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Celver, Jeremy Phillip. "Molecular mechanisms of opioid receptor regulation by GRK and arrestin /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/6299.
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Wilham, Laura Elizabeth. "The role of [beta]-arrestin in agonist-induced down-regulation of the M₁mAChR." CONNECT TO THIS TITLE ONLINE, 2006. http://etd.lib.umt.edu/theses/available/etd-03022007-104437/.
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Parisis, Nikolaos. "Identification of PAR-2-regulated ERK substrates and (Beta)-arrestin-interacting proteins in invasive breast cancer cells." Thesis, University of Essex, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520107.
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Bengreed, Amal H. I. "Characterisation of P2Y receptor-mediated contractile signalling and its regulation by G protein coupled receptor kinases and arrestin proteins in a rat bladder smooth muscle." Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42795.
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ATP released from parasympathetic nerves can mediated bladder contraction, can activate purinergic P2Y/Gq/11-coupled G protein-coupled receptors (GPCR) expressed on detrusor (bladder) smooth muscle cells (DSMC). P2Y/Gq/11 signalling activates phospholipase C (PLC) and increases intracellular calcium concentrations to induce contraction. DSMC contractile GPCR activity is tightly regulated to prevent inappropriate contraction/incontinence. Additionally, GPCRs activity is regulated by G protein-coupled receptor kinase (GRK) and arrestin proteins, it is likely that they play a similar role in DSMC, and may help to maintain continence. Combining confocal imaging, calcium-sensitive dyes and selective P2X and P2Y receptor agonists/antagonists, showed that after 3-4 days in culture DSMC calcium signals were mediated by P2Y1 and P2Y2, but not P2X, P2Y4 or P2Y6 receptors. Repeated agonist additions indicated a desensitization of P2Y1 and P2Y2 activated phospholipase C (PLC)/Ca2+ signals, which was restored when the washout period between agonist challenges was increased. Transfection of DSMC with dominant-negative, catalytically inactive GRK mutants, which block endogenous GRK function, showed that P2Y1 and P2Y2 receptor stimulated calcium signalling was selectively regulated by GRK3 and GRK2, respectively. Furthermore, desensitization of P2Y1 and P2Y2 receptor PLC/Ca2+ was attenuated following RNAi-mediated knockdown of arrestin2 or arrestin3, suggesting both arrestins were able to regulate P2Y1/2 receptor signalling. To mimic the effects of obstructive bladder, DSMC were mechanically stretched which resulted in increased GRK2, and decreased GRK3/5/6 expression. These data show that DSMC express functional P2Y1/P2Y2 receptors which mediate purinergic agonist PLC/Ca2+ signalling, implying roles for P2Y1 and P2Y2 in bladder contraction and voiding. Furthermore, P2Y1 and P2Y2 receptor are selectively regulated by GRK and arrestin proteins, which suggests that GRK and arrestin proteins play an important role in the regulation of bladder tone. Furthermore, since GRK expression following mechanical stretch this may in turn affect GPCR signalling and produce dysregulation of DSMC contraction observed during incontinence.
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Davidson, Reshma. "Regulation of Septum Formation by Two Novel Proteins Art1 and Bga1 in Fission Yeast Cytokinesis." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469185292.
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Fessart, Delphine. "Regulation of the endocytic adaptor proteins [beta] arrestin and AP-2 during clathrin-mediated internalization of Angiotensin II type 1 receptor." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102501.
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G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors. They transduce the signals mediated by a diverse range of signalling molecules, including ions, amines, and peptides, as well as photons, to mediate intracellular functions. These receptors play a fundamental role in many physiological responses such as cardiovascular functions. To remain responsive to their environment, cells must find a way to rapidly desensitize and resensitize their activated GPCRs. Desensitization of receptors, for instance, involves the phosphorylation of receptors by G protein-coupled receptor kinase (GRKs) followed by the recruitment of betaarrestin. This interferes with the binding of the G protein (the signalling effector). betaarrestin then targets the receptors to the clathrin endocytosis pathway, and serves as an adaptor linking receptors to other signalling pathways. Internalization of receptors serves not only to remove desensitized receptors from the plasma membrane, but also to engage receptors in the resensitization pathway.The internalization of Angiotensin II (Ang II) type 1 receptor (AT1R) is controversial and poorly described. Therefore, our laboratory studies the mechanisms behind AT1R internalization. The agonist-induced internalization of AT1R begins with the formation of a complex including betaarrestin, the clathrin adaptor AP-2, and the tyrosine protein kinase, c-Src. In turn, this c-Src recruitment regulates the clathrin-mediated internalization of AT1R by controlling the formation of endocytic complexes during endocytosis. Indeed, the recruitment of c-Src is involved in the dissociation of AP-2 during receptor internalization. Based on our evidence that AP-2 and c-Src can be found in the same complex, we suggested that AP-2 could be phosphorylated by c-Src. Indeed, we found that Ang II induced the c-Src-mediated tyrosine phosphorylation of the beta-subunit of AP-2 (beta2-adaptin). We were able to map one of the tyrosines in beta2-adaptin and assess its role in regulating the binding of its principal partner: betaarrestin. The phosphorylation state of beta2-adaptin dictates its association profile with betaarrestin: when phosphorylated it reduces its binding to betaarrestin. Finally, we proposed a model for AT1R internalization. Overall, these studies are significant because they allow a better understanding of the underlying mechanism that regulates the initial steps of clathrin-coated vesicle endocytosis of AT1R.
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Armando, Sylvain. "Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leur effecteurs." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20208/document.
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Les récepteurs couplés aux protéines G (RCPG) sont la famille de récepteurs membranaires la plus représentée chez les vertébrés, et la plus grande cible thérapeutique chez l'Homme. L'évolution du paradigme initial qui énonçait une stœchiométrie récepteur : protéine G : effecteur de 1 :1 :1 sera présentée sur le modèle des récepteurs aux chimiokines CXCR4 et CCR2. Grâce à la technique de transfert d'énergie par bioluminescence (BRET), les travaux réalisés durant cette thèse montrent (1) que c'est par un couplage alternatif de CXCR4 à Gα13 au lieu de la voie classique Gαi que les cellules de cancer du sein migrent pour former des métastases, (2) que la désensibilisation de CXCR4 implique le recrutement d'une combinaison définie de protéines (GRK et arrestines) permettant l'arrêt sélectif des multiples voies engagées en réponse à l'agoniste, et (3) que le protomère CXCR4 a un rôle déterminant dans l'engagement de la protéine Gαi et le recrutement de la β-arrestine par l'hétéro-oligomère CXCR4/CCR2 lorsque CCR2 est activé. Dans cette dernière et principale étude, les résultats montrent également que le dimère CCR2 peut s' assembler au dimère CXCR4 pour former un tétramère, et que l'activation de CCR2 influence la conformation du dimère CXCR4. Les phénomènes de coopérativité et d'activation asymétrique déjà rapportés pour cet hétérodimère pourraient donc impliquer l'interaction de quatre protomères. En conclusion les travaux effectués durant cette thèse démontrent une régulation supplémentaire de l'activité des récepteurs chimiokines au niveau de leur structure quaternaire, de leur signalisation, et de l'arrêt de cette signalisationG protein coupled receptors (GPCR) are the most represented cell surface receptors among vertebrates, and the major therapeutic target in humans. The initial paradigm stating a 1 :1 :1 stoichiometry for receptor :G protein :effector has evolved to a more complex model, as illustrated here with the example of the chemokine receptors CXCR4 and CCR2. Bioluminescence resonance energy transfer (BRET) was used to demonstrate that (1) CXCR4 is able to couple Gα13 instead of Gαi to promote breast cancer metastasis, (2) the multiple pathways engaged by stimulation of CXCR4 are selectively desensitized by the specific recruitment of a defined combination of proteins (GRKs and arrestins) and (3) the CXCR4 protomer plays a crucial role during Gαi engagement and β-arrestin recruitment by the CXCR4/CCR2 heterodimer upon CCR2 activation. In this last and main study, the results shown also demonstrate that CCR2 dimers could assemble with CX CR4 dimers into hetero-tetramers, and that CCR2 activation leads to a conformational change in the CXCR4 dimer. Former results showing cooperativity and asymmetric activation of a simple CXCR4/CCR2 heterodimer could then be applied to a tetramer. To conclude, the work done during this thesis demonstrates a more sophisticated regulation of chemokine receptors than previously suspected at 3 different levels: quaternary structure of the protomers, G protein signalling, and signalling termination
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RAZAGHI, AHMAD. "Les proteines apparentees a l'antigene-s/arrestine : une nouvelle famille de proteines." Paris 6, 1992. http://www.theses.fr/1992PA066592.
