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1
Hirose, Masayuki. "Phosphorylation and recruitment of Syk by ITAM-based phosphorylation of tamalin". Kyoto University, 2004. http://hdl.handle.net/2433/145291.
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2
Napper, Scott. "Phosphorylation sites of HPr". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0020/NQ43518.pdf.
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3
Craig, Timothy James. "Phosphorylation of exocytotic proteins". Thesis, University of Liverpool, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406719.
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4
Ackerley, Steven. "Neurofilament transport and phosphorylation". Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289881.
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5
Cleverly, Karen Elizabeth. "Investigation of neurofilament phosphorylation". Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267652.
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6
Chaubey, Mark. "Phosphorylation of endocytic proteins". Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615671.
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7
Thurston, Barbara. "Protein Phosphorylation in Archaea". Diss., Virginia Tech, 1997. http://hdl.handle.net/10919/30617.
Testo completoAbstract (sommario):
Protein phosphorylation constitutes an important
mechanism for cellular regulation in both Eucarya and
Bacteria. All living organisms evolved from a common
progenitor; this implies that protein phosphorylation as
a means of regulation also exists in Archaea.
Previously, in the sulfur-dependent archaeon
Sulfolobus solfataricus a gene was cloned encoding a
protein-serine/threonine phosphatase that was similar
to eucaryal protein-serine/threonine phosphatases
type 1, 2A, and 2B. To identify protein phosphatases
in other archaeons, oligonucleotides encoding
conserved regions of eucaryal protein-serine/threonine
phosphatases were used in the polymerase chain
reaction to amplify genomic DNA from the
methanogenic archaeon Methanosarcina thermophila.
From the PCR reaction a fragment of DNA was
isolated that encoded a portion of a protein
phosphatase. Using this DNA fragment as a probe,
the entire phosphatase gene was isolated. The amino
acid sequence of the phosphatase encoded by this
gene displayed greater than 30% identity with
eucaryal protein-serine/threonine phosphatase type 1.
The gene encoding the Methanosarcina phosphatase
was expressed in Escherichia coli. The expressed
protein exhibited protein serine phosphatase activity
that was sensitive to inhibitors of eucaryal
phosphatases such as okadaic acid, microcystin,
calyculin, and tautomycin. In order to identify potential
endogenous substrates of archaeal
protein-serine/threonine phosphatases and kinases, a
study was initiated to characterize the most prominent
phosphoproteins in S. solfataricus. Cell extracts were
incubated with [g-32P] ATP, MgCl2, and MnCl2,
and the proteins in the extracts were separated by
SDS-PAGE. Autoradiography of the gels revealed
four prominent phosphoproteins with apparent
molecular masses of 35, 46, and 50 kDa. N-terminal
sequence analysis and enzymatic assays of the 35 kDa
phosphoprotein identified this phosphoprotein as the
a-subunit of succinyl-CoA synthetase. N-terminal
sequence analysis and enzymatic assays revealed that
the 50 kDa phosphoprotein was a hexosephosphate
mutase. Neither the 50 kDa nor the 35 kDa
phosphoprotein appeared to be the target of protein
kinases or phosphatases. Therefore, while
protein-serine phosphatases exist in Archaea, the
targets of these phosphatases have yet to be
determined.Ph. D.
8
Martins, Filipa de Sá. "Abeta dependent tau phosphorylation". Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7647.
