLC2+/+-STZ) and vehicle-treated controls of both genotypes (DLC2-/--Veh and DLC2+/+-Veh. Similar responses were seen from the von Frey filament test, where the DLC2-/--STZ group exhibited mechanical allodynia compared to the DLC2+/+-STZ group and both control groups.
Dorsal root ganglia (DRG) were dissected from these four groups of mice for qPCR screening and protein analysis. DLC2-/--STZ mice showed significantly higher gene expression of the voltage-gated sodium channel Nav 1.9 compared to DLC2+/+-STZ mice, while there was a strong trend of increased levels in the DLC2-/--STZ group compared to both non-diabetic groups. Western blot analysis of the DRG from these mice shows increased levels of COX-2 expression of DLC2-/--STZ mice compared to DLC2+/+-Veh, and elevated levels of phosphorylated ERK (pERK) in DLC2-/--Veh and both diabetic groups compared to DLC2+/+-Veh.
Overall, diabetic DLC2-/- mice have more severe painful diabetic neuropathy, with thermal hyperalgesia and mechanical allodynia. Increased RhoA activity and pERK, which are known to be involved in regulation, transcription and trafficking of sodium channels, may lead to increased Nav1.9 mRNA levels and activation. Localized mainly to nociceptors of the DRG, Nav1.9 is known to play a role in sensitizing neurons through lowering the threshold for action potentials, possibly leading to the observed heightened pain response. Additionally, elevated COX-2 levels in DLC2-/--STZ mice may lead to further deficits through activation of inflammatory responses. Future studies will further investigate how these mechanisms are involved in the altered pain response from diabetes.published_or_final_version
Anatomy
Master
Master of Philosophy
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Callis, Thomas. "Characterization of genes required for RhoA signaling during epithelial morphogenesis in Drosophila". Honors in the Major Thesis, University of Central Florida, 2003. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/685.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucfBachelors
Arts and Sciences
Biology
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Cooper, Katherine. "Analysis of RhoA GTPase and the cytoskeleton in planar polarity of Drosophila". Thesis, University of Sheffield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401119.
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Megrelis, Laura. "Caractérisation de Fam65b, un nouvel inhibiteur de RhoA, impliqué dans la réponse des lymphocytes T en aval de CCR7". Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB092/document.
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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 GTPasesThe 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
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Gluth, Markus [Verfasser], Wolfgang [Akademischer Betreuer] Uckert, Franz [Akademischer Betreuer] Theuring i Daniel C. [Akademischer Betreuer] Baumgart. "Untersuchungen zum Einfluss von RhoA und der RhoA Effektorkinase PKN auf die TNF-induzierte Barrieredysfunktion in humanen intestinalen Epithelzellen / Markus Gluth. Gutachter: Wolfgang Uckert ; Franz Theuring ; Daniel C. Baumgart". Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://d-nb.info/1023725754/34.
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Osaki, Juliana Harumi. "O papel de RhoA e Rac1 GTPases nas respostas celulares após danos no DNA induzidos por radiação ionizante gama". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22092015-075415/.
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O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-HomólogaThe mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination