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Ravichandran, Yamini. "Cdc42 isoforms : localization, functions and regulation." Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS405.
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Les mutations sont responsables de diverses pathologies du développement, en particulier chez les patients atteints de maladies rares ou pour lesquels il n’y a pas de diagnostic clinique clair. Cdc42 est une protéine clé pour la polarité cellulaire, une étape cruciale de nombreux processus cellulaires, comme la migration cellulaire, la division cellulaire ou la réponse immunitaire. Les mutations de Cdc42 entrainent une variété de pathologies, par exemple des dérégulations de la croissance ou de la morphologie faciale ainsi que des anomalies immunologiques, hématologiques et du développement neuronal. Les fonctions de Cdc42 reposent en grande partie sur la localisation de cette protéine dans la cellule. La comparaison des différentes formes de Cdc42 et de certaines formes mutantes montrent que les derniers acides aminés de la protéine jouent un rôle clé dans sa localisation et donc dans sa fonction. Nous avons centré notre étude sur l’identification : 1) des acides aminés essentiels à la localisation de la protéine ; et 2) de nouveaux mécanismes de régulation de Cdc42 responsables de sa localisation intracellulaire. Nous avons aussi montré que les deux isoformes jouent des rôles différents au cours de la migration cellulaire. Ce travail devrait nous permettre de mieux comprendre les pathologies liées aux mutations de Cdc42Mutations in proteins cause diverse developmental disorders, particularly for individuals with rare diseases or for whom a unifying clinical diagnosis is unknown. Cdc42 is one such protein; vital for establishing cell polarity, a crucial step in many biological processes such as cell migration, division and immune responses. Not surprisingly, mutations in Cdc42 cause a range of diseases such as growth dysregulation, facial dysmorphism and neurodevelopmental, immunological, and hematological abnormalities. In vertebrates there are two isoforms of Cdc42. The first being the ubiquitous isoform, has almost exclusively been studied and the role of the second isoform, being the brain isoform, is largely unknown. We have shown that the two isoforms are localized differently in cells. The ubiquitous isoform is mostly found in the cell cytoplasm and at the plasma membrane, while the Brain isoform localizes at the Golgi apparatus and on intracellular vesicles. We have also shown that the two isoforms carry out different functions during cell migration, suggesting that the differences between these two isoforms which only differs by the last 10 amino acids are responsible for their distinct localisation and function. Interestingly, a mutation in the C-ter sequence of Cdc42 ubiquitous isoform alters Cdc42 localisation and causes a generalized pustular psoriasis disease. Two main objectives have been studied in this project 1) the impact of the last amino acids of the protein in Cdc42 localization; and 2) new regulatory mechanisms of Cdc42 responsible for its intracellular localization. These findings will bring a better understanding of pathologies related to Cdc42 mutations
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Lu, Ruifeng, and Jean M. Wilson. "Rab14 specifies the apical membrane through Arf6-mediated regulation of lipid domains and Cdc42." NATURE PUBLISHING GROUP, 2016. http://hdl.handle.net/10150/622499.
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The generation of cell polarity is essential for the development of multi-cellular organisms as well as for the function of epithelial organs in the mature animal. Small GTPases regulate the establishment and maintenance of polarity through effects on cytoskeleton, membrane trafficking, and signaling. Using short-term 3-dimensional culture of MDCK cells, we find that the small GTPase Rab14 is required for apical membrane specification. Rab14 knockdown results in disruption of polarized lipid domains and failure of the Par/aPKC/Cdc42 polarity complex to localize to the apical membrane. These effects are mediated through tight control of lipid localization, as overexpression of the phosphatidylinositol 4-phosphate 5-kinase a [PtdIns(4) P5K] activator Arf6 or PtdIns(4) P5K alone, or treatment with the phosphatidylinositol 3-kinase (PtdInsI3K) inhibitor wortmannin, rescued the multiple-apical domain phenotype observed after Rab14 knockdown. Rab14 also co-immunoprecipitates and colocalizes with the small GTPase Cdc42, and Rab14 knockdown results in increased Cdc42 activity. Furthermore, Rab14 regulates trafficking of vesicles to the apical domain, mitotic spindle orientation, and midbody position, consistent with Rab14' s reported localization to the midbody as well as its effects upon Cdc42. These results position Rab14 at the top of a molecular cascade that regulates the establishment of cell polarity.
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Murali, Arun [Verfasser]. "Role of XIAP in ubiquitin mediated regulation of Cdc42 and other Rho GTPases / Arun Murali." Mainz : Universitätsbibliothek Mainz, 2019. http://d-nb.info/1191286649/34.
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Francis, Monika K. "Regulation of GRAF1 membrane sculpting function during cell movement." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-111213.
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All eukaryotic cells rely on endocytic events to satisfy a constant need for nutrient and fluid uptake from their surroundings. Endocytosis-dependent turnover of cell surface constituents also serves to control signal transduction and establish morphological changes in response to extracellular stimuli. During endocytosis, distinct protein machineries re-sculpt the plasma membrane into vesicular carriers that enclose molecules that are to be taken up into the cell. Besides those produced from the canonical clathrin-mediated endocytic machinery, it is becoming increasingly clear that other membrane carriers exist. The indisputable connection between the function of these uptake systems and various disease states, highlights why it is so important to increase our knowledge about the underlying molecular machineries. The aim of this thesis was therefore to characterise the function of GRAF1, a protein suggested to be a tumour suppressor due to that the gene has been found to be mutated in certain cancer patients. My work focused on understanding how this protein operates during formation of clathrin-independent carriers, with possible implications for disease development. Previous in vitro studies showed that GRAF1 harbours a GTPase activating domain to inactivate Rho GTPase Cdc42, a major actin cytoskeleton regulator. Herein, microscopy based approaches used to analyse HeLa cells demonstrated the importance of a transient interaction between GRAF1 and Cdc42 for proper processing of GRAF1-decorated carriers. Although GRAF1-mediated inactivation of Cdc42 was not vital for the budding of carriers from the plasma membrane, it was important for carrier maturation. In addition, studies of purified GRAF1 and its association with lipid bilayers identified a membrane scaffolding-dependent oligomerisation mechanism, with the ability to sculpt membranes. This was consistent with the assumption that GRAF1 possesses an inherent banana shaped membrane binding domain. Remarkably, this function was autoinhibited and in direct competition with the Cdc42 interaction domain. Finally, other novel GRAF1 interaction partners were identified in this study. Interestingly, many of these partners are known to be associated with protein complexes involved in cell adherence, spreading and migration. Although never actually seen localising to mature focal adhesions that anchor cells to their growth surface, dynamic GRAF1 carriers were captured travelling to and from such locations. Moreover, GRAF1 was recruited specifically to smaller podosome-like structures. Consistent with this, the tracking of GRAF1 in live cells uncovered a clear pattern of dynamic carrier formation at sites of active membrane turnover – notably protrusions at the cell periphery. Furthermore, the silencing of GRAF1 gave rise to cells defective in spreading and migration, indicating a targeting of GRAF1-mediated endocytosis to aid in rapid plasma membrane turnover needed for morphological changes that are a prerequisite for cell movement. Since these cells exhibited an increase in active Rab8, a GTPase responsible for polarised vesicle transport, the phenotype could also be explained by a defect in Rab8 trafficking that results in hyperpolarisation. Taken together, the spatial and temporal regulation of GRAF1 membrane sculpting function is likely to be accomplished via its membrane binding propensity, in concert with various protein interactions. The importance of GRAF1 in aiding membrane turnover during cell movement spans different functional levels – from its local coordination of membrane and actin dynamics by interacting with Cdc42, to its global role in membrane lipid trafficking.