Testo completoAbstract (sommario):
Mestrado em Biomedicina MolecularAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by the presence of two histopathological hallmarks: the extracellular amyloid plaques (APs) composed of beta-amyloid protein (Abeta) and intracellular neurofibrillary tangles (NFTs), containing hyperphosphorylated tau protein. Therefore, Abeta and tau are important molecules associated with AD and evidence suggests that Abeta may initiate the hyperphosphorylation of tau, which by disrupting neuronal network leads to the process of neurodegeneration. In the present study, using rat primary cortical and hippocampal neuronal cultures, it was shown that exposure to aggregated Abeta1-42 for prolonged periods decreased tau phosphorylation at Ser202 and Thr205 residue, but in contrast increased at Ser262 residue. Tau hyperphosphorylation in AD may be related to alterations in signal transduction pathways involving tau phosphorylation, such as an imbalance in the regulation of protein kinases (PKs) and protein phosphatases (PPs). Thus it is also important to determine which specific PKs and PPs are involved in this process. We observed the involvement of PP1 in the dephosphorylation of tau at Ser202 and Thr205, and the involvement of PP1 and PP2A at the Ser262 residue. An important aspect of tau metabolism are its binding proteins, and to date many such proteins have already been described both in vitro and in vivo. The interactome of tau is shaped by its phosphorylation and so is crucial to map the crosstalk between normal and pathologically hyperphosphorylated tau. By co-immunoprecipitation we intend to identify proteins that interact with tau and more specifically with phosphorylated tau (p-Tau). Furthermore the effect of Abeta on this interactome should be forthcoming, which is relevant for AD tau pathology.
A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada pela presença de duas características histopatológicas: as placas senis na matriz extracelular compostas por Beta-amilóide (Abeta) e as tranças neurofibrilhares intracelulares contendo proteína tau hiperfosforilada. Assim, o Abeta e a proteína tau são importantes moléculas associadas à DA e evidências sugerem que o Abeta possa mediar a hiperfosforilação da tau levando á disrupção da rede neuronal e consequentemente ao processo de neurodegeneração. No presente estudo, em culturas primárias neuronais de córtex e hipocampo de rato, verificou-se que a exposição a Abeta1-42 agregado por longos períodos diminui a fosforilação da tau nos resíduos Ser202 e Thr205 e, em contraste, aumenta a fosforilação no resíduo Ser262. Pensa-se que a hiperfosforilação da tau na DA pode estar relacionada com alterações nas vias de sinalização celular envolvidas no processo de fosforilação da tau, tais como alterações na regulação das cinases e das fosfatases. Deste modo, é também de extrema importância determinar as cinases e fosfatases envolvidas neste processo. Por tratamento de neurónios corticais com diferentes concentrações de ácido ocadéico (AO), um inibidor das fosfatases, verificamos o envolvimento da PP1 na desfosforilação da tau nos resíduos Ser202 e Thr205, bem como o envolvimento da PP1 e PP2A na desfosforilação do resíduo Ser262. Um outro aspecto importante do metabolismo da tau são as proteínas de ligação, e actualmente já foram descritas várias proteínas que interagem com a tau in vitro e in vivo. O interactoma da tau é regulado pela sua fosforilação e portanto é crucial estabelecer uma relação entre a tau normal e a tau patológica hiperfosforilada no que diz respeito às proteínas de ligação. Por co-imunoprecipitação de neurónios corticais pretendemos identificar proteínas de ligação à tau e especificamente à tau fosforilada, e ainda avaliar o efeito do Abeta neste interactoma. O interactoma da tau dependente da fosforilação e do Abeta é de particular relevância para a compreensão da DA.
9
Rardin, Matthew James. "Reversible phosphorylation in mitochondria". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3331484.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed Dec. 16, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
10
Wang, Huachun. "Protein phosphorylation regulation in Arabidopsis". Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/5896.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on July 18, 2008) Vita. Includes bibliographical references.
11
Carr, M. D. "NMR studies of oxidative phosphorylation". Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382584.
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12
Wells, Nicholas James. "Phosphorylation of human topoisomerase II". Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318833.
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13
Russell, Elinor Kate. "Engineering phosphorylation responsive peptide complexes". Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515786.
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14
Morris, Lorna Josephine. "Regulation of E2F by phosphorylation". Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398675.
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15
Selitsianos, Dimitrios. "Approaches to asymmetric chemical phosphorylation". Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413042.
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Turner, A. M. "Protein phosphorylation in Rhodomicrobium vannielii". Thesis, University of Warwick, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383358.
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Thornhill, Paul. "Neurofilament phosphorylation and axonal transport". Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272216.
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Davis, Daniel Robert. "Modulation of neuronal tau phosphorylation". Thesis, King's College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299450.
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19
Kousar, Rehana. "Regulation of eIF2B by phosphorylation". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/regulation-of-eif2b-by-phosphorylation(82439f9a-9ed1-4708-a87f-f0bcc6f4b64e).html.