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L'antigene-s (arrestine ou proteine 48 k) a ete considere comme un constituant specifique des photorecepteurs de la retine et des cellules de l'organe pineal, qui derivent phylogenetiquement de photorecepteurs. Dans les batonnets retiniens, il participe a la regulation de la phototransduction en se liant a la rhodopsine photoactivee et phosphorylee. L'antigene-s apparait aujourd'hui comme l'un des membres d'une famille de proteines qui auraient des fonctions regulatrices semblables dans differents systemes de transduction. 1) nous purifions a partir de glande pineale de buf une proteine dont les proprietes, y compris le pouvoir d'induire l'uveo-retinite auto-immune experimentale, semblent identiques a celles de l'antigene-s de retine. 2) nous caracterisons et purifions des proteines immunoreactives avec des anticorps diriges contre l'antigene-s de retine dans differentes cellules et tissus: erythrocytes d'oiseau et de poisson, myocarde de buf, rein de buf et de rat, plaquettes sanguines humaines, cellules de tabac et chlamydomonas. 3) nous montrons que ces proteines apparentees de differentes origines partagent la propriete de se lier a la rhodopsine phosphorylee. Il semble que des membres de cette famille de proteines soient presents dans l'ensemble du monde vivant. Leur connaissance progressera avec l'isolement de leurs genes et l'etude de leurs fonctions, qui sont actuellement entrepris
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Lake, David Jonathan. "A human alpha-arrestin protein with a potential role in cargo protein trafficking within the endocytic system." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13335/.
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β-Arrestins are essential adaptors for G protein-coupled receptor (GPCR) trafficking. Evolutionary ancestors of the β-arrestins – dubbed α-arrestins – are present in yeast/fungi and, similar to β-arrestins, recognise cargo proteins and mediate their intracellular trafficking. Mammalian α-arrestins include five largely uncharacterised arrestin domain-containing (ARRDC1-5) proteins that display a predicted arrestin structure; the current study focuses on human ARRDC2. Confocal microscopy of exogenous, fluorescent protein-tagged ARRDC2 in U2OS cells in combination with compartment-specific markers indicated that ARRDC2 is dynamically distributed throughout the plasma membrane and endocytic system, predominantly to late endosomes/lysosomes. Anti-ARRDC2 immunostaining in several primary cell lines broadly supported this conclusion. ARRDC2 contains two proline-rich (PPxY) motifs that in other α-arrestins have been reported to mediate interactions with WW domain-containing NEDD4 family E3 ubiquitin ligases. Coimmunoprecipitation indicated that ARRDC2 is able to interact with several NEDD4 E3s via its PPxY motifs, and confocal microscopy suggested that this interaction may influence the subcellular targeting of the ligases. Ubiquitination of ARRDC2 was detected by coimmunoprecipitation, although this modification was independent of ARRDC2 interaction with NEDD4 E3s. ARRDC2 colocalised with agonist-stimulated, internalised GPCRs (β2-adrenergic receptor (β2AR) and δ-opioid receptor (δOR)) and colocalisation analysis indicated that this involved compartmental redistribution of ARRDC2 to receptor-containing early/recycling endosomes, suggesting a specific effect. Interaction of ARRDC2 with δOR was detected using coimmunoprecipitation, and confocal analysis suggested that ARRDC2 may influence δOR and β2AR intracellular trafficking. ARRDC2 was also found to oligomerise with itself and the β-arrestins. Confocal microscopy showed that ARRDC2 overexpression can induce the redistribution of β-arrestin1 to ARRDC2-positive vesicles, and a punctate bimolecular fluorescence complementation (BiFC) signal was detected between ARRDC2 and β-arrestin2. From this, it is speculated that α-/β-arrestins may function cooperatively or competitively to mediate discrete GPCR sorting events in the endocytic pathway.
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Witty, Marie-France. "Role of the adaptor protein, beta-arrestin1, in the Notch signaling pathway." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/446.
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The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.
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Hirata, Cristiane Lumi. "Thioredoxin interacting protein (Txnip) forms redox sensitive high molecular weight nucleoprotein complexes." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/264647.
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Goh, Poh. "Roles of protein kinase C and arrestin in migration of cells via CXCR4/CXCL12 signalling axis." Thesis, University of East Anglia, 2018. https://ueaeprints.uea.ac.uk/67806/.
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Aim: The chemokine system not only coordinates leukocyte migration in immunity and inflammation, but it is also implicated in the pathogenesis of many human diseases, including cancer. The expression of chemokines and their receptors is altered in many malignancies and leads to aberrant chemokine receptor signalling. Emerging evidence indicates that the tumour microenvironment has critical roles in all aspects of cancer biology, including growth, angiogenesis, metastasis and progression. One of the important representatives of this system are the chemokine ligand CXCL12 and its receptor, CXCR4 as they are most commonly found on human and murine cancer cells. Our aims are to study and understand if there are any differences in activation of signalling molecules in the downstream signalling cascades in CXC- chemokine receptors in different cell types, and to identify the importance of different effector proteins in migration of cells; the two proteins of interest include Protein Kinase C (PKC) and arrestins. Methodology: Experimentation was undertaken in MCF-7 breast cancer cells and Jurkat leukemic T-lymphocytes which both naturally express the chemokine receptor CXCR4. Small molecule inhibition and protein overexpression was used in chemotaxis and calcium release assays to measure cellular responses. Immunocytochemistry was used to determine the effect of protein blocking and protein overexpression on receptor internalisation, protein localisation and the formation of cellular structures associated with migration. Results: Inhibition of PKC has no effect on Jurkat cell migration, but it blocks MCF-7 cell migration showing that there is a difference in the usage of PKC in different cell types. Arrestin 3 is important for migration in both suspension Jurkat cells and adherent breast cancer MCF-7 cells. Conclusion: Our study shows that CXCL12-induced migration may be arrestin 3 mediated. We have also shown that activation of signalling molecules needed for CXCL12-induced migration can differ between different cell lines. Overall, the research in this thesis has identified potential signalling molecules that can be targeted to interfere with migration of cells.
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Lortie, Karine. "The growth-arrest-specific protein gas7 potentiates neuronal differentiation." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26701.
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The growth-arrest-specific gas7 protein is involved in neuronal development. Its role in neuronal differentiation and its potential neuroprotective activity were investigated in PC 12 and NT2 cells. gas7 overexpression in PC12 cells promoted neurite outgrowth and potentiated nerve growth factor-induced expression of the neuronal markers betaIII-tubulin, synaptotagmin, alpha7 subunit of the acethylcholine receptor, and dihydropyrimidinase related protein-3. This effect was exerted independently of cellular proliferation, as gas7 did not affect cell cycle progression. Endogenous gas7 expression was induced during neuronal differentiation of NT2 cells with retinoic acid, suggesting a role for gas7 in neuronal development. Finally, gas7 overexpression in PC 12 cells did not protect against toxicity triggered by oxygen-glucose deprivation, the calcium ionophore A23187 or sodium nitroprusside. The ability of gas7 to potentiate neuritogenesis and neuronal differentiation makes it a potential therapeutic target to promote re-establishment of neuronal connections in the injured or diseased brain, such as following stroke.
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Rao, Deepa Prema. "The role of growth arrest-specific 6 in venous thromboembolism /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112349.
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Background. Growth-arrest specific 6 (gas6) is a novel vitamin-K dependent protein whose role in venous thromboembolism was recently characterized in murine models. Gas6 is suggested to be a prothrombotic protein capable of mediating thrombus stability. However, the association between gas6 and venous thromboembolism has yet to be elucidated in humans. The present work aims to delineate the existence of such an association in humans and propose a mechanism by which gas6 expression is related to venous thromboembolic disease.Methods. To analyze the association between gas6 and venous thromboembolism, a highly specific ELISA method was used to measure plasma gas6 levels in 306 patients with a history of deep-vein thrombosis (DVT) and 89 control volunteers. Medication history, comorbid conditions and DVT characteristics were documented for the purposes of statistical analyses. Median gas6 levels were compared between the subgroups, and prevalence rate ratios were calculated. Human umbilical vein endothelial cells were used to measure the effect of gas6 treatment on the expression of various mediators of coagulation. Murine thrombosis models were developed to serve as in vivo models for thrombosis.
Results. The median levels of gas6 were 28.21 ng/ml in patients compared to 26.15 ng/ml in controls (p=0.01). After adjustment for age, sex, comorbidity and medications, DVT patients had a PRR of 2.5 (95% CI 1.36 to 4.61, p=0.003) compared with controls. Within the DVT subgroup, median gas6 levels were significantly higher in those with cancer-associated (vs. unprovoked or secondary) DVT (p<0.001) and in those with more extensive DVT (p=0.037), while levels were significantly lower in those taking warfarin (vs. no warfarin) (p=0.03). Preliminary results with endothelial cell cultures are inconclusive with regards to the effect of gas6 on endothelium derived mediators of coagulation.