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Mutavchiev, Delyan Rumenov. "Regulation of fission yeast cell polarity by stress-response pathways." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29006.
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Cell polarisation is a key biological process crucial for the functioning of essentially all cells. Regulation of cell polarity is achieved through various processes determined by both internal and external factors. An example of the latter is that cell polarity can be disrupted or lost as a consequence of a variety of external stresses. When facing such stresses, cells adapt to unfavourable conditions by activating a range of molecular signalling pathways, collectively termed ‘stress response’. Despite the connections between external stress and cell polarity, whether stress-response signalling regulates cell polarisation and what the molecular basis for such regulation remains an open question. The fission yeast Schizosaccharomyces pombe presents an excellent biological platform to study the complexity of cell polarity regulation on a systematic level. This study is aimed at understanding the functional relationship between stress-response signalling and maintenance of cell polarity in this model organism. The findings presented in this thesis set the basis for establishing a functional link between the activation of the S.pombe stress-response pathway and the activity of the master regulator of cell polarity- the Rho GTPase Cdc42. Here, I describe experiments that identify an active involvement of the stress-response mitogen-activated kinase (MAPK) Sty1 in the dispersal of active Cdc42 from the sites of growth. This new role for Sty1 occurs independently from its involvement in transcription regulation and other previously identified signalling pathways involving Sty1. Furthermore, I also find that Sty1’s involvement in Cdc42 regulation has direct implications for fission yeast physiology as it is essential for the maintenance of cellular quiescence upon nitrogen starvation. This thesis also focuses on identifying the targets of Sty1 orchestrating the active Cdc42 disruption. Here, I describe a candidate-based approach, where I investigate the role of proteins from the Cdc42 regulatory network during Sty1 activation. Additionally, I present a global phospho-proteomics approach to identify novel targets of Sty1 and offer preliminary findings which might explain Sty1’s involvement in Cdc42 regulation.
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Langer, Torben [Verfasser]. "Der Einfluss des Tumorsuppressorproteins Merlin auf die Regulation der beiden Rho-GTPasen Rac2 und Cdc42 / Torben Langer." Ulm : Universität Ulm. Medizinische Fakultät, 2013. http://d-nb.info/1036215121/34.
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Ye, Xiangcang. "Role of a CDC42 homologous gene in the regulation of cell polarity and morphogenic transitions in Wangiella dermatitidis /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Primeau, Martin. "Novel mechanisms of regulation of the Cdc42 GTPase- activating protein CdGAP/ARHGAP31, a protein involved in cell migration and adhesion." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96901.
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The Rho GTPases form a family of enzymes that control numerous cellular processes including cell migration and proliferation through effects on the cytoskeleton, membrane trafficking and cell adhesion. The activity of these molecular switches is modulated by GTPase-activating proteins (GAPs), a group of negative-regulators which includes Cdc42-GTPase-Activating Protein (CdGAP). This protein specifically negatively-regulates the Rho GTPases Cdc42 and Rac1. In this study, we show that CdGAP is regulated by lipid-, protein- and intramolecular-interactions. First, we demonstrate that a polybasic region (PBR) of CdGAP preceding the GAP domain and found in numerous Rho family GAPs is required for CdGAP specific association with phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). We show that the binding of PI(3,4,5)P3 is required for CdGAP-mediated GAP activity in vitro, and that an intact PBR is required for its CdGAP-mediated GAP activity in vivo. Second, we characterize the binding site for the negative-regulator of CdGAP Intersectin-1 located in the Basic-Rich (BR) domain of CdGAP. We present evidence that this interaction mediated by the SH3D domain of Intersectin requires one to three lysine residues located in the Basic-Rich (BR) domain of CdGAP. Thirdly, we show that CdGAP is negatively-regulated by its C-terminal domain. This observation is part of a study that links two human CdGAP gene mutations to a syndrome which presents a combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). In this syndrome, the deletion-mutant gene products which lack the residual amino-acid of CdGAP at its C-terminus have an increased activity compared to wild-type proteins. We show that this C-terminus can bind to the GAP domain of CdGAP, providing a model to explain how the absence of the C-terminus induces this syndrome. In summary, this work provides novel insight into understanding the mechanisms of regulation of CdGAP, a protein involved in cell migration and adhesion with unexpected roles related to human diseases.Les Rho GTPases forment une famille d'enzymes qui contrôlent de nombreux processus cellulaires, tels que la migration cellulaire et la prolifération, grâce à leurs effets sur le cytosquelette, le trafic membranaire et l'adhésion cellulaire. L'activité de ces interrupteurs moléculaires est modulée par les protéines activatrices de GTPases (GAPs), un groupe de régulateurs négatifs qui inclu CdGAP (Cdc42-GTPase activating protein). Cette protéine régule négativement les Rho GTPases Cdc42 et Rac1 de façon spécifique. Dans la présente étude, nous montrons que CdGAP est régulée par des interactions lipidiques, protéiques et intramoléculaire. Premièrement, nous démontrons qu'une région polybasique (PBR), précédant le domaine GAP et retrouvée dans plusieurs GAP de la famille Rho, est requise pour l'association spécifique de CdGAP avec le phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). Nos résultats suggèrent que l'activation des GAP requiert la liaison du PI(3,4,5)P3 à CdGAP dans un contexte in vitro et un PBR intact pour que CdGAP provoque ses effets GAP-dépendants dans un contexte in vivo. Deuxièmement, nous caractérisons le site de liaison du régulateur négatif de CdGAP Intersectin-1. Ce site est localisé dans le domaine riche en résidus basiques (BR) de CdGAP. Nous suggérons que cette interaction, médiée par le domaine SH3D d'Intersectin, requiert de un à trois résidus lysine dans le domaine BR de CdGAP. Troisièmement, nous montrons que CdGAP est régulé de manière négative par son propre domaine C-terminal. Cette observation fait partie d'une étude qui associe deux mutations humaines du gène CdGAP à un syndrôme présentant une combinaison d'aplasie cutis congenita (ACC) et de malformation des doigts et des orteils (TTLD). Les gènes mutants produisent des protéines tronquées qui ont une activité GAP supérieure à la protéine de type sauvage. Nous montrons que ce C-terminal peut lier le domaine GAP de CdGAP, supportant un modèle expliquant comment l'absence du C-terminal induit ce syndrome. En bref, ce travail présente un nouvel aperçu des mécanismes de régulation de CdGAP, une protéine impliquée dans la migration cellulaire et dans l'adhésion des cellules en plus d'être directement impliquée dans une maladie humaine.
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Ofo, Enyinnaya. "Flourescent biosensor-based, Cdc42 activity imaging for understanding the regulation of Epidermal Growth Receptor (EGFR) signalling in head and neck cancer." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/flourescent-biosensorbased-cdc42-activity-imaging-for-understanding-the-regulation-of-epidermal-growth-receptor-egfr-signalling-in-head-and-neck-cancer(32081fef-10f1-4a3e-ac33-67afbbf78376).html.
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The Epidermal Growth Factor Receptor (EGFR) is overexpressed in several solid tumours including squamous cell carcinoma of the head & neck (SCCHN). Drugs that directly block the action of EGFR are currently available. However, a major unanswered question is; how best to select patients most likely to respond to these new treatments, as the response rate to EGFR-targeted mono-therapy in SCCHN, as well as other solid tumours, such as lung and colorectal cancer, is very low. Resistance to EGFR therapy may stem from aberrant receptor trafficking. Cdc42 is known to affect EGFR downregulation by sequestering c-Cbl, preventing it from catalyzing receptor ubiquitination. The aim of this project is to determine the role of Cdc42 in mediating cancer cell response to EGFR and ErbB-family targeted therapy. Using optical imaging techniques such as, Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FUM) I have analysed Cdc42 activity in Squamous Carcinoma Cell Lines after EGFR tyrosine kinase inhibitor (TKI) treatment, using the FRET biosensor Raichu-Cdc42. I have demonstrated that conversely, Raichu-Cdc42, and consequently endogenous Cdc42 activity increases significantly following EGFR TKI treatment. Further investigation revealed that the serine/threonine kinase, c-Jun NH2 Terminal Kinase 1 (JNK1) may modulate Cdc42 activity via a negative feedback mechanism, and JNK1 in turn regulates EGFR ubiquitination and downregulation. I have also demonstrated for the first time protein-protein interactions in pathological SCCHN tissue between members of the ErbB receptor family (EGFR and HER2) and between PKCa and Ezrin, using FRET and FUM. The novel results from this thesis provides further knowledge on factors influencing EGFR downregulation in SCCHN, that could account for the resistance to EGFR targeted therapies observed in a clinical setting. In addition the optical proteomic assays could be translated into new diagnostic/predictive tests, potentially allowing us to improve the outcome for head and neck cancer patients.
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Bretou, Marine. "Regulation of the dynamics of the fusion pore : importance of the SNARE protein synaptobrevin 2 and of the Rho GTPase Cdc42." Paris 7, 2010. http://www.theses.fr/2010PA077157.
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L'exocytose nécessite la formation d'un pore de fusion. Le pore initial est étroit ; seules de petites molécules sont libérées. Quand le pore s'élargit les macromolécules sont libérées. J'ai étudié le rôle de deux protéines sur la dilatation du pore: la protéine SNARE synaptobrévine 2 (Syb2), et la Rhô GTPase Cdc42. L'assemblage des SNAREs fournirait l'énergie nécessaire à la fusion. L'insertion d'un espacer dans le domaine juxtamembranaire de Syb2 ne modifie pas la fréquence des événements d'exocytose détectés par ampérométrie à 1|jM [Ca2+], mais empêche l'apparition d'une composante de sécrétion à de plus fortes [Ca2+]. Les événements peuvent être classés en deux groupes, liés à la vitesse et au degré de dilatation des pores; l'allongement de Syb2 réduit la population de pics rapides, mais n'affecte pas celle des pics lents. Les événements lents seraient dus à un assemblage partiel des SNAREs, alors que ceux dits rapides résulteraient d'un assemblage plus serré, assurant ainsi une dilatation rapide du pore. Cdc42 contrôle la dynamique de l'actine. Diminuer son expression dans les cellules BON réduit le nombre de granules fusionnant complètement avec la membrane, mais n'affecte pas leur recrutement et leur liaison à la membrane. Réduire l'expression de Cdc42 diminue le nombre de hauts pics dus à une dilatation rapide et complète des pores, et augmente le nombre de pieds seuls, dus à des pores ne s'élargissant pas. L'augmentation de tension de la membrane corrige les effets dus à l'absence de Cdc42 ; sa diminution par dépolymérisation de l'actine imite les effets obtenus en son absence. Cdc42 contrôlerait la dilatation du pore en modulant la tension de membraneExocytosis ends with the formation of a fusion pore. The initial pore is narrow, only small molecules flow through it. The pore then enlarges, releasing larger secretory products. I studied the role of two proteins on the dilation of the pore: the SNARE protein synaptobrevin 2 (Syb2), and the Rho GTPase Cdc42. Zippering of SNAREs in opposed membranes might give energy to catalyze fusion. Inserting a linker between the SNARE core and the transmembrane domain of Syb2 did not modify the frequency of exocytotic events detected by amperometry at 1|jM free [Ca2+] but prevented the occurrence of an extra component of release at higher [Ca2+]. Analysis of these events led to their classification into two groups, due to the rate and extent of dilation of the pore; lengthening Syb2 reduced the population of fast spikes, leaving the slow one unchanged. Slow fusion events might be due to a partial zippering of the SNAREpin while fast fusion events require a tight one, i. E. A short intermembrane distance to assure rapid dilation of the pore. Cdc42 controls actin dynamics. TIRFM experiments showed that its silencing in BON cells reduced the number of granules undergoing full fusion, with little effect on their recruitment and docking at the membrane. Using amperometry, we showed that this silencing reduced the number of high spikes due to fast and complete dilation of the pore, and increased stand-alone foot signals reflecting pores failing to enlarge. Increasing membrane tension rescued the effects of silencing while decreasing it through actin depolymerization mimicked Cdc42 silencing. Cdc42 might control fusion pore dilation by modulating membrane tension
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Meir, Michael [Verfasser], and Nicolas [Akademischer Betreuer] Schlegel. "Bedeutung der desmosomalen Adhäsion und Rolle der Rho-GTPasen RhoA, Rac1 und Cdc42 für die Regulation der Darmbarriere / Michael Meir. Betreuer: Nicolas Schlegel." Würzburg : Universität Würzburg, 2013. http://d-nb.info/1107802563/34.
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Leung, Daisy W. "Biochemical and biophysical characterization of the allosteric equilibrium of the Wiskott-Aldrich Syndrome protein." Access to abstract only; dissertation is embargoed until after 12/20/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=131.
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Annan, Robert Bruce. "Roles and regulation of «Saccharomyces cerevisiae» Rho-type GTPases Rho5p and Cdc42p." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32262.
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The eukaryotic Rho family of GTPases acts as central regulators of numerous processes, including the polarization of cell morphology, membrane transport, transcription, and MAPK pathway signaling. As GTPases, Rho proteins act as molecular switches which, in the GTP-bound form, transduce upstream signals to a variety of downstream effectors, thereby generating appropriate cellular responses. As central nodes of signaling, they are subject to strict regulation, and the regulation of the GTP-GDP cycle of Rho members has been well characterized. Rho GTPases participate in a variety of pathways, though in many cases the mechanisms are still poorly understood. This study examines the roles and regulation of two Rho GTPases in Saccharomyces cerevisiae, Rho5p and Cdc42p. First, we describe the regulation of Rho5p signaling by phosphorylation and ubiquitination, the first instance of post-translational regulation of a yeast Rho-type GTPase. This regulation is mediated by a module involving the Npr1p kinase and its inhibitor Msi1p. We also identify Rgd2p as the RhoGAP for Rho5p in vivo, and demonstrate a genetic interaction between RHO5 and STE50: a STE50 deletion combined with expression of an activated RHO5 allele results in osmotic sensitivity. Next, we describe a role for Rho5p in the activation of the cAMP-PKA pathway. Expression of an activated allele of RHO5 generates a number of phenotypes associated with activated cAMP-PKA pathway signaling, and also suppresses the lethality of a strain deleted for two other cAMP-PKA activators, Δras1 Δras2. A mechanism by which Rho5p may activate the pathway is suggested by the observed two-hybrid interaction between Rho5p and adenyLa famille eucaryote des GTPases Rho agit à titre de régulateur principal de nombreux processus biologiques, incluant la polarisation de la morphologie cellulaire, le transport membranaire, la transcription et la voie de signalisation MAPK. En tant que GTPases, les protéines Rho jouent un rôle de controlleurs moleculaires qui, sous la forme liée au GTP, transmettent les signaux en amont à une variété d'effecteurs en aval, générant ainsi les réponses cellulaires appropriées. Étant des acteurs principaux de la signalisation, les protéines Rho sont sujettes à une régulation précise. La régulation du cycle GTP-GDP par les membres de la famille Rho a été largement caractérisée. Cependant, les GTPases Rho participent à une variété de mécanismes qui, dans plusieurs cas, sont encore aujourd'hui sous-étudiés. Cette étude examine plus particulièrement les rôles et la régulation de deux GTPases Rho chez la levure Saccharomyces cerevisiae, Rho5p and Cdc42p. Dans un premier temps, nous décrivons la régulation de la signalisation de Rho5p par la phosphorylation et l'ubiquitination, ceci étant le premier exemple de régulation post-traductionnelle d'une GTPase de type Rho chez la levure. Cette régulation est arbitrée par un module impliquant la kinase Npr1p et son inhibiteur MSi1p. Nous démontrons également une interaction génétique entre RHO5 et STE50. En effet, une délétion au niveau de STE50 combinée à l'expression d'un allèle activé de RHO5 résulte en une sensibilité osmotique. De plus, nous identifions Rgd2p comme étant le RhoGAP pour Rho5p in vivo. Dans un deuxième temps, nous décrivons un rôle pour Rho5p dans l'activation d
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Aldharee, Hitham Abdulrahman. "Role of ERK3 in Regulating RhoGDI1-PAKs Signaling Axis." Wright State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=wright1498007023965276.
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Böhm, Stefanie. "The Cdc48 Shp1 complex mediates cell cycle progression by positive regulation of Glc7." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-154660.
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Rumpf, Sebastian. "Regulation des Abbaus von Cdc48-Substraten durch die antagonistischen Aktivitäten von Ufd2 und Ufd3." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-54278.
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Jensen, Bryan. "Regulation of the G1 to S-phase transition in S. cerevisiae by CDC4 /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/10257.
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Ewert-Krzemieniewska, Katarzyna. "Investigation into the regulation of DNA repair by the S. pombe cell cycle kinase Cdc2-cyclinB." Thesis, Bangor University, 2009. https://research.bangor.ac.uk/portal/en/theses/investigation-into-the-regulation-of-dna-repair-by-the-spombe-cell-cycle-kinase-cdc2cyclinb(71f3241d-b969-4ff1-9b16-47161f91b755).html.
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The work presented in this thesis proposes two novel functions for S. pombe Cdc2 in the cell cycle-dependent coordination of DNA recombination: (i) during unperturbed cell cycle and (ii) in response to camptothecin (CPT)-induced DNA breaks. In unperturbed cells, in vivo elevated Cdc2 activity causes problems during DNA replication that lead to an increase in spontaneous gene conversion between sister chromatids and enhanced loss of a non-essential minichromosome. Data presented here suggest that Cdc2 regulates the anti-recombinogenic activity of the Srs2 DNA helicase to prevent such spontaneous gene conversion events in S phase. Both proteins associate with PCNA in distinct protein complexes, which may allow them to regulate DNA repair in S phase. Elevated Cdc2 activity leads to constitutive phosphorylation of the checkpoint kinase Chk1, indicating that the inability to regulate Srs2 DNA helicase causes DNA replication lesions, which engage the G2-M checkpoint. Cells with elevated Cdc2 activity are specifically sensitive to the Topoisomerase I (Top1) poison CPT. The camptothecin sensitivity of cdc2.1w mutant cells increases in the absence of Tyrosyl-DNA-phosphodiesterase (Tdp1), which cleaves immobilised Top1 releasing it from the 3'-end of DNA in S phase. As during the unperturbed cell cycle, Cdc2 appears to regulate Srs2 DNA helicase under these circumstances. Srs2 may unwind the blocked 3'-strand in the absence of Tdp1 to allow a nuclease to access the damaged site. Both Mus81 and Rad16 are potential candidates since both nucleases act in the Cdc2-dependent pathway in response to CPT. Although all "wee" mutants with elevated Cdc2 activity are defective in this CPT repair pathway, both Wee1 and Mik1 kinase may perform independent repair functions.
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Köhler, Tim. "Regulation of growth and development by the small GTPase Cdc42p and the transcription factor Tec1p in Saccharomyces cerevisiae." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969652704.
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Böhm, Stefanie [Verfasser], and Stefan [Akademischer Betreuer] Jentsch. "The Cdc48 Shp1 complex mediates cell cycle progression by positive regulation of Glc7 / Stefanie Böhm. Betreuer: Stefan Jentsch." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1032131713/34.
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Mavrakis, Konstantinos J. "Functional analysis of DEF6 and def8 revealed DEF6 as a novel activator of Rac, Cdc42 and Rho GTPases regulating cell morphology." Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404145.
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Gao, Yanzhe. "Regulation of The DNA Unwinding Element Binding Protein DUE-B in The Cell." Wright State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1355250568.
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Bryant, Victoria. "CMG Helicase Assembly and Activation: Regulation by c-Myc through Chromatin Decondensation and Novel Therapeutic Avenues for Cancer Treatment." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6191.
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The CMG (Cdc45, MCM, GINS) helicase is required for cellular proliferation and functions to unwind double-stranded DNA to allow the replication machinery to duplicate the genome. Cancer cells mismanage helicase activation through a variety of mechanisms, leading to the potential for the development of novel anti-cancer treatments. Mammalian cells load an excess of MCM complexes that act as reserves for new replication origins to be created when replication forks stall due to stress conditions, such as drug treatment. Targeting the helicase through inhibition of the MCM complex has sensitized cancer cells to drugs that inhibit DNA replication, such as aphidicolin and hydroxyurea. However, these drugs are not used in the clinical management of cancer. We hypothesized that the effectiveness of the clinically relevant drugs gemcitabine and 5-FU against pancreatic cancer cells, and oxaliplatin and etoposide against colorectal cells, could be increased through co-suppression of the MCM complex. The oncogene c-Myc also leads to the mismanagement of CMG helicases in part due to a non-transcriptional role in overactivating replication origins and causing DNA damage. We sought to elucidate the mechanism by which Myc causes overactivation of CMG helicases.
Herein we demonstrate that co-suppression of reserve MCM complexes in pancreatic or colorectal cancer cell lines treated with clinically applicable chemotherapeutic compounds causes significant loss of proliferative capacity compared with cells containing the full complement of reserve MCMs. This is in part due to an inability to recover DNA replication following drug exposure, leading to an increase in apoptosis. Targeting of Myc to genomic sites induced large-scale decondensation of higher order chromatin that was required for CMG helicase assembly and activation at reserve MCM complexes. The physiological mediators of Myc, GCN5 and Tip60, are required for the chromatin unfolding and Cdc45 recruitment.
We conclude that depletion of the reserve MCM complexes causes chemosensitization of multiple human tumor cell types to several chemotherapeutic drugs used in the clinical management of human cancer. This argues for the development and use of anti-MCM drugs in combination with chemotherapeutic compounds, which has the potential to increase the therapeutic index of existing clinical compounds. We have also identified a previously unknown role for Myc in normal cell cycle progression whereby DNA replication initiation is regulated through the assembly and activation of CMG helicases on Myc-mediated open chromatin regions. Our results also provide new mechanistic insight into Myc oncogenic transformation in which overstimulation of DNA replication could result in genomic instability and provide an explanation for Myc driven oncogenic transformation.
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Quaresma, Paula Gabriele Fernandes 1987. "Estudo da regulação da proteína CDC2-Like Kinase (Clk2) em hipotálamo e fígado de camundongos controles e obesos = CDC2-Like Kinase (Clk2) hypothalamic and hepatic regulation in lean and obese mice." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311558.
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Orientador: Patrícia de Oliveira PradaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-21T14:12:45Z (GMT). No. of bitstreams: 1 Quaresma_PaulaGabrieleFernandes_M.pdf: 949361 bytes, checksum: 237d0acc0b5bc5358adba045381b4a92 (MD5) Previous issue date: 2012
Resumo: O hipotálamo é um órgão crucial na regulação do balanço energético por integrar sinais hormonais e nutricionais de órgão periféricos. O hormônio produzido pelo pâncreas - insulina - e o hormônio derivado de células adiposas - leptina- reconhecidamente, agem no SNC controlando a ingestão alimentar e o gasto energético. Recentemente foi demonstrado que a fosforilação em treonina 343 da proteina Cdc2-like kinase 2 (Clk2) é induzida pela sinalização de PI3-q/Akt no fígado. Esta regulação envolve a repressão de genes que controlam a gliconeogênese e produção de glicose hepática, levando a hipoglicemia. Porém, não há informações de que a insulina ou a leptina podem regular a Clk2 no hipotálamo in vivo. Camundongos das linhagens Swiss, db/db e C57/BL6J com oito semanas de idade foram utilizados nos experimentos. Nossos resultados mostraram que a Clk2 é expressa e regulada por insulina e leptina em hipotálamo e também que a inibição da Clk2 causou aumento da adiposidade e ingestão alimentar, diminuição do gasto energético e alterações na expressão de neuropeptídeos e do metabolismo de glicose. Além disso, a fosforilação no sítio treonina 343 da Clk2 está diminuída em animais com obesidade induzida por dieta e geneticamente obesos (db/db). A avaliação da gliconeogênese hepática em animais com a proteína Clk2 inibida via ICV mostrou uma tendência ao aumento da produção hepática de glicose, revelando uma possível participação da proteína Clk2 no controle hipotalâmico da gliconeogênese hepática. Sendo assim, podemos sugerir que a Clk2 hipotalâmica é importante no controle do balanço energético pois sua inibição acarreta obesidade acompanhada por aumento da ingestão alimentar e diminuição do gasto energético, e também podemos sugerir um papel no controle hipotalâmico da produção hepática de glicose
Abstract: The hypothalamus plays an important role in the regulation of whole-body energy balance by integrating nutrients and hormones signals from peripheral inputs. The pancreatic hormone - insulin - and the adipocyte hormone - leptin - are known to act in the CNS controlling food intake and energy expenditure. Leptin and insulin signaling regulate anorexigenic neuropeptide expression. Recently, it was shown that Cdc2-like kinase 2 (Clk2) threonine 343 phosphorylation is induced by PI3K/Akt signaling in the liver. This regulation is involved in the repression of gluconeogenic gene expression and hepatic glucose output leading to hypoglycemia. Thus, it was not shown if insulin or leptin are able to regulate Clk2 threonine 343 phosphorylation in the hypothalamus in vivo. Swiss, db/db and C57/BL6J mice eight-weeks-old were used to proceed the experiments. Our data show that Clk2 is expressed and regulated by insulin and leptin in hypothalamus and hypothalamic Clk2 inhibition increased adiposity and food intake, decreased energy expenditure and disrupted neuropeptides expressions and glucose metabolism. Indeed, Clk2 threonine 343 phosphorylation is impaired in the hypothalamus of DIO and db/db mice. Hepatic gluconeogenesis was evaluated and showed increase in ICV inhibited Clk2 mice, it is plausible that Clk2 participates of hypothalamic control of hepatic gluconeogenesis. We suggest that hypothalamic Clk2 is crucial to control energy balance because its inhibition triggers obesity accompanied by increased food intake, decreased energy expenditure and increased hepatic gluconeogenesis
Mestrado
Clinica Medica
Mestra em Ciências
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Winters, Zoe Ellen. "The role of p53 in the regulation of Cdc2 and cyclin B1 in radiation-induced G2 arrest in human cells." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299469.
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Birot, Adrien. "Regulation of fission yeast cohesin by the Cyclin Dependent Kinase PeF1." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0386/document.