Testo completoAbstract (sommario):
The ability to sense and respond to environmental cues is crucial for the survival of all organisms. This response is often manifested by exerting control at different levels of gene expression, i.e. transcription, translation and post translation levels. Global control of protein synthesis is frequently exercised at the initial step of translation initiation and is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. The formation of ternary complex (TC) is considered first step of translation initiation and depends on the recycling of inactive eIF2-GDP to active eIF2-GTP form. This nucleotide exchange reaction is catalyzed by the eukaryotic initiation factor-2B (eIF2B). eIF2B is composed of a regulatory sub-complex of alphaβdelta subunits and a catalytic sub-complex of the γε subunits. The guanine nucleotide exchange activity of eIF2B is regulated by phosphorylation of eIF2alpha and additionally in mammalian cells, by direct phosphorylation of eIF2B at multiple sites in ε subunit, where most of the catalytic activity of eIF2B resides. Recent unpublished studies in the Pavitt laboratory identified novel phosphorylation sites by Mass Spectrometry in γ and ε subunits of eIF2B catalytic sub-complex. In order to study the functional significance of these phospho-sites for translation initiation, Site Directed Mutagenesis (SDM) was performed to generate Ser to Ala mutants. All mutations are viable and have no significant growth defect on rich or minimal media; however the significance of these sites in yeast growth became apparent by growing yeast in different stress conditions (e.g. Rapamycin, Torin1, amino acid starvation and 1-butanol). Effects on the phosphorylation pattern at these sites were monitored by using custom generated phospho-specific antibodies. All phosphorylation events appear independent of the eIF2alpha kinase (Gcn2p in yeast). The phosphorylation of ε-S528 depends on the presence of ε-S525. This study finds that addition of rapamycin, Torin1, amino acid starvation and butanol, which each inhibits global translation initiation, alters the phosphorylation pattern at ε-S435, ε-S525 and ε-S528 sites. Linking growth to phosphorylation, it appears that phosphorylation at ε-S435 and ε-S525 is directly proportional to growth. Phosphorylation of ε-S435 is necessary for effect of eIF2alpha-Ser51 phosphorylation on protein synthesis while phosphorylation of ε-S528 seems to be a target of various mechanisms. This study also suggests that eIF2Bε may be a key player of the cell cycle progression and phosphorylation changes can serve as marker for the regulation of eIF2B activity. The kinases responsible for phosphorylation at these sites are not yet known in yeast. Further investigation is required to find the functional significance of alterations in phosphorylation pattern to definitively establish eIF2Bε phosphorylation as a mechanism for regulating eIF2B activity in yeast. Models are presented to account for the results obtained that show how phosphorylation of eIF2Bε at these sites may contribute to the control of protein synthesis.
20
Zhao, Y. "Pot1 phosphorylation regulates telomere function". Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1380712/.
Testo completoAbstract (sommario):
The telomere is a conserved nucleoprotein structure at the ends of eukaryotic chromosomes. It is essential for maintenance of genomic stability: on the one hand, it suppresses DNA damage response and protects the natural chromosome ends from repair activities; on the other hand, it recruits telomerase, the specialized reverse transcriptase, to counteract the end-replication problem. The telomeric G-strand ssDNA-binding protein Pot1 plays a crucial role in both of these functions. In fission yeast S. pombe, inhibition of Pot1 induces rampant 5’ resection and loss of telomere signal in a single cell cycle. It was recently shown that spPot1 interacts with, and is phosphorylated by, the master cell cycle regulator DDK. Alleles of a V5-tagged version of pot1+ were constructed with mutations at the putative phosphorylation sites, which reside in the N-terminal OB-fold DNA binding domain of Pot1 {Kuznetsov, 2008 #6380}. The goal of this study was to determine the molecular mechanism by which phosphorylation of Pot1 regulates telomere function. We found that the phospho-deficient mutants of Pot1 induce telomere elongation, checkpoint activation, and deregulation of ssDNA generation, suggesting reduced association with the ssDNA. Our data point to a model in which cell cycle-regulated Pot1 phosphorylation coordinates telomere replication and telomerase activity in different cell cycle phases. Furthermore, we showed that the C-terminal V5-tagging of Pot1 also affects its functions, suggesting an additional layer of complexity governing Pot1 function.
21
Gonçalves, Joana Araújo Monteiro Antão. "Multiple phosphorylation of Histatin 1". Master's thesis, Universidade de Aveiro, 2005. http://hdl.handle.net/10773/4726.
Testo completoAbstract (sommario):
Mestrado em Métodos Biomoleculares AvançadosAs histatinas pertencem a uma família de peptídeos básicos ricos em histidinas que se encontram na saliva humana (Oppenheim et al., 1988). As principais histatinas, designadas histatinas 1 e 3, são codificadas pelos genes HTN1 e HTN2, localizados no cromossoma 4q13 (Sabatini et al., 1993). A histatina 3 é submetida a uma fragmentação pré-secretória, presumivelmente pela acção de uma convertase furin-like, e cria pelo menos 24 diferentes histatinas menores. Pelo contrário, a histatina 1, um peptídeo de 38 resíduos de amino ácidos, que comparte largamente a sua sequência com a histatina 3, não é submetida a fragmentação, provavelmente devido à ausência da sequência consenso da convertase (Castagnola et al., 2004). A histatina 3 e alguns dos seus fragmentos, em particular a histatina 5, têm propriedades antifungícas e antimicrobianas potentes, enquanto que o papel da histatina 1 ainda não foi bem estabelecido. Este estudo descreve os resultados obtidos na caracterização estrutural dos derivados poli-fosforilados da histatina 1 detectados por HPLC-ESI MS e MALDI-TOF-MS na saliva humana.
Histatins belong to a family of basic histidine-rich peptides found in human saliva (Oppenheim et al., 1988). The major histatins, namely histatins 1 and 3, are codified by the genes HTN1 and HTN2, localized on chromosome 4q13 (Sabatini et al., 1993). Histatin 3 is submitted to a pre-secretory fragmentation, presumably by the action of a furin-like convertase, and it generates at least 24 different minor histatins. On the contrary, histatin 1, a peptide of 38 amino acid residues, largely sharing its sequence with histatin 3, is not submitted to fragmentation, probably due to the lack of the convertase consensus sequence Castagnola et al., 2004). Histatin 3 and some of its fragments, particularly histatin 5, show powerful antifungal and antimicrobial properties, whereas the role of histatin 1 has not been established. This study describes the results on the structural characterization of poly-phosphorylated derivatives of histatin 1 detected by HPLC-ESI MS and MALDI-TOF-MS in human saliva.
22
Quezada, Cindy Maria Richards John Gray Harry B. "Histidine phosphorylation in bacterial chemotaxis /". Diss., Pasadena, Calif. : California Institute of Technology, 2003. http://resolver.caltech.edu/CaltechETD:etd-05302003-145522.
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Nicoll, James B. "TR Phosphorylation & Nuclear Import". W&M ScholarWorks, 2001. https://scholarworks.wm.edu/etd/1539626303.
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Nadeau, Robert J. "Sprouty Regulation of Tyrosine Kinase Signal Transduction is Governed by Tyrosine Phosphorylation: A Functional Role for Sprouty2 N- and C- Terminal Tyrosines". Fogler Library, University of Maine, 2006. http://www.library.umaine.edu/theses/pdf/NadeauRJ2006.pdf.
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Groslambert, Marine. "Etude de l'impact fonctionnel des modifications post-traductionnelles dans l'activation de l'inflammasome NLRP3". Thesis, Lyon, 2019. http://www.theses.fr/2019LYSEN022.