Conclusions. Elevated plasma gas6 is associated with venous thromboembolism. The etiology of the clot influences detected levels of gas6, with the highest levels seen in cancer-patients. Furthermore, increasing clot burden correlates with elevated levels of gas6. A mechanistic explanation for how gas6 modulates this association is in its preliminary stages, and is worth pursuing.
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Crosby, Meredith Ellen. "E2F4 is a critical molecule involved in the cell cycle arrest reponse following ionizing radiation." Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1136934596.
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Thesis (Ph. D.)--Case Western Reserve University, 2006.[School of Medicine] Department of Environmental Health Sciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.
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Mariggio, Stefania Pasqua. "Regulation of immune receptor functional responses by G protein-coupled receptor kinases (GRKs) and arrestins." Thesis, Open University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343747.
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Raehal, Kirsten Michele. "Opioid-Induced Side Effects in Beta-arrestin2 adn G Protein-Coupled Receptor Kinase Knockout Mice." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1236884585.
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Raehal, Kirsten M. "Opioid-induced side effects in beta-arrestin2 and G protein-coupled receptor kinase knockout mice." Columbus, Ohio : Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1236884585.
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Kemp, Hilary A. "A complex of six FAR proteins required for pheromone arrest and mating /." view abstract or download file of text, 2003. http://wwwlib.umi.com/cr/uoregon/fullcit?p3113011.
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Thesis (Ph. D.)--University of Oregon, 2003.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 94-104). Also available for download via the World Wide Web; free to University of Oregon users.
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Schmid, Cullen L. "Differential regulation of serotonin 2A receptor responsiveness by agonist-directed interactions with beta-arrestin2." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1300287547.
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Bhattacharya, Sarbani [Verfasser]. "Structural and functional studies of growth arrest and DNA-damage proteins / Sarbani Bhattacharya." Berlin : Freie Universität Berlin, 2010. http://d-nb.info/1023959607/34.
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Sehat, Bita. "SUMO and ubiquitin; the yin and yang of IGF-1R function /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-360-3/.
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Morais, Carla Patrícia Amorim Carneiro de. "A atividade do NHE3 em túbulo proximal é inibida pela sinalização enviesada do receptor de angiotensina II tipo 1/beta-arrestina." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5166/tde-20042016-113857/.
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Os receptores medeiam a maioria das respostas fisiológicas em resposta a diversidade de estímulos. A ativação da sinalização mediada pelo receptor de angiotensina II tipo 1 é o principal responsável pelos efeitos do hormônio angiotensina II (Ang II) nos tecidos alvo. No rim concentrações fisiológicas de Ang II aumentam a atividade no túbulo proximal da isoforma 3 do trocador de Na+/H+ (NHE3). Este efeito é crucial para a manutenção do volume extracelular e pressão arterial. Evidências recentes mostraram que a ativação seletiva da sinalização enviesada da beta-arrestina/ receptor AT1 induz diurese e natriurese independentemente da sinalização via proteína G. Neste estudo testamos a hipótese de que a sinalização enviesada do receptor AT1/ beta-arrestina inibe a atividade do NHE3 no túbulo proximal, bem como investigar os possíveis mecanismos moleculares que medeio este efeito. Para tal, nós determinamos os efeitos do composto TRV120023, que se liga ao receptor AT1, bloqueando o acoplamento da proteína G e estimulando a sinalização da beta-arrestina, na função do NHE3 in vivo e in vitro. A atividade do NHE3 foi medida quer em túbulo proximal nativo, por meio de microperfusão estacionária, bem como em uma linha celular de túbulo proximal de gamba (OKP), por meio de recuperação de pH intracelular dependente de Na+. Os nossos resultados mostram que o TRV120023 na concentração de 10-7 M inibe marcadamente a atividade do NHE3 em túbulo proximal quer in vivo quer in vitro, sendo que este efeito é completamente abolido nas células silenciadas para a beta-arrestina 1 e 2 através de RNA de interferência. Adicionalmente, a estimulação do NHE3 pela Ang II é completamente suprimida pelo TRV120023 quer in vivo quer in vitro. A inibição do NHE3 pelo TRV120023 foi associada com a diminuição do NHE3 expresso na superfície da membrana plasmática em células OKP e com a redistribuição entre o corpo e a base das microvilosidades em túbulo proximal de rato. A diminuição do NHE3 na superfície da membrana plasmática em células OKP estava associado com um aumento na internalização do NHE via endocitose mediada por clatrina. A inibição do NHE3 mediada pela beta-arrestina não envolve a sinalização do receptor AT2, cAMP/ PKA, Akt e ERK1/2. Estes achados indicam que a sinalização enviesada do receptor AT1/beta-arretina inibe a atividade do NHE3 em túbulo proximal, pelo menos em parte, devido a alterações na localização subcelular do NHE3Cell surface receptors mediate most of our physiological responses to an array of stimulus. The triggering of the angiotensin II type I (AT1) receptor signaling is the major control point in the regulation of the ultimate effects of the peptide hormone angiotensin II (Ang II) on its target tissue. In the kidney physiological concentrations of Ang II upregulate the activity of proximal tubule Na+/H+ exchanger isoform 3 (NHE3). This effect is crucial for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the betaarrestin-biased AT1 receptor signalingpathway induces diuresis and natriuresis independent of G-protein mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule as well as investigate the underlying molecular mechanisms mediating this effect. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G protein coupling, and stimulates beta-arrestin signaling, on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. Our results showed that 10-7 MTRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro, and the effect was completely abolished in OKP cells silenced for beta-arrestin 1 and 2 by small interference RNA. Additionally, stimulation of NHE3 by Ang II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. The decreased surface NHE3 in OKP cells was associated with an increase in NHE3 internalization via clathrin mediated endocytic. Beta-arrestin mediated NHE3 inhibition did not involve AT2 receptor, cAMP/ PKA, Akt and ERK1/2 signaling. These findings indicate that biased signaling of the AT1 receptor/beta-arrestin pathway inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization
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Costa, Mariana Fernandes Alves. "Molecular remodelling of the spindle architecture during metaphase arrest in oocytes." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31255.
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Oocytes of most species assemble and maintain a functional bipolar spindle in the absence of centrosomes. Strikingly, after bipolar spindle formation, oocytes arrest in metaphase for several hours before fertilisation. How the dynamic spindle maintains its bipolarity during this long arrest is poorly understood. I hypothesise that the bipolar spindle is stably maintained by changes in the distribution of microtubule-associated proteins (MAPs) on the spindle during the long oocyte arrest. To test this, I generated transgenic flies expressing GFP-tagged microtubule-associated proteins (MAPs), and found that 13 out of 24 proteins change localisation between early and late oocytes. I refer to these changes in MAP localisation after establishment of bipolarity as 'spindle maturation'. In order to identify the molecular mechanisms triggering MAP relocalisation, I manipulated the kinase activity of the cell cycle regulator Cdk1 by over-expressing non-degradable cyclin A or B, the major activators of Cdk1. Their expression prevented re-localisation of distinct sets of MAPs, and disrupted spindle bipolarity and accurate chromosome segregation in oocytes. Kinesin-6 Pavarotti/MKlp1 localised strongly to the spindle equator in late oocytes, whilst nearly always absent from this region in early oocytes. The localisation of Pavarotti to the spindle equator in late oocytes was reduced when cyclin B is over-expressed in oocytes, suggesting a role for Cdk1/cyclin B complex in regulating Pavarotti localisation. Indeed, a Pavarotti/Mklp1 mutant non-phosphorylatable by Cdk1 prematurely localised to the meiotic spindle and disrupted spindle bipolarity. Moreover, removal of Pavarotti from the metaphase-I spindle by RNAi induced spindle defects in oocytes. Therefore, it is likely that the microtubule cross-linking activity of Pavarotti enhances the stability of the metaphase-I spindle during the long arrest. Consistent with this, I found that the microtubule density in the spindle equator is higher in late oocytes. Altogether, I propose that remodelling the molecular architecture of the spindle during the long oocyte arrest is important to stabilise the bipolar spindle without centrosomes.
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Azoulay, Eric. "Induction of apoptosis or cell cycle arrest by two human wildtype variants of the p53 protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0031/MQ64313.pdf.
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Zhang, Yuan. "An investigation of p53’s differential activation of cell cycle arrest and apoptosis." Thesis, Curtin University, 2008. http://hdl.handle.net/20.500.11937/1577.
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The p53 tumour suppressor protein lies at the hub of a very complex network of cellular pathways including apoptosis, cell cycle arrest, DNA repair and cellular senescence. However, the mechanism of why and how p53 switches between apoptosis and cell cycle arrest, thereby determining a cell’s fate, remains a mystery to us. To enable us to investigate this ability of p53 to switch between cell cycle arrest and apoptosis, we developed a model which demonstrates similar p53 expression patterns but different functional outcomes. Treating cells with Cisplatin (a common chemotherapeutic drug) and Nutlin-3 (an MDM-2 inhibitor) results in similar high levels of p53 accumulation but different cellular responses. Cisplatin-treated cells undergo apoptosis while Nutlin-treated cells enter cell cycle arrest. Using this model, we explored the localization of p53 and in particular a C-terminal Ser 392 moiety in an attempt to identify how p53 is able to preferentially activate cell cycle arrest or apoptotic pathway.