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Le complexe cohésine est un complexe protéique en forme d'anneau composé de quatre sous-unités essentielles très conservées: Smc1, Smc3, Rad21 et Scc3. Par sa capacité à encercler les molécules d’ADN, les cohésines participent à de nombreux processus cellulaires tels que la ségrégation des chromosomes, la signalisation et la réparation des dommages à l’ADN, la régulation de la transcription et l'organisation du génome. Pour assurer ces différentes fonctions biologiques les cohésines doivent être finement régulées à la fois dans le temps et l’espace. Ces régulations reposent en partie sur le contrôle de leur association à la chromatine (capture de l’ADN). Cela nécessite l'action d'un «facteur de chargement » composé de deux protéines conservées et essentielles, Mis4 et Ssl3 chez la levure S. pombe. Comment ce complexe régule la capture de l’ADN par l’anneau de cohésine dans l'espace et le temps demeure à ce jour très mal compris. Afin d’identifier des régulateurs de l’association des cohésines à la chromatine, nous avons réalisé un crible génétique visant à rechercher des suppresseurs de la mutation thermosensible mis4-367. Ce crible a conduit à l’identification de la Cyclin-Dependent Kinase Pef1 qui agit comme un régulateur négatif de la cohésion des chromatides soeurs en contrôlant vraisemblablement négativement l’association des cohésines à la chromatine. De forts arguments expérimentaux indiquent que Pef1 exerce sa fonction en régulant directement la phosphorylation de la sous-unité Rad21 du complexe cohésine. De façon intéressante, via un autre crible génétique, nous avons identifié la phosphatase Pph3/Psy2 qui joue un rôle dans l’établissement de la cohésion des chromatides soeurs en contrôlant la déphosphorylation de Rad21.Ensemble, ces données suggèrent que le contrôle de l’état de phosphorylation de la sous-unité Rad21 du complexe cohésine joue un rôle central dans le processus de cohésion chez la levure S. PombeCohesin is a highly conserved ring-shaped protein complex made of four essential subunits: Psm1, Psm3, Rad21 and Psc3. By its ability to capture DNA molecules within its ring-like structure, cohesion plays a key role in many cellular processes such as chromosome segregation, DNA damage signalling and repair, transcriptional gene regulation and nuclear organization. To ensure all of its biological functions, cohesin must be tightly regulated in space and time. This regulation relies in part on the control of cohesin binding to chromatin (DNA capture). Cohesin recruitment to chromatin requires the action of a “loading complex” made of two conserved and essential proteins named Mis4 and Ssl3 in the fission yeast. How this complex regulates where and when DNA capture by the cohesin ring must occur remains poorly understood. To identify regulators of cohesin binding to chromatin we have performed a genetic screen for suppressors of the thermosensitive mutation mis4-367. This genetic screen has led to the identification of the cyclin-dependent-kinase Pef1 that acts as a negative regulator of sister chromatids cohesion may be bynegatively controlling cohesin binding to chromatin. Strong experimental evidences indicate that Pef1 exerts its function at least in part by directly phosphorylating the Rad21 subunit of the cohesin complex. Interestingly, a genetic screen made in parallel identified the Pph3/Psy2 phosphatase as implicated in the establishment of sister chromatid cohesion by regulating Rad21 dephosphorylation. Strikingly, the control of Rad21 phosphorylation status appears central to the cohesion process in the fission yeast S. pombe
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Bhaduri, Samyabrata. "Regulation of CDK1 Activity during the G1/S Transition in S. cerevisiae through Specific Cyclin-Substrate Docking: A Dissertation." eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/871.
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Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.
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Kommajosyula, Naveen. "Regulation of DNA Replication Origins in Fission Yeast: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/436.
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Cells need to complete DNA replication in a timely and error-free manner. To ensure that replication is completed efficiently and in a finite amount of time, cells regulate origin firing. To prevent any errors from being transmitted to the next generation, cells have the checkpoint mechanism.
The S-phase DNA damage slows replication to allow the cell to repair the damage. The mechanism of replication slowing by the checkpoint was not clear in fission yeast, Schizosaccharomyces pombe, at the start of my thesis. The downstream targets of the DNA damage checkpoint in fission yeast were also unclear. I worked on identifying the downstream targets for the checkpoint by studying if Cdc25, a phosphatase, is a target of the checkpoint.
Work from our lab has shown that origin firing is stochastic in fission yeast. Origins are also known to be inefficient. Inefficient origins firing stochastically would lead to large stretches of chromosome where no origins may fire randomly leading to long replication times, an issue called the random gap problem. However, cells do not take a long time to complete replication and the process of replication itself is efficient. I focused on understanding the mechanism by which cells complete replication and avoid the random gap problem by attempting to measure the firing efficiency of late origins.
Genome-wide origin studies in fission yeast have identified several hundred origins. However, the resolution of these studies can be improved upon.
I began a genome-wide origin mapping study using deep sequencing to identify origins at a greater resolution compared to the previous studies. We have extended our origin search to two other Schizosaccharomyces species- S. octosporus and S. japonicus.There have been no origin mapping studies on these fission yeasts and identifying origins in these species will advance the field of replication.
My thesis research shows that Cdc25 is not a target of the S-phase DNA damage checkpoint. I showed that DNA damage checkpoint does not target Cdc2-Y15 to slow replication. Based on my preliminary observation, origin firing might be inhibited by the DNA damage checkpoint as a way to slow replication. My efforts to measure the firing efficiency of a late replicating sequence were hindered by potentially unidentified inefficient origins firing at a low rate and replicating the region being studied. Studying the origin efficiency was maybe further complicated by neighboring origins being able to passively replicate the region. To identify origins in recently sequenced Schizosaccharomyces species, we initiated the genome-wide origin mapping. The mapping was also done on S. pombe to identify inefficient origins not mapped by other mapping studies. My work shows that deep sequencing can be used to map origins in other species and provides a powerful tool for origin studies.
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Borgne, Annie. "Etude de la regulation de cdc2/cycline b a la transition prophase/metaphase de l'ovocyte d'etoile de mer. Caracterisation des effets de la roscovitine, un nouvel inhibiteur chimique de cdk." Paris 6, 1998. http://www.theses.fr/1998PA066422.
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La transition prophase/metaphase du cycle cellulaire est regulee par cdc2/cycline b (ou mpf, m-phase promoting factor). En prophase, la kinase se trouve sous la forme d'un complexe inactif, cdc2 etant phosphorylee sur thr-14 et tyr-15 (forme t p-y p) et la cycline b etant non phosphorylee. L'activation de la kinase se deroule en deux etapes concomitantes : 1) dephosphorylation de cdc2 par la phosphatase cdc25, qui active la kinase (forme t-y), 2) phosphorylation de la cycline b, qui permet la translocation du complexe dans le noyau ou se trouvent ses substrats. En utilisant l'ovocyte d'etoile de mer comme modele cellulaire, nous avons montre que la dephosphorylation de cdc2 in vivo et in vitro se deroule en deux etapes, la dephosphorylation de la thr-14 precedant celle de la tyr-15. La forme transitoire de cdc2 (t-y p) est partiellement active. Ces resultats laissent supposer que la forme t-y p peut etre impliquee dans l'amplification auto-catalytique du complexe. Nous avons egalement etudie la regulation de la phosphorylation de la cycline b. Nous avons mis au point une methode de detection de l'activite cycline b kinase in vitro. Nos resultats montrent que : 1) la phosphorylation de la cycline b n'est pas requise pour l'activite kinase du complexe, 2) cdc2 est responsable de la phosphorylation (shift) de la cycline b en metaphase, 3) la phosphorylation de la cycline b est une reaction intra-mpf. Enfin, nous avons caracterise les effets biochimiques et cellulaires de la roscovitine, un nouvel inhibiteur chimique de cdk. Ce derive de purine inhibe specifiquement cdc2, cdk2 et cdk5 et possede un pouvoir inhibiteur 10 fois superieur a l'olomoucine. La roscovitine empeche la replication de l'adn dans les extraits d'ufs de xenope et provoque l'arret du cycle en g 2/m dans les ovocytes, embryons et lignees de cellules de mammiferes testees. La cible physiologique en prophase de l'inhibiteur est probablement le complexe cdc2/cycline b.