Testo completoAbstract (sommario):
L'inflammation est un processus déclenché suite à la détection de pathogènes et de dommages tissulaires. Elle conduit à la sécrétion de cytokines pro-inflammatoires par les cellules immunitaires innées ainsi qu'au déclenchement de la pyroptose, mort cellulaire pro-inflammatoire. NLRP3 est une protéine senseur de stress cellulaire régulant le déclenchement de ces processus via la formation d'une plateforme multiprotéique appelée inflammasome. L'activation non contrôllée de NLRP3 conduit au développement d'une maladie auto-inflammatoire appelée CAPS (Cryopyrin associated periodic syndrome). De plus, l'inflammasome est impliqué dans le développement et la sévérité des symptômes de nombreuses maladies multifactorielles (diabète de type 2, athérosclérose, maladie de Parkinson et d'Alzheimer, sclérose en plaques, cancers...). Les mécanismes régulant l'activation de NLRP3 ne sont pas encore compris, mais les modifications post-traductionnelles de NLRP3 sont impliquées dans ce processus. Notre laboratoire a identifié différents sites d'ubiquitination et de phosphorylation sur NLRP3 par des approches biochimiques. Via la création de lignées cellulaires NLRP3 knock out reconstituées pour exprimer NLRP3 muté sur les résidus précédemment identifiés et de souris NLRP3 knock-in par la technique de CRISPR/Cas9, le travail de thèse a consisté en l'étude de l'impact fonctionnel de ces modifications. Ces résultats montrent que les substitutions de deux lysines identifiées comme étant ubiquitinées conduisent à une dérégulation de l’activation de l'inflammasome NLRP3 dans les cellules primaires. Un nouveau point de contrôle de l'activation de NLRP3 a ainsi pu être mis en lumièreInflammation is triggered after the sensing of pathogens or tissue damages. This process leads to the secretion of pro-inflammatory cytokines by innate immune cells and to the triggering of a pro-inflammatory form of cell death called pyroptosis. NLRP3 protein is a sensor of cellular stress and regulates the triggering of these events through the formation of a multiproteic platform called inflammasome. NLRP3 activation has to be tightly controlled as its deregulation leads to the development of an auto-inflammatory disease called CAPS (Cryopyrin associated periodic syndrome). Moreover, NLRP3 inflammasome is associated with the development and the severity of numerous multifactorial diseases (type 2 diabetes, atherosclerosis, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, cancers…). The mechanisms involved in the regulation of NLRP3 activation are not fully understood. Recently, post-translational modifications of NLRP3 were shown to be important for the regulation of NLRP3 activation. Our lab has identified several phosphorylation and ubiquitination sites on this protein through biochemical studies. This phD work aims to identify the functional impact of these modifications. Thus, the generation of reconstituted cell lines expressing NLRP3 mutated on the previously identified residues and the generation of NLRP3 knock in mice via CRISPR/Cas9 technology were performed. The results show that substitution of two lysine residues previously identified as ubiquitinated leads to the deregulation of NLRP3 inflammasome activation in primary cells. This work highlights a new point of control in NLRP3 activation
26
Koss, David John. "Phosphorylation based Models of Neurodegenerative Diseases". Thesis, University of Aberdeen, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485668.
Testo completoAbstract (sommario):
Aberrant protein phosphorylation is a hallmark of neurodegenerative diseases, such as Alzheimer's disease (AD) and tauopathies. Pathological phosphorylation is commonly observed as intracellular tau containing deposits correlating with neuronal death. Tau is a cytoskeletal protein, key to the stabilisation of the microtubules (MT), important ,for structural integrity and protein trafficking. Tau's ability to promote MTs is regulated by protein phosphorylation by kinases/phosphatases. In spontaneous AD, the activity/expression of kinases are enhanced w$ilst phosphatases are decreased. Alterations in phosphorylation have significant regulatory effects on neuronal signalling which may lead to deficits in neuronal signalling prior to neuronal death. Using cultured rat hippocampal neurons and pharmacological inhibitors of serine/threonine (Okadaic acid and Cantharidin) and tyrosine (Sodium orthovanadate) phosphatases, the consequences of increased phosphorylation for neuronal signalling were investigated. Using Fura-2 Ca2+ imaging, serine/threonine phosphatase inhibition was determined to exert bidirectional modulation on neuronal excitability. Low level short duration inhibition enhanced, whilst str9nger or more prolonged inhibition suppressed Ca2 + responses towards stimulation. Suppression of excitability was coincident with' a reduction in receptor expression and an increase in cytoskeletal phosphorylation. The data mimic changes in signalling utilising alternative means of modelling AD, suggesting that common ' phosphorylation cascades may be active within these models and in the disease process. Acute inhibition of tyrosine phosphatases evoked a prolonged Ca2 + response dependent on the activation of trans-plasma membrane Ca2+ channels. Prolonged inhibition of tyrosine phosphatases enhanced neuronal excitability. A novel sponge toxin polymeric 1,3-alkylpyridinium salts was employed as an agent for inducing tau over-expression in cultured neurons and further Ca2 + imaging studies conducted. Modulation on excitability was observed to be dependent on the level oftau expression. Overall, this thesis furthers the understanding of phospho-regulation of neuronal signalling ~oth physiologically, and pathologically and provides preliminary data for novel roles of tau in neuronal signalling regulation.