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Muchhala, Karan Hitesh. "An investigation on the role of β-arrestin 2, protein kinase C and sex on the mechanism of morphine tolerance in the mouse ileum." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6074.
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Opioids such as morphine are frequently used in the clinic to treat pain. However, the perennial bane of chronic opioid use is the rapid development of tolerance to the analgesic effects but delayed development of tolerance to the respiratory depressant and constipating effects. As constipation is one of the most common opioid-related adverse effects in humans, it is important to delineate mechanisms that drive opioid tolerance in the ileum and lack of it in the colon. The overarching goal of this thesis was to investigate mechanisms of morphine tolerance in the ileum by comparing the mechanism of morphine tolerance in ileum myenteric plexus neurons and dorsal root ganglia (DRG) neurons. Myenteric plexus neurons are integral to the motor function of the ileum, whereas DRG neurons are important components of peripheral nociceptive sensation. We also examined the mechanism of morphine tolerance development to small intestinal transit and to antinociception at the systemic level in male and female mice. Studies presented in this dissertation demonstrate that the mechanism of morphine tolerance in the mouse ileum is not contingent on b-arrestin 2. In fact, tolerance in the small intestine might be mediated by a b-arrestin 2-independent mechanism following protein kinase C-induced phosphorylation of the m-opioid receptor. We also demonstrate that morphine tolerance to antinociception is not solely dependent on b-arrestin 2, and is mediated by b-arrestin 2-dependent and-independent mechanisms. Lastly, we have shown how sex of the animal can influence mechanisms underlying the development of morphine tolerance. Collectively, the findings presented here increase our understanding of the mechanisms by which morphine tolerance develops in the ileum and to antinociception.
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Mayall, Stephen James. "Cyclins in the slime mould Dictyostelium discoideum." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243862.
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Zhang, Yuan. "An investigation of p53’s differential activation of cell cycle arrest and apoptosis." Curtin University of Technology, School of Pharmacy, 2008. http://espace.library.curtin.edu.au:80/R/?func=dbin-jump-full&object_id=118347.
Full textAbstract:
The p53 tumour suppressor protein lies at the hub of a very complex network of cellular pathways including apoptosis, cell cycle arrest, DNA repair and cellular senescence. However, the mechanism of why and how p53 switches between apoptosis and cell cycle arrest, thereby determining a cell’s fate, remains a mystery to us. To enable us to investigate this ability of p53 to switch between cell cycle arrest and apoptosis, we developed a model which demonstrates similar p53 expression patterns but different functional outcomes. Treating cells with Cisplatin (a common chemotherapeutic drug) and Nutlin-3 (an MDM-2 inhibitor) results in similar high levels of p53 accumulation but different cellular responses. Cisplatin-treated cells undergo apoptosis while Nutlin-treated cells enter cell cycle arrest. Using this model, we explored the localization of p53 and in particular a C-terminal Ser 392 moiety in an attempt to identify how p53 is able to preferentially activate cell cycle arrest or apoptotic pathway.
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Sivko, Gloria S. BS DV M. "Characterization and regulation of C/EBPδ in human mammary epithelial cell G0 growth arrest." The Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1082580020.
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Aikio, M. (Mari). "Novel roles for basement membrane collagens:isoform-specific functions of collagen XVIII in adipogenesis, fat deposition and eye development, and effects of the collagen IV-derived matricryptin arresten on oral carcinoma growth and invasion." Doctoral thesis, Oulun yliopisto, 2013. http://urn.fi/urn:isbn:9789526203188.
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Abstract Collagen XVIII is an evolutionarily conserved, ubiquitously-expressed basement membrane (BM) proteoglycan produced in three isoforms, the individual roles of which are largely unknown. The physiological in vivo roles of these collagen XVIII isoforms are studied here using novel genetically modified mouse strains deficient in either the short or the medium/long isoforms of the molecule. In addition, the effects of keratin-14-driven overexpression of the thrombospondin-1 (Tsp-1) –like domain, which is common to all three collagen XVIII isoforms, are studied. The findings underline the importance of the short collagen XVIII isoform in the eye, as its absence was sufficient to cause the aberrant vascularisation of the retina previously reported in mice lacking all isoforms of collagen XVIII. In addition, an excess of the collagen XVIII Tsp-1 domain led to serious eye abnormalities, possibly by interfering with the functions of the full-length collagen XVIII produced in mice. Collagen XVIII was also shown to contribute to adipogenesis in an isoform-specific manner, in that a lack of the medium/long isoforms of collagen XVIII led to impaired adipocyte maturation and the subsequent reduction in the adipocyte number induced liver steatosis and hypertriglyceridaemia. Hence this work establishes a new extracellular matrix ECM-directed mechanism contributing to control over the multistep adipogenesis programme and points to the functional consequences of its impairment for ectopic fat deposition. The enzymatic remodelling of ECM components results in molecules with novel biological activities. Arresten is a collagen IV(α1)-derived fragment with anti-angiogenic properties which was originally described as not having any direct anti-tumour effects on cancer cells themselves. The present data revealed novel inhibitory roles for arresten in oral squamous carcinoma cell proliferation, survival, motility and invasion. Since arresten is a potent inhibitor of angiogenesis, the data generated here further underline the possibility for using it as a therapeutic agent in cases of cancerTiivistelmä Kollageeni XVIII on tyvikalvojen proteoglykaani ja yksi harvoista evoluutiossa konservoituneista kollageeneista. Se esiintyy elimistössä kolmena isomuotona, joiden biologiset tehtävät ovat vielä jokseenkin epäselviä. Tässä tutkimuksessa selvitettiin kollageeni XVIII:n isomuotojen fysiologista merkitystä hyödyntäen uusia hiirilinjoja, joilta kollageeni XVIII:n lyhyt tai kaksi pisintä varianttia oli geneettisesti inaktivoitu. Poistogeenisten hiirimallien rinnalle tehtiin kaikille varianteille yhteistä trombospondiini-1 (Tsp-1)-domeinia yli-ilmentävä hiirilinja. Tämän väitöskirjatutkimuksen avulla saatiin uutta tietoa kollageeni XVIII:n ja etenkin sen lyhimmän variantin tärkeästä roolista silmässä. Aikaisemmat tutkimukset ovat osoittaneet kollageeni XVIII:n puutteen häiritsevän silmän verkkokalvon verisuonituksen normaalia kehittymistä. Tässä työssä havaittiin, että pelkästään lyhyen isomuodon puute riitti altistamaan hiiret muutoksille verkkokalvon suonituksessa. Tsp-1-osan ylimäärän havaittiin lisäksi alistavan hiiret muutoksille silmän rakenteessa, mahdollisesti häiritsemällä silmässä jo olemassa olevan kollageeni XVIII:n toimintaa. Tässä työssä havaittiin myös uusi yhteys kollageeni XVIII:n ja rasvasolujen kypsymisen välillä. Verrokkihiiriin verrattuna muodostuvan rasvakudoksen havaittiin jäävän merkittävästi vähäisemmäksi poistogeenisillä hiirillä, joilta kollageeni XVIII:n pitkät isomuodot olivat geneettisesti inaktivoitu. Heikentynyt rasvakudoksen muodostuminen lisäsi triglyseridien kertymistä hiiren verenkiertoon ja maksaan. Tutkimustulos on merkittävä avaus soluväliaineen merkityksestä rasva-aineenvaihdunnalle ja kannustaa lisätutkimuksilla selvittämään, onko kollageeni XVIII:lla yhteys myös ihmisen metaboliseen oireyhtymään. Soluväliaineen komponenttien entsymaattinen muokkaus tuottaa usein molekyylejä, joilla on uusia isäntämolekyyleistä poikkeavia ominaisuuksia. Tässä työssä tutkittiin yhden tällaisen molekyylin, tyvikalvokollageenin IV hajoamistuotteen, arrestenin, suoria vaikutuksia syöpäsoluille. Arrestenin tiedettiin entuudestaan estävän syöpäkasvainten verisuonten uudismuodostusta koe-eläimillä. Työssä osoitettiin, että arresten vaikutti endoteelisolujen lisäksi myös itse syöpäsoluihin estäen niiden lisääntymistä ja vähentäen niiden elinkykyä ja liikkuvuutta, mikä tekee arrestenista entistä houkuttelevamman ehdokasmolekyylin lääkekehitystyöhön
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Fujinaga, Takuji. "Isoflurane inhalation after circulatory arrest protects against warm ischemia reperfusion injury of the lungs." Kyoto University, 2007. http://hdl.handle.net/2433/135895.