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Vendrell, Arasa Alexandre. "SCF cdc4 regulates msn2 and msn4 dependent gene expression to counteract hog1 induced lethality." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7153.
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L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1.També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut.
Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation.
We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.
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Perl, Abbey Leigh. "Leveraging Small Molecule Activators of Protein Phosphatase 2A (PP2A) toElucidate PP2As Role in Regulating DNA Replication and Apoptosis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1574418174603893.
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Grewal, Navneet. "Identification of amino acids involved in Cdc42-calmodulin interaction and regulation of Cdc42 activation." 2015. http://hdl.handle.net/1993/30716.
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Cdc42 is a member of Rho family of Ras GTPase superfamily and has been shown to regulate actin cytoskeleton re-organization and filopodia formation. Calmodulin (CaM) is a calcium modulating protein and regulates calcium dependent signal transduction pathways in the cell. According to CaM target database analysis, amino acid region 151-163 of Cdc42 has a potential CaM binding domain that interacts with CaM. In the present work, we have investigated putative CaM binding region in Cdc42. In addition the role of basic amino acids K153 and K163 within this region in Cdc42 interaction with CaM and effect on Cdc42 activity was elucidated.
GST-Cdc42 M (Δ151-163), GST-Cdc42K153A and GST-Cdc42K163A mutants were generated. Binding assay experiments showed that amino acid region 151-163 in Cdc42 is an important regulatory domain for CaM binding. Results also demonstrated that K163A mutant showed significantly reduced binding to CaM, whereas Cdc42 K153A showed reduced but non-significant decrease in its interaction with CaM.
A previous study in our laboratory has shown that CaM plays critical role in maintaining basal activity of Cdc42 suggesting that K153A and K163A mutants may play a role in regulating this basal activity. In CHRF 288-11 cells expressing mutant forms of Cdc42 (K153A & K163A), basal activation was significantly decreased as compared to wild type Cdc42. The decrease in basal activity in Cdc42 mutants was not due to an inability to bind GTP.
In summary, the results demonstrated that K163 in Cdc42 is a critical amino acid for CaM interaction and in the regulation of basal activity of Cdc42.October 2015
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Elsaraj, Sherif. "Regulation of Rac1 and Cdc42 GTPases through direct interaction with calmodulin." 2006. http://hdl.handle.net/1993/20344.
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Meir, Michael. "Bedeutung der desmosomalen Adhäsion und Rolle der Rho-GTPasen RhoA, Rac1 und Cdc42 für die Regulation der Darmbarriere." Doctoral thesis, 2013. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85111.
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Eine intakte Darmbarriere ist überlebensnotwendig. Bei einigen Erkrankungen kann eine Störung der Darmbarriere zur Translokation von Bakterien aus dem Lumen des Darmes in den menschlichen Körper führen, die septische Entzündungsprozesse auslösen können. In dieser Arbeit untersuchten wir zum einen die Bedeutung der desmosomalen Adhäsion für die Darmbarriere und zum anderen die Rolle der Rho-GTPasen in der Regulation der Darmbarriere. Für unsere Untersuchungen charakterisierten wir Caco2 Zellen, von denen wir nachweisen konnten, dass sie ein geeignetes Modell für die Darmbarriere sind. Wir konnten zeigen, dass Caco2 Zellen 14 Tage nach ihrer Konfluenz einen vollständigen Schlussleistenkomplex ausbilden und funktionell ähnlich Permeabilitäseigenschaften, wie die Mukosa von Ratten ex vivo aufweisen. Um die Bedeutung der desmosomalen Adhäsion zu klären, applizierten wir einen gegen die Extrazellulärdomäne von Dsg2 gerichteten Antikörper. Dieser Antikörper war spezifisch in der Lage Dsg2 vermittelte Adhäsion zu blockieren. Nach Applikation des Dsg2 ED konnten wir eine Fragmentierung der Occludensproteine, sowie eine gestörte Barrierefunktion mit erhöhter Permeabilität und erniedrigtem transepithelialen Widerstand nachweisen. Damit konnten wir zeigen, dass die Dsg2 vermittelte Adhäsion essentiell für die Aufrechterhaltung der Darmbarriere ist. Des Weiteren untersuchten wir die Rolle der Rho-GTPasen. Wir veränderten die Aktivität der Rho-GTPasen durch Applikation von bakteriellen Toxinen, wie CNF-1, CNF-y, Toxin B, C3-TF und LT sowie Mediatoren, wie Y27632 und quantifizierten die Änderung anschließend durch die Aktivitätsmessung der Rho-GTPasen mittels GLISA. In Immunfluoreszenzen konnten wir zeigen, dass sowohl eine Steigerung als auch eine Erniedrigung der Aktivität von RhoA mit einer Fragmentierung der Occludensproteine einhergeht, während die Adherens Junktionen unbeeinflusst bleiben. Diese morphologische Veränderung korreliert mit einer signifikant erhöhten Permeabilität und einem erniedrigtem transepithelialem elektrischen Widerstand. Im Gegensatz dazu, konnten wir zeigen, dass eine Erhöhung der Aktivität von Rac1 und Cdc42 in der Immunfluoreszenz zu keinen sichtbaren Veränderungen führt, die funktionellen Ergebnisse, mit einem erhöhten transepithelialen elektischen Widerstand und einer erniedrigten Permeabilität auf eine Stabilisierung der Barriere hinweisen. Eine Erniedrigung der Aktivität von Rac1 und Cdc42 führt hingegen zu einer Destabilisierung der Barriere. Morphologisch führte die Verringerung der Aktivität von Rac1 durch LT zu einer Reduzierung der Occludensproteine an den Zellgrenzen und zu einer diffuseren Färbung des Adherens Junktionsprotein E- Cadherin. Zum anderen zeigte sich in diesem Fall eine deutliche Reduzierung der Barrierefunktion mit einem erniedrigten transepithelialen elektrischen Widerstand und einer erhöhten Permeabilität. Letzlich konnte diese Arbeit durch ihre Erkenntnisse einen Teil dazu beizutragen, dass die komplexe Regulation der Darmbarriere besser verstanden wird. Dieses bessere Verständnis soll künftig zur Entwicklung neuer Therapieoptionen für Patienten dienen, die unter den septischen Folgen einer Störung der Darmbarriere leidenThe integrity of intestinal barrier function is essential. In some diseases intestinal barrier breakdown can lead to contamination by bacteria in the human body. In our research we investigated on the one hand the relevance of desmosomal adhesion and the role of Rho GTPases in regulation of intestinal barrier function. For our research we characterized Caco2 cells. We could reveal, that 14 days after confluence Caco2 cells form the "terminal bar" and show a barrier function similar to rat muccosa. To address the relevance ofdesmosomal adhesion we applied an antibody directed against the extracellular domain (Dsg2 ED) in order to test whether impaired Dsg2-mediated adhesion affects intestinal epithelial barrier functions in vitro. This antibody was capable to specifically block Dsg2 interaction. The application of Dsg2 ED led to a fragmentation in tight junction proteins and an impaired barrier function as revealed by an increase of permeability and a decrease of transepithelial electrical resistance. We could show that Dsg2 mediated adhesion is essential for intestinal barrier function. Second we investigated the role of the Rho-GTPases. We modulated the activity of Rho-GTPases by the application of bacterial toxins like CNF-1, CNF-y, toxin B, C3-TF and LT and mediators like Y27632. We could show that an increase as well as a decrease of RhoA activity led to a fragmentation of tight junction proteins as revealed by immunostaining. These morphologic changes correlated with a significant increase of permeability as well as a decreased transepithelial electrical resistance. Apart from that an increased activation of Rac1 and Cdc42 led to a stabilization of intestinal barrier function with an increase of transepithelial electrical resistance and a decrease in permeability. A destabilization of intestinal barrier function was shown after a reduction of Rac1 and Cdc42 activity. Under these conditions we could observe a fragmentation of tight junction and adherens junction proteins. Furthermore decreased Rac1 and Cdc42 activity led to an increased permeability and a decreased transepithelial electrical resistance. In the final analysis this publication led to new insights in the complex regulation of intestinal barrier functions and therefore can possible lead to new targets in the therapy of impaired intestinal barrier function
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Diederich, Wendy-Anneliese [Verfasser]. "Apolipoprotein AI und HDL3 inhibieren die Migration humaner Monozyten durch Induktion der Cholesterinefflux und Regulation von CDC42 / vorgelegt von Wendy-Anneliese Diederich." 2006. http://d-nb.info/980289513/34.
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Köhler, Tim. "Regulation of Growth and Development by the Small GTPase Cdc42p and the Transcription Factor Tec1p in Saccharomyces cerevisiae." Doctoral thesis, 2003. http://hdl.handle.net/11858/00-1735-0000-0006-AE6F-C.
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Deplazes, Joëlle [Verfasser]. "Die Rolle der kleinen Rho-GTPasen Rac1, Rho und Cdc42 in der Regulation der Motilität und Invasion von Tumorzellen mit einer Mutation in E-Cadherin / Joëlle Deplazes." 2009. http://d-nb.info/995844062/34.
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Kalwat, Michael Andrew. "F-Actin regulation of SNARE-mediated insulin secretion." Thesis, 2013. http://hdl.handle.net/1805/3624.
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Indiana University-Purdue University Indianapolis (IUPUI)In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.
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Weidmann, Rolf Günter [Verfasser]. "Endothel und Regulation der Inflammation : Überexpression inaktiver Mutanten der kleinen GTP-bindenden Proteine RhoA/Rac1/Cdc42 inhibiert die LPS-induzierte Expression von Interleukin-8/CXCL8 in humanen mikrovaskulären Endothelzellen / von Rolf Günter Weidmann." 2005. http://d-nb.info/978803736/34.
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Pollok, Sibyll [Verfasser]. "Untersuchungen zur Regulation des humanen Replikationsfaktors Cdc45 / von Sibyll Pollok." 2007. http://d-nb.info/985809736/34.
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Rumpf, Sebastian [Verfasser]. "Regulation des Abbaus von Cdc48-Substraten durch die antagonistischen Aktivitäten von Ufd2 und Ufd3 / vorgelegt von Sebastian Rumpf." 2006. http://d-nb.info/980478286/34.
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Kolawa, Natalie J. "Proteomic Analysis of the Cdc48/Ubx Network Identifies a Role for Ubx2 in the Regulation of Lipid Biosynthesis." Thesis, 2013. https://thesis.library.caltech.edu/7761/1/kolawa_natalie_2013_thesis.pdf.
Full textAbstract:
Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.
Current models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.
Here we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubxΔ mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.
As validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2Δ cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2Δ cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.
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Thattikota, Yogitha. "Regulation of chromosome condensation in Saccharomyces cerevisiae during mitosis." Thèse, 2017. http://hdl.handle.net/1866/19316.
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Köhler, Tim [Verfasser]. "Regulation of growth and development by the small GTPase Cdc42p and the transcription factor Tec1p in Saccharomyces cerevisiae / vorgelegt von Tim Köhler." 2003. http://d-nb.info/969652704/34.
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Van, Zijl Magdalena Catherina. "In vitro effects of 2-methoxyestradiol, an endogenous estrogen, on MCF-12A and MCF-7 cell cycle progression." Diss., 2006. http://hdl.handle.net/2263/26594.
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2-Methoxyestradiol (2ME) is an endogenous estrogen metabolite with antiproliferative and antiangiogenic properties. 2ME also plays an active role in the induction of apoptosis, especially in cancerous cells. These properties have been confirmed by various in vitro and in vivo studies and render 2ME a potential antitumor agent. The mechanism of action of 2ME, however, is not yet fully elucidated and it is believed that multiple mechanisms are involved that may be dependent on cell type. The aim of this study was to investigate the differential effects of 2ME on cell growth, morphology and spindle formation in the non-tumorigenic MCF-12A breast cell line and the tumorigenic MCF-7 breast cell line. In dose-dependent studies, cell growth was determined spectrophotometrically. Light microscopy was used to investigate the morphological changes induced by 2ME and its effect on spindle formation was investigated by means of indirect immunofluorescence. The estrogen receptor status of the MCF-12A cells was confirmed with immunocytochemistry. In order to investigate the effect of 2ME on the length of the cell cycle, cells were blocked in early S-phase with hydroxyurea, then allowed to continue through the cell cycle and mitotic indices determined at regular time intervals. Checkpoint kinase and Cdc2 kinase assays were used to determine the effect of 2ME on relevant cell cycle kinases. Although 2ME inhibited cell growth in both cell lines, the MCF-7 cells were inhibited from much lower concentrations and growth inhibition was more pronounced than in the MCF-12A cells. Treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent, or not as prominent in the MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells. Although the two cell lines differ in their estrogen receptor status, this could not explain the differential effects, for 2ME has a very low affinity for the estrogen receptor. 2ME had no effect on the length of the cell cycle, but blocked MCF-7 cells in mitosis. There were no significant alterations in the phosphorylation status of Cdc25C after 2ME treatment. However, Cdc2 activity was increased to a greater extend in the MCF-7 cells than in the MCF-12A cells. Therefore, it is suggested that exposure to 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest, which may result in the induction of apoptosis. The tumorigenic MCF-7 cells are especially sensitive to 2ME treatment compared to the normal MCF-12A cells. 2ME shows potential for the treatment of breast cancer. Selecting the concentration of 2ME that has maximum inhibitory effect on tumorigenic, but minimal effect on normal cells is crucial in its possible application as antitumor agent. Furthermore, research concerning the differential action mechanisms of 2ME is essential to create a better understanding regarding the treatment of cancer and may possibly contribute to the development and/or improvement of novel chemotherapeutic agents.Dissertation (MSc (Physiology))--University of Pretoria, 2008.
Physiology
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