27
Javad, Safaei Mehranpour. "Modelling human cell protein phosphorylation networks". Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52983.
Testo completoAbstract (sommario):
Defects in cell signalling networks are linked to over 400 human diseases. My thesis research aimed to model these networks in more detail to facilitate understanding of their architecture and operations under normal and pathological conditions. The various protein levels in diverse normal human cell and tissue types were inferred from their mRNA expressions, and their up/down-regulation was also investigated in about 300 human cancer cell lines and 50 types of human cancers. This was based on meta-analyses of gene microarray measurements deposited in the USA National Center for Biotechnology Information's Gene Expression Omnibus database. I identified proteins that were commonly or uniquely expressed in normal and cancerous human cells and tissues. The co-expression patterns of proteins were used to predict potential interactions, but there was not a strong correlation between high co-expression and actual direct protein-protein interactions documented in the scientific literature. With respect to the post-translational regulation of proteins, my research efforts primarily targeted protein phosphorylation, which is the most predominant type of reversible covalent modification of proteins. Complex protein phosphorylation networks emerge through the interplay of protein kinases, protein phosphatases, phosphorylation site-dependent binding proteins, and their phosphoprotein substrates. I modelled the interactions of protein kinases with substrate proteins and inhibitory compounds. Nearly a million human phosphosites were predicted, and each of these was tested in silico as substrates for 500 human protein kinases. The interactions of over 550 known protein kinase inhibitory drugs with the 500 protein kinases were also tested. These predicted interactions were compared with empirical data from other on-line protein-protein and protein-drug interaction databases. The human phosphosites were also analysed with respect to their protein conservation in over 20 other diverse species, and it was found that threonine phosphosites in protein kinases that were activatory were particularly well conserved in evolution. Finally, probabilistic graphical models were developed to model the most probable structure of substrate phosphosites for specific protein kinases. The discussed probabilistic graphical model, gave more theory justifications for the protein-protein interaction modelling that was presented in the earlier parts of the thesis.Science, Faculty of
Computer Science, Department of
Graduate
28
Eijsden, Rudy Gerardus Elisabeth van. "Microarray analysis of oxidative phosphorylation disorders". [Maastricht] : Maastricht : Maastricht University ; University Library, Universiteit Maastricht [host], 2008. http://arno.unimaas.nl/show.cgi?fid=10708.
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Cumming, D. V. E. "Nuclear protein phosphorylation in rat liver". Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375227.
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Sapountzi, Vasileia. "Study of TIP60 regulation by phosphorylation". Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424155.
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Carter, Simon Mark. "Phosphorylation of the cardiac ryanodine receptor". Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432739.
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Patek, Samantha Clare. "Androgen receptor phosphorylation in prostate cancer". Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/38914/.