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Houri, Nadia. "Study of ERK12 MAP kinases activation by the bradykinin type 2 receptor : characterization of beta-arrestin scaffolding function in the temporal regulation of ERK12 activation induced by the B2R." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112637.
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G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors. The beta-arrestins, adaptor proteins involved in GPCR desensitization, may also act as scaffolds for signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. The MAPK family, which includes the extracellular-signal regulated kinases (ERK) 1 and 2, promotes cellular differentiation and proliferation. Herein, the activation of ERK1/2 upon stimulation of the GPCR bradykinin type 2 receptor (B2R) with bradykinin was examined. Various B2R mutants with modified C-termini were employed to examine the temporal kinetics of ERK1/2. One of these receptor mutants displayed a loss of beta-arrestin binding as well as greatly enhanced ERK1/2 activation, compared to the wild-type receptor, when a cluster of serine/threonine residues important for B2R internalization was mutated. The other receptor mutants exhibited a correlation between their affinity for beta-arrestin and the intensity of ERK1/2 activation. Data from a mouse embryonic fibroblast cell line null for beta-arrestin suggested that beta-arrestin is involved in late-phase ERK1/2 activation by the B2R. These data point to the involvement of beta-arrestin in the activation of the ERK1/2 MAPKs through the B2R.
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Pokela, M. (Matti). "Predictors of brain injury after experimental hypothermic circulatory arrest:an experimental study using a chronic porcine model." Doctoral thesis, University of Oulu, 2003. http://urn.fi/urn:isbn:951427105X.
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Abstract
There is a lack of reliable methods of evaluation of brain ischemic injury in patients undergoing cardiac surgery. The present study was, therefore, planned to evaluate whether serum S100β protein (I), brain cortical microdialysis (II), intracranial pressure (III) and electroencephalography (EEG) (IV) are predictive of postoperative death and brain ischemic injury in an experimental surviving porcine model of hypothermic circulatory arrest (HCA).
One hundred and twenty eight (128) female, juvenile (8 to 10 weeks of age) pigs of native stock, weighing 21.0 to 38.2 kg, underwent cardio-pulmonary bypass prior to, and following, a 75-minute period of HCA at a brain temperature of 18°C. During the operation, hemodynamic, electrocardiograph and temperature monitoring was performed continuously. Furthermore, metabolic parameters were monitored at baseline, end of cooling, at intervals of two, four and eight hours after HCA and before extubation. Electroencephalographic recording was performed in all animals, serum S100β protein measurement in 18 animals, cortical microdialysis in 109 animals, and intracranial pressure monitoring in 58 animals. After the operation, assessment of behavior was made on a daily basis until death or elective sacrifice on the seventh postoperative day.
All four studies showed that these parameters were predictive of postoperative outcome. Animals with severe histopathological injury had higher serum S100β protein levels at every time interval after HCA. Analysis of cortical brain microdialysis showed that the lactate/glucose ratio was significantly lower and the brain glucose concentration significantly higher among survivors during the early postoperative hours. Intracranial pressure increased significantly after 75 minutes of HCA, and this was associated with a significantly increased risk of postoperative death and brain infarction. A slower recovery of EEG burst percentage after HCA was significantly associated with the development of severe cerebral cortex, brain stem and cerebellum ischemic injury.
In conclusion, serum S100β protein proved to be a reliable marker of brain ischemic injury as assessed on histopathological examination. Cerebral microdialysis is a useful method of cerebral monitoring during experimental HCA. Low brain glucose concentrations and high brain lactate/ glucose ratios after HCA are strong predictors of postoperative death. Increased intracranial pressure severely affected the postoperative outcome and may be a potential target for treatment. EEG burst percentage as a sum effect of anesthetic agent and ischemic brain damage is a useful tool for early prediction of severe brain damage after HCA. Among these monitoring methods, brain cortical microdialysis seems to be the most powerful one in predicting brain injury after experimental hypothermic circulatory arrest.
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Zhang, Tong. "The investigation of growth-arrest-specific 2 protein functions in cell division and its cytoskeleton binding mechanisms." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116848.
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The eukaryotic cell cytoskeleton plays many important roles in cells and their coordination is crucial for these important biological processes. Their functions are partially achieved by the actin-microtubule (MT) cross-linking proteins. Growth-arrest-specific (Gas) 2 was originally identified in murine fibroblasts under growth-arrest conditions. Gas2 protein contains putative actin- and MT-binding domains, which makes it a strong candidate to be an actin-MT cross-linking protein. However, the mechanisms of Gas2 cytoskeleton binding and its roles in cell division are still unclear. This thesis is focused on these questions. In Chapter 2, we have determined the Gas2 protein functions in Xenopus laevis embryo cell division and oocyte wound healing process. We have found that the full-length (FL) Gas2 protein and its MT-binding Gas2 domain, but not the actin-binding calponin homology (CH) domain, inhibit cell division and result in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that FL-Gas2 function is mediated by binding MTs. To investigate the mechanism of Gas2-induced cell division arrest, we have used a wound-induced contractile array assay and showed that FL-Gas2 stabilizes MTs. In Chapter 3, we have further investigated the FL-Gas2 protein cytoskeleton binding mechanisms in HeLa cells. The Gas2 domain itself significantly changes MTs morphology into a long loop-like bundled network. The FL-Gas2 facilitates MT polymerization and changes the dynamic behaviors of MTs. There are three protein kinase C (PKC) phosphorylation sites in the FL-Gas2 protein, and phosphomimetic point mutations reveal that all three are involved in the FL-Gas2 protein functional changes. Size-exclusive chromatography finally reveals that the FL-Gas2 protein forms a dynamic complex, which presumably has an important role in its cytoskeleton binding properties. Taken together, our data suggest that the non-phosphorylated FL-Gas2 protein forms a dynamic complex to bundle MTs, but its ability to cross-link F-actin and MTs is achieved by the phosphorylated FL-Gas2 protein. We propose that Gas2 function is mediated by binding and bundling MTs, leading to cell division arrest.Le cytosquelette participe à de nombreuses fonctions dans les cellules eucaryotes et sa bonne coordination est essentielle au bon fonctionnement de ces fonctions biologiques. Ces fonctions sont partiellement accomplies grâce aux protéines de liaisons des filaments actine-microtubule (MT). La protéine Gowth-arrest-specific (Gas) 2 a été originellement identifiée dans des fibroblastes murins en arrêt de croissance. Le fait que la protéine Gas2 contienne des domaines putatifs d'intéractions avec actine et les microtubules supporte l'hypothèse qu'elle agit comme protéine de liaison des filaments actine-microtubule. Cependant, les mécanismes que Gas2 utilise pour interagir avec le cytosquelette et participer à la division cellulaire ne sont pas connus. Cette thèse répond à ces questions.Dans le deuxième chapitre, nous avons démontré que la protéine Gas2 participe à la division cellulaire de l'embryon et à la cicatrisation de la plaie de l'ovocyte chez le Xenopus laevis. Nous avons trouvé que la protéine entière (FL) Gas2 et son domaine d'intéraction avec les microtubules, mais pas son domaine d'intéraction avec actine homologue a calponine (CH), inhibent la division cellulaire et résultent en la formation de cellules multinucléées. Nous déduisons du fait que le domaine de Gas2 puisse causer un arrêt de la division cellulaire à lui seul que la protéine entière FL-Gas2 fonctionne grâce à l'intéraction avec les microtubules. Pour étudier comment Gas2 induit l'arrêt de la division cellulaire, nous avons utilisé un essai d'induction de contraction par cicatrisation et démontré que FL-Gas2 stabilise les microtubules. Dans le troisième chapitre, nous avons étudié en détails comment la protéine FL-Gas2 interagit avec le cytosquelette dans les cellules HeLa. Le domaine de Gas2 change dramatiquement la morphologie des microtubules, celles-ci forme un réseau de boucles longues et regroupées. La protéine FL-Gas2 favorise la polymérisation des microtubules et change leur comportement dynamique. Il existe trois sites de phosphorylation par la protéine kinase C (PKC) dans FL-Gas2. Des points de mutations imitant la phosphorylation de ces trois sites ont démontré que les trois sites participent aux changements de fonctionnalité de FL-Gas2. La chromatographie par exclusion de taille a démontré que FL-Gas2 forme un complexe dynamique qui doit jouer un rôle important dans l'intéraction de la protéine avec le cytosquelette.En conclusion, nos résultats suggèrent que la protéine FL-Gas2 non-phosphorylée forme un complexe pour regrouper les microtubules, et qu'elle crée une liaison entre F-actine et les microtubules lorsqu'elle est phosphorylée. Nous formulons hypothèse que Gas2 agit en intéragissant et en regroupant les microtubules ce qui cause un arrêt de la division cellulaire.
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Wong, Kam-wai, and 黃錦偉. "The molecular mechanism of mitotic arrest induced by a novel diterpenoid pseudolaric acid B and a novel gene encoding RNA-bindingprotein 22." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B37679090.
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Wong, Kam-wai. "The molecular mechanism of mitotic arrest induced by a novel diterpenoid pseudolaric acid B and a novel gene encoding RNA-binding protein 22." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B37679090.
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Theuer, Stefanie [Verfasser]. "Die Rolle von growth arrest specific protein 6 im Aldosteron induzierten Endorganschaden : Untersuchungen an einem Knockout-Mausmodell / Stefanie Theuer." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2013. http://d-nb.info/1042658005/34.
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Chen, Dong. "Function of Insulin-like Growth Factor Binding Protein 7 (IGFBP7) in Hepatocellular Carcinoma." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2821.
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Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.
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Inamdar, Kaushik. "Role of I-BAR proteins and membrane curvature in HIV-1 assembly and release." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT032.
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Lors de l'assemblage et du bourgeonnement du VIH, la membrane plasmique subit une courbure entraînée par l'auto-assemblage du VIH-1 Gag au site d'assemblage vers l'extérieur de la cellule. Cependant, la multimérisation de Gag peut ne pas être suffisante et Gag peut avoir besoin de recruter des facteurs cellulaires pour induire une courbure de membrane locale. Nous avons récemment rapporté que la libération de particules de VIH-1 Gag dépendait de l'activation de la voie de signalisation Rac1 / IRSp53 / Wave2 / Arp3 dans les cellules Jurkat T et les lymphocytes sanguins primaires. Ce complexe cellulaire, lorsqu'il est activé, est recruté dans la membrane plasmique cellulaire et favorise le recrutement de la ramification de l'actine, la polymérisation de l'actine et le remodelage de la membrane dans les lamellipodes. En particulier, la protéine IRSp53 contient un domaine I-BAR capable d'induire une courbure membranaire via la reconnaissance du phospholipide de membrane plasmique PI (4,5) P2. Ce phospholipide est également spécifiquement reconnu par le domaine Matrix N-terminal de Gag et est un cofacteur lipidique du ciblage de Gag vers la membrane plasmique et de l'assemblage du VIH-1. Ce projet de recherche visait à caractériser les mécanismes cellulaires et moléculaires de l'implication de l'IRSp53 dans l'assemblage du VIH-1 dans les cellules T CD4 et HEK293T. Nos résultats montrent que l'IRSp53 est associé à des particules virales et que son renversement par l'ARNsi diminue la libération du VIH-1 dans les lymphocytes T et les cellules T HEK293. La microscopie électronique des cellules renversées pour IRSp53 a révélé un phénotype frappant de bourgeons viraux arrêtés à un stade précoce de l'assemblage. L'immunoprécipitation de l'IRSp53 a montré une conversion indépendante de p6 de HIV-1 Gag, indiquant une complexation intracellulaire de Gag et de l'IRSp53. Le fractionnement cellulaire et la flottation de la membrane ont montré que le recrutement de l'IRSp53 vers la membrane cellulaire double lors de l'expression du VIH-1 Gag. La microscopie PALM / STORM à molécule unique bicolore et les analyses ultérieures ont montré l'IRSp53 à proximité des amas Gag. Nous avons également trouvé une incorporation spécifique de l'IRSp53 dans les particules de VIH-1 Gag par rapport à d'autres protéines I-BAR, un phénomène dépendant de son domaine I-BAR. Comme le domaine I-BAR est impliqué dans la courbure de la membrane, nous avons ensuite sondé cet aspect de l'implication de l'IRSp53 dans l'assemblage du VIH-1. L'analyse des images de microscopie électronique a révélé un défaut de courbure pour les bourgeons des cellules assommées IRSp53. Des études concomitantes dans des systèmes GUV in vitro sans cellule ont également indiqué un rôle pour la courbure de la membrane induite par IRSp53 dans la liaison à la membrane Gag. Ces résultats confirment le rôle essentiel de l'IRSp53 dans les premiers stades de l'assemblage du VIH-1. Enfin, comme l'IRSp53 est un acteur essentiel dans l'échafaudage des protéines de signalisation de l'actine, nous avons établi un rôle pour le Rac1 activé dans le recrutement de la membrane IRSp53 en aval du VIH-1 Gag, et testé l'implication du RacGEF Tiam1 dans la production de particules du VIH-1. La microscopie à super résolution révèle également la présence de nanostructures d'actine sur les sites d'assemblage du VIH-1. Tous ces résultats mettent en évidence le rôle nouveau et essentiel de la protéine I-BAR à courbure membranaire IRSp53, et la mobilisation de l'actine corticale, dans les phases tardives de la réplication du VIH-1During the HIV assembly and budding, the plasma membrane undergoes a curvature driven by HIV-1 Gag self-assembly at the assembly site towards the exterior of the cell. However, the multimerization of Gag may not be sufficient and Gag may need to recruit cell factors for inducing local membrane curvature. We recently reported that the HIV-1 Gag particle release was dependent on the activation of the signalling pathway Rac1/IRSp53/Wave2/Arp3 in Jurkat T cells and primary blood lymphocytes. This cellular complex, when activated, is recruited to the cell plasma membrane and promotes the recruitment of actin branching, actin polymerisation and membrane remodelling in lamellipodia. In particular, the protein IRSp53 contains an I-BAR domain capable of inducing membrane curvature via the recognition of the plasma membrane phospholipide PI(4,5)P2. This phospholipid is also specifically recognized by the N-terminal Matrix domain of Gag and is a lipidic cofactor of Gag targeting to the plasma membrane and of HIV-1 assembly. This research project aimed at characterizing the cellular and molecular mechanisms of IRSp53 involvement in HIV-1 Gag assembly in CD4 T cells and HEK293T cells. Our results show that IRSp53 is associated with viral particles and that its knockdown by siRNA decreases HIV-1 release in T lymphocytes and HEK293 T cells. Electron microscopy of cells knocked down for IRSp53 revealed a striking phenotype of viral buds arrested at an early stage of assembly. Immunoprecipitation of IRSp53 showed a p6 independent pulldown of HIV-1 Gag, indicating intracellular complexing of Gag and IRSp53. Cellular fractionation and membrane flotation showed that IRSp53 recruitment to cellular membrane doubles upon expression of HIV-1 Gag. Dual colour single molecule PALM/STORM microscopy and subsequent analyses showed IRSp53 in close proximity to Gag clusters. We also found specific incorporation of IRSp53 in HIV-1 Gag particles as compare to other I-BAR proteins, a phenomenon dependent on its IBAR domain. As the I-BAR domain is involved in membrane curvature, we then probed this aspect of IRSp53 involvement in HIV-1 assembly. Analysis of electron microscopy images revealed a curvature defect for buds from IRSp53 knocked out cells. Concomitant studies in cell free in vitro GUV systems also indicated a role for IRSp53 induced membrane curvature in Gag membrane binding. These results affirm the essential role of IRSp53 in the early stages of HIV-1 assembly. Finally as IRSp53 is a vital player in scaffolding actin signaling proteins, we established a role for activated Rac1 in IRSp53 membrane recruitment downstream of HIV-1 Gag, and test the involvement of the RacGEF Tiam1 in HIV-1 particle production. Super resolution microscopy also reveals the presence of actin nanostructures at HIV-1 assembly sites. All these results highlight the novel and essential role of the membrane curving I-BAR protein IRSp53, and the mobilization of cortical actin, in the late phases of HIV-1 replication
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Deye, Nicolas. "Cardiac Arrest-Induced Brain Injury : Diagnostic And Prognostic Values of Circulating Biomarkers." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC150.
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Le pronostic de l’arrêt cardiaque (AC) reste dramatique. Diagnostiquer sa cause rapidement et prédire précocement son pronostic ("pronostication") de manière fiable permettrait de mieux guider les traitements initiaux, en évitant de traiter futilement les patients avec faible probabilité d’évolution favorable ou à l’inverse de permettre d’intensifier le traitement de patients avec forte probabilité d’évolution favorable. Les biomarqueurs, dont l’utilité diagnostique et pronostique reste débattue, semblent actuellement insuffisamment sensibles et précis, surtout dans les 1ères heures après la reprise de l’activité circulatoire spontanée (RACS). Dans l’algorithme pronostique, seule la Neuron Specific Enolase (NSE) est validée après le 3ème jour post-AC et en 2ème intention. Notre première étude a montré que la valeur diagnostique des biomarqueurs "spécifiques" des lésions cérébrales en post-AC (protéine S100B : S100 et surtout NSE) était insuffisante, à l’admission en réanimation, pour étayer précisément le diagnostic de cause neurologique d’AC. Si la coronarographie précoce est l’outil diagnostique de référence de l’AC de probable cause cardiaque, les biomarqueurs ne peuvent remplacer le scanner cérébral pour diagnostiquer une cause neurologique d’AC. La deuxième étude a évalué, au 1er jour post-AC, S100 et NSE avec 2 témoins d’œdème cérébral proposés comme outils pronostiques : le diamètre de l’enveloppe du nerf optique (DENO) par échographie et le rapport de dédifférenciation substance grise / substance blanche (DSG/B) par scanner cérébral. Même si une relation directe ne peut être affirmée formellement entre ces paramètres, l’élargissement du DENO à J1 post-AC était corrélé aux lésions cérébrales, surtout l’œdème cérébral et les lésions neuronales suspectés sur l’élévation de la NSE (à l’admission et à J1) et la baisse de DSG/B. Si NSE, DSG/B et DENO à J1 étaient liés, S100, plus spécifique de la glie, n’était pas corrélée au DENO ni au DSG/B. NSE et S100 à l’admission, à J1 et J2 post-RACS et DENO à J1 étaient associées à la mortalité hospitalière. La troisième étude évaluait la valeur pronostique des biomarqueurs à la phase précoce de l’AC (NSE et S100 étant prélevées en médiane 220 min après la RASC). S100, réalisée en aveugle des cliniciens, était le biomarqueur le plus précis à l’admission en réanimation pour prédire correctement le pronostic défavorable à la sortie de l’hôpital et à 3 mois après AC, par rapport au lactate, pH et créatininémie, et surtout à la NSE. Les variations de S100 dans le temps permettaient d’affiner cette prédiction. S100 à l’admission était un facteur indépendant du pronostic défavorable à la sortie de l’hôpital, avec la durée sans massage cardiaque, le rythme initial non-choquable, le lactate initial et la présence de convulsion clinique. Selon les recommandations, la pronostication nécessite théoriquement d’être différée et multimodale, les biomarqueurs seuls n’étant pas recommandés, surtout précocement. Les biomarqueurs ne peuvent constituer une alternative, en comparaison à l’imagerie, pour l’aide diagnostique de la cause d’AC. A l’inverse, certains biomarqueurs comme la S100 après admission pourraient facilement et spécifiquement discriminer les AC ayant une certitude de pronostic défavorable. Associée à d’autres outils prédictifs clinico-radiologiques, la S100 pourrait être incorporée dans des algorithmes permettant de guider les thérapeutiques initiales. Une pronostication correcte précoce pourrait éviter des traitements invasifs inutiles, ou au contraire optimiser certaines thérapeutiques agressives. Le choix de méthodes recommandées et automatisées de contrôle ciblé de la température, très efficaces mais invasives et onéreuses, ou l’indication d’utiliser -ou pas- une assistance cardio-circulatoire extra-corporelle pourrait bénéficier d’une telle stratégie précoce de sélection des patientsOutcome of cardiac arrest (CA) remains dramatic. To quickly diagnose the cause of CA and establish a reliable outcome prediction (prognostication) as early as possible could help to guide initial treatments. It could avoid futile treatments in patients with low chance of survival or of good neurological recovery, or conversely allow treatment optimization in patients expected to have a high likelihood of good neurological outcome. Usefulness of biomarkers to guide clinicians in finding the CA diagnosis and helping prognostication is debated. Biomarkers are considered as not sensitive and accurate enough, especially within the first hours after return of spontaneous circulation (ROSC). Their use is only recommended in prognostication for Neuron Specific Enolase (NSE) as a second line tool and after the third day from CA. Our first study confirmed that biomarkers “specific” of brain injury (S100B protein: S100 and moreover NSE) cannot sufficiently discriminate the neurological cause of CA on ICU admission. If early coronary angiogram is the standard for diagnosing a probable cardiac cause of CA, biomarkers cannot replace brain computed-tomography (CT) in CA from a neurological cause. The second study evaluated, during the 1st day after ROSC, the link between biomarkers (S100 and NSE) and 2 surrogates of brain oedema recently proposed as outcome predictors: echography of the optic nerve sheath diameter (ONSD), and grey to white matter attenuation ratio (GWR) on brain CT-scan. Even though we cannot conclude on a definitive relationship between these parameters, ONSD enlargement at day 1 was associated with specific brain damage after CA, such as brain oedema and mostly axonal injuries, as reflected by increases in NSE (on admission and at day 1) and low GWR measurements. Whereas NSE, GWR and ONSD at day 1 were correlated, S100, which is more specific of glial injuries, did not reach significance. NSE and S100 on admission, at days 1 and 2 after ROSC, as well as ONSD at day 1, were associated with survival at hospital discharge. The third study evaluated the prognostic value of several biomarkers in the early phase after CA (NSE and S100 being sampled at median 220 min after ROSC). S100, blinded to physicians, was the biomarker with the best accuracy after ICU admission to correctly predict unfavourable outcome at hospital discharge and at 3 months after CA, compared with all other biomarkers such as lactate, pH, creatinine, and especially NSE. S100 variations during the first day after admission refined prognostication. Initial S100 was an early independent predictive factor associated with unfavourable outcome at hospital discharge, with the no-flow duration, initial lactate value, initial non-shockable rhythm, and the presence of clinical seizure. According to guidelines, prognostication theoretically needs to be delayed and multimodal, biomarkers alone not being recommended especially in the early phase after CA. Biomarkers cannot seem to be an alternative option compared to imaging to precisely diagnose the CA cause. By contrast, some biomarkers, such as S100 after admission, could easily and specifically discriminate CA patients with certainty of unfavourable outcome. Associated with other predictive tools (clinical or using imaging), biomarkers could interestingly be incorporated in early decisional algorithms to optimally guide initial therapies. This correct patient classification could help to avoid unuseful treatments versus to maximize aggressive therapies. The choice of recommended servo-controlled targeted temperature management devices, very efficient but invasive and expensive, or the indication -or not- of a cardio-circulatory assist device implementation should be guided in the early stage after ROSC using this simple strategy of patient selection
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Roopchand, Diana Elizabeth. "Human adenovirus E4orf4 protein induces premature mitotic arrest by a PP2A-dependent mechanism leading to cell death in Saccharomyces cerevisiae." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86049.
Full textAbstract:
The E4orf4 protein of human adenovirus kills transformed cancer cell lines by a p53-independent mechanism. Accumulating evidence suggests that the mechanism of E4orf4-induced death is distinct from apoptosis and occurs via some novel pathway. Previous studies have shown that E4orf4 can interact with the mammalian Balpha subunit of protein phosphatase 2A (PP2A) and that this interaction is important for E4orf4-induced cell death. As PP2A is highly conserved across eukaryotic species we chose to study the effects of E4orf4 in a genetically tractable organism, Saccharomyces cerevisiae, in an effort to elucidate the mechanism of E4orf4-mediated cell death. E4orf4 expression is lethal in yeast cells and this toxicity is dependent on E4orf4 having a functional interaction with Cdc55, the yeast homolog of Balpha. Through its interaction with the B regulatory subunit, which determines substrate specificity, E4orf4 may inhibit or promote the dephosphorylation of selected PP2A substrates.E4orf4 expression can induce high levels Clb2-Cdc28 activity and mitotic arrest in a Cdc55-dependent manner. Since E4orf4 targets only the Cdc55-containing pool of PP2A, the E4orf4-induced mitotic arrest suggests that PP2A-Cdc55 plays a direct role in regulating exit from mitosis. Two anaphase-promoting complexes (APCs) control mitotic exit, APCCdc20 and APCHct1. We find that E4orf4 induces premature APCCdc20 activity resulting in the premature degradation of Pds1 and Scc1 in a Cdc55-dependent manner. In contrast, E4orf4 did not induce APCHct1 as evidenced be the stability of its substrates, Cdc20, Clb2 and Cdc5 as well as the hyperphosphorylation of Hct1. E4orf4 prevents Cdc55-containing PP2A complexes from localizing normally, another mechanism by which E4orf4 may modulate PP2A activity towards substrates. We propose that E4orf4 promotes mitotic arrest by acting as a Cdc55-specific inhibitor of PP2A and that PP2A plays a role in controlling the timing of anaphase by regulating APCCdc20 activity.
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Guo, Yang. "Overexpression of Heat-Shock Protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through s-phase arrest and apoptosis." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-176979.
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Hassumani, Daniel O. "Expression of Growth Arrest and DNA Damage Protein 45-alpha (gadd45-alpha) and the CCAAT/enhancer binding protein-delta (C/EBP-delta) in Fishes Exposed to Heat and Hypoxia." Thesis, Portland State University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=1536122.
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The cellular stress response (CSR) is one of the most highly conserved mechanisms among all organisms. Cellular stress can be defined as damage or the threat of damage to proteins, macromolecules and/or DNA. The response to damage can involve cell cycle regulation, protein chaperoning, DNA repair or, if macromolecular damage is too severe, apoptotic mechanisms can be initiated. This thesis details experiments that were designed to examine the cellular response to non-lethal environmental stressors at the protein level, using two fish species as study models. Two proteins that can cause cell cycle arrest and apoptosis mechanisms were examined. Expression of the CCAAT enhancer binding protein-delta (C/EBP-δ) was examined in the zebrafish, Danio rerio, exposed to acute, non-lethal hypoxic conditions. While C/EBP-δ was expressed constitutively in control individuals during all time points, exposure to hypoxic conditions did not have a consistent significant effect on C/EBP-δ expression (two-way ANOVA, P>0.05) in zebrafish white muscle tissue. In a second study, the expression of the growth arrest and DNA damage 45-alpha protein (gadd45-α), a mediator of cell cycle arrest and perhaps apoptosis was examined in heat-stressed liver tissue of an extremely cold-adapted Antarctic fish, Trematomus bernacchii. Gadd45-α levels were higher in fish exposure to 2°C across all time points (one-way ANOVA; P<0.05). The findings in these two studies expand our understanding of the CSR and how two genes that are involved in cell cycle regulation respond to acute, non-lethal environmental stress.
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Hassumani, Daniel Omar. "Expression of Growth Arrest and DNA Damage Protein 45-alpha (gadd45-alpha) and the CCAAT/enhancer binding protein-delta (C/EBP-delta) in Fishes Exposed to Heat and Hypoxia." PDXScholar, 2013. https://pdxscholar.library.pdx.edu/open_access_etds/943.
Full textAbstract:
The cellular stress response (CSR) is one of the most highly conserved mechanisms among all organisms. Cellular stress can be defined as damage or the threat of damage to proteins, macromolecules and/or DNA. The response to damage can involve cell cycle regulation, protein chaperoning, DNA repair or, if macromolecular damage is too severe, apoptotic mechanisms can be initiated. This thesis details experiments that were designed to examine the cellular response to non-lethal environmental stressors at the protein level, using two fish species as study models. Two proteins that can cause cell cycle arrest and apoptosis mechanisms were examined. Expression of the CCAAT enhancer binding protein-delta (C/EBP-[delta]) was examined in the zebrafish, Danio rerio, exposed to acute, non-lethal hypoxic conditions. While C/EBP-[delta] was expressed constitutively in control individuals during all time points, exposure to hypoxic conditions did not have a consistent significant effect on C/EBP-[delta] expression (two-way ANOVA, P>0.05) in zebrafish white muscle tissue. In a second study, the expression of the growth arrest and DNA damage 45-alpha protein (gadd45-[alpha], a mediator of cell cycle arrest and perhaps apoptosis was examined in heat-stressed liver tissue of an extremely cold-adapted Antarctic fish, Trematomus bernacchii. Gadd45-[alpha] levels were higher in fish exposure to 2°C across all time points (one-way ANOVA; P
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Sercovich, Mark J. "Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr)-mediated cell cycle arrest : an analysis of current mechanistic models /." Download the thesis in PDF, 2006. http://www.lrc.usuhs.mil/dissertations/pdf/sercovich2006.pdf.
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Magnin, Florence Magnin Florence. "How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle : the multiple interactions of Rep78 with cellular regulatory, replication and rcombination proteins /." [S.l.] : [s.n.], 2009. http://library.epfl.ch/theses/?nr=4486.
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Brignole, Claudine. "The adenovirus E4orf4 protein induces G2 / M arrest and cell death by inhibiting PP2A phosphatase activity regulated by the B Alpha subunit /." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81604.
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The human adenovirus E4orf4 protein is toxic in human tumor cells and yeast. In both cases, interaction of E4orf4 with the Balpha/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) appears to be critical for cell killing; however, the effect of E4orf4 binding on PP2A is not known. Balpha is one of several B subunits that form holoenzymes with A and C subunits and regulate PP2A substrate specificity. In the present studies the effects of E4orf4 on PP2A activity was examined, as well as the role of PP2A in E4orf4-mediated cell death. Previously it was shown that binding of E4orf4 had little effect on the dephosphorylation of a universal peptide substrate by purified PP2A, however this study demonstrates that E4orf4 reduced PP2A activity to varying degrees against two substrates, phosphorylase a and histone H1, the latter known to represent a class of substrate against which Balpha-containing PP2A complexes are highly active. Expression of E4orf4 was also found to result in the accumulation of hyperphosphorylated forms of two PP2A substrates, 4EBP-1 and p70S6K (Balpha specific) in vivo, indicating a significant inhibition of PP2A activity against these substrates. The importance of such inhibition in cell killing by E4orf4 was supported by the fact that low levels of okadaic acid (OA) or expression of the heat stable I1 PP2A inhibitor, both of which inhibit PP2A activity globally, enhanced E4orf4-induced cell death. Furthermore, E4orf4 killing was reduced by overexpression of a Y307F mutant form of the PP2A catalytic Calpha subunit, which is insenstitive to downregulation by tyrosine phosphorylation. E4orf4 was found to induce a significant G2/M arrest, an effect that was enhanced both by treatment with OA, which alone at high levels has similar effects, and by expression of simian virus 40 small T antigen (SV40-ST), which binds to PP2A holoenzymes and preferentially replaces the Balpha subunit. These results suggest that E4orf4 induce
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Labasque, Marilyne. "La calmoduline, un partenaire du récepteur 2C de la sérotonine essentiel à la signalisation indépendante des protéines G et dépendante des arrestines du récepteur." Montpellier 1, 2008. http://www.theses.fr/2008MON1T016.
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Le récepteur 2C de la sérotonine (récepteur 5-HT2C) est un récepteur couplé aux protéines G (RCPG) principalement exprimé dans le système nerveux central qui joue un rôle majeur dans les états anxio-dépressifs. Il est également la cible de nombreux composés thérapeutiques incluant la plupart des classes d'antidépresseurs. Le récepteur 5-HT2C interagit avec de nombreuses protéines intracellulaires différentes des protéines G. Celles-ci incluent les arrestines et la calmoduline (CaM). J'ai démontré que l'activation de la cascade des MAP kinases ERK1,2 par le récepteur 5-HT2C était indépendante des protéines G auxquelles il est couplé mais requérait les β-arrestines1,2 et la CaM. L'association physique de la CaM, au domaine C-terminal (motif 1-10 localisé à la région juxtamembranaire) du récepteur 5-HT2C est essentielle à cette signalisation dépendante de la β-arrestine. Mes travaux démontrent également que la CaM favorise le recrutement de la β-arrestine au récepteur 5-HT2C pour former un complexe protéique incluant probablement le récepteur 5-HT2C, un dimère de CaM et la β-arrestine. Le rôle de la CaM dans la signalisation indépendante des protéines G a été établi non seulement dans un système d'expression hétérologue (cellules HEK-293) mais également dans des contextes cellulaires authentiques (neurones corticaux, cellules épithéliales choroïdiennes). La régulation de cette cascade par l'activité constitutive du récepteur et différentes classes d'antidépresseurs (tricycliques, tétracycliques, ISRS, dérivés du mCPP) a enfin été étudiée.
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Byrne, Tara. "The evaluation of the effects of the Mitotic Arrest Deficiency Protein 2 (MAD2) and the miR-433 microRNA on chemoresistance and cancer prognosis." Thesis, Queen's University Belfast, 2017. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725493.
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Chemoresistance is a major obstacle in the treatment of high-grade serous ovarian cancer (HGSOC) hence identification of molecular markers of chemoresistance is of crucial importance. The primary focus of this thesis is to investigate the prognostic potential of the miR-433 microRNA target, mitotic arrest deficiency 2 protein (MAD2). In order to do this, MAD2 expression was evaluated in three HGSOC cohorts and was found not to correlate with survival. An association between MAD2 expression in neoadjuvant treated HGSOC patients and survival was evident. MAD2 levels therefore may be of prognostic value in this subgroup of patients. We previously described miR-433 as a critical cell cycle regulator and mediator of cellular senescence. In an attempt to determine whether miR-433 infers a survival advantage under chemotherapeutic selective pressures, a miR-433 overexpressing cell line was developed. Here, we have shown that higher miR-433 expression was associated with an increased resistance to platinum, which was unrelated to loss of MAD2 expression. Moreover, cells which remained viable following exposure to high doses of chemotherapy expressed higher levels of miR-433. Our data suggests that chemotherapy may not be driving the transcriptional up-regulation of miR-433 but rather selecting a population of cells with high miR-433 expression that escape chemotherapeutic kill. IHC analysis by our group of the miR-433 target, histone deacetylase 6 (HDAC6), previously, confirmed that its expression was significantly associated with a decrease in overall survival in HGSOC patients. In this thesis, the inhibition of HDAC6 in HGSOC cells did not attenuate apoptotic responses to paclitaxel. Therefore we suggest that HDAC6 inhibitors should not be recommended in conjuction with paclitaxel for HGSOC treatment. In conclusion, miR-433 and its target proteins play an important role in chemoresistance in HGSOC and could be measured or manipulated for potential therapeutic benefit.