Testo completoAbstract (sommario):
Prostate cancer is the most common male cancer in the UK. Although incidence is increasing, prostate cancer mortality is decreasing, mainly owing to the over diagnosis of disease that would not have become clinically apparent during the patient’s lifetime. The gold-standard for prostate cancer diagnosis is transrectal ultrasound guided biopsy of the prostate. Whilst prostate biopsy can inform on diagnosis, it’s prognostic ultiltiy is poor. Currently clinicians lack pathological biomarkers to differentiate between patients with prostate cancer who have indolent disease that can be safely managed with surveillance strategies, and those who will go onto develop aggressive disease which requires early radical curative treatment. Phosphorylation of the androgen receptor has been extensively investigated in relation to prostate cancer development and progression. Androgen receptor phosphorylation has been shown to regulate cellular localisation, transcriptional activity, cell growth and sensitivity to androgens in prostate cancer. However, only a small number of studies have investigated the prognostic significance of androgen receptor phosphorylation, and only consider a limited number of serine residues in clinical specimens. The research presented in this thesis sought to investigate the prognostic and predictive significance of AR phosphorylation at serine 578 in hormone-naïve prostate cancer. It was hypothesised that pARS578 would be associated with poor outcomes in prostate cancer and may be utilised as a prognostic marker at diagnosis in prostate cancer and predict response to drug treatment with a PKC inhibitor. It was also hypothesised that PKC, the putative kinase for phosphorylation at serine 578, would be associated with poor outcomes and may offer a potential therapeutic target in prostate cancer. In the current study, the phosphorylation site of primary interest was serine 578. Scansite 2.0, an online kinase search tool, predicted that PKC is the putative kinase mediating phosphorylation at serine 578 on the androgen receptor. Phosphorylation of the androgen receptor at serine 578 has been linked with increased AR transcriptional activity, cell growth, nuclear cytoplasmic shuttling, modulation of other AR phosphorylation sites and DNA-repair mechanisms. The prognostic significance of androgen receptor phosphorylation at serine 81 was also investigated in this study. Serine 81 is phosphorylated in response to DHT via an alternative pathway to that of serine 578. Serine 81 phosphorylation is associated with increased androgen receptor transcriptional activity and increased cell growth in prostate cancer. It was therefore hypothesised that androgen receptor phosphorylation at serine 578 and serine 81 would be associated with poor outcome measures in prostate cancer. Immunohistochemical analysis was performed in a cohort of 105 hormone-naïve prostate cancer patients undergoing active surveillance, representing a cohort of patients with low-risk disease, as defined by current clinical markers such as PSA and Gleason score at diagnosis. Nuclear PKC expression was significantly associated with pARS578 expression in the clinical specimens, supporting the prediction of Scnasite 2.0 that PKC is the kinase responsible for phosphorylation of the AR at this site. High cytoplasmic expression of pARS81 was associated with decreased time to intervention (HR 2.76 (95% CI 1.1-7.3), p=0.032). There was no association between pARS578 and time to intervention in this cohort. Analysis of combined expression of both phosphorylation sites revealed an association between high dual expression of cytoplasmic pARS81 and cytoplasmic pARS578 and decreased time to treatment intervention (HR 2.35 (95% CI 1.2-4.6), p=0.031). These results suggest a synergistic prognostic effect when these two phosphorylation sites are combined and identifies a sub-population of low-risk prostate cancer patients who are at increased risk of disease progression. A second study was conducted to investigate if these results could be replicated in a cohort of prostate cancer patients with all stages of disease at diagnosis. Immunohistochemical analysis in 90 hormone-naïve prostate cancer patients found that high expression of nuclear pARS81 (HR 2.1 (95% CI 1.1 – 4.2), p=0.030), nuclear pARS578 (HR 2.24 (95% CI 1.0-4.9), p=0.036) and cytoplasmic pARS578 (HR 4.54 (95% CI 2.0-10.4), p= < 0.001) was associated with decreased disease survival. Furthermore, high expression of cytoplasmic pARS578 was associated with decreased time to biochemical relapse (HR 2.1 (95% CI 1.0-4.2), p=0.034) and decreased disease-specific survival following biochemical relapse (HR 3.2 (95% CI 1.0-9.9), p=0.034). Dual expression of nuclear, cytoplasmic and total pARS81 and pARS578 were all associated with decreased-disease specific survival, suggesting that there is a sub-population of prostate cancer patients who may benefit from dual targeted therapy with androgen deprivation therapy and PKC inhibitors. A validation cohort of 243 hormone-naïve prostate cancer patients with all stages of disease was utilised to verify the results of the second cohort. Unfortunately, due to technical issues and time constraints, IHC could not be completed for the phosphorylation sites of interest in all patients. Despite this, high expression of cytoplasmic pARS578 was significantly associated with decreased time to biochemical relapse (HR 2.9 (95% CI 1.0-8.2), p=0.037) and trended towards an association with decreased overall survival (p=0.076). Interestingly, dual expression of high cytoplasmic pARS81 and cytoplasmic pARS578 was associated with decreased overall survival (HR 2.1 (95% CI 1.3-3.3) p=0.001) despite neither phosphorylation site independently predicting decreased overall survival. Lastly, a study to develop a technique for isolation, propagation and characterisation of primary prostate cancer cells from TRUS biopsy specimens was undertaken. Two primary prostate cell cultures were developed which were confirmed to have a malignant luminal epithelial cell phenotype with a functional AR using flow cytometry, RT-PCR and immunofluorescence. This technique is of high translational relevance, as it provides a model with potential to identify biomarkers to predict individual patient’s response to prostate cancer therapies. Overall these results suggest that androgen receptor phosphorylated at serine 81 and serine 578 are associated with poor outcomes in prostate cancer and are potential targets for new drug therapies. Additional studies are required to validate these results in a larger multi-centre cohort of prostate cancer patients before either of these phosphorylation sites can be utilised as a biomarker in clinical practice.
33
Hauser, Anett. "Chemical Approaches to Elucidate Lysine Phosphorylation". Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22287.
Testo completoAbstract (sommario):
Reversible Phosphorylierung ist die bekannteste posttranslationale Modifikation (PTM) und die O-Phosphomonoester von Serin, Threonin und Tyrosin galten lange als die einzigen relevanten Vertreter. Vor kurzem wurden erste Erkenntnisse über die biologische Relevanz von labilen Phosphorylierungen veröffentlicht, z.B. die Phosphorylierung von Histidin, Arginin und Cystein sowie die Pyrophosphorylierung von Serin und Threonin. Auch die Aufklärung von Phospho-Lysin (pLys) wurde mit der Etablierung einer chemoselektiven Synthese zur Darstellung ortsselektiv phosphorylierter Lysinpeptide und der Entwicklung massenspektrometrischer Protokolle zur eindeutigen pLys-Identifizierung in Angriff genommen. Dennoch wurde bisher kein endogenes pLys beschrieben oder eingehende Untersuchungen mit interagierenden Enzymen durchgeführt.
In dieser Arbeit werden mehrere Ansätze zur Erweiterung des Wissens über pLys vorgestellt. Dazu gehören das Design einer alternativen Syntheseroute zu pLys-Peptiden und die Entwicklung sowie Evaluierung von zwei stabilen Analoga als Bausteine für die Peptidsynthese. Weiterhin wurde die Protonierung des Phosphoramidatstickstoffs untersucht. In systematischen Enzymaktivitätsassays wurden die Wechselwirkungen zwischen Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) und verschiedenen Phospho-Substraten untersucht. Dabei zeigte sich eine ausgeprägte Selektivität für pLys, eine hohe Sequenzabhängigkeit der LHPP-Aktivität und ein klares Bindungsmotiv. Darüber hinaus wurden proteomische Methoden hinsichtlich ihrer Eignung für pLys-Peptide evaluiert. Im Laufe dieser Untersuchung wurden mehrere pLys-Immunogene für die Generierung von monoklonalen anti-pLys-Antikörpern und ein Workflow für die Histontrennung und -analyse entwickelt. Des Weiteren wurde die chelationsverstärkte Fluoreszenz von markierten Phospho-Peptiden als Werkzeug zur Bestimmung des Phosphorylierungsgrades in Enzymaktivitäts- oder Stabilitätsassays untersucht.Reversible phosphorylation is the most prominent post-translational modification (PTM) and the O-phosphomonoesters of serine, threonine and tyrosine have been considered as the only notable forms for long time. Recent developments have paved the way to insights into the biological relevance of labile phosphorylations, e.g. phosphorylation of histidine, arginine and cysteine as well as pyrophosphorylation of serine and threonine. Also, the elucidation of phospho-lysine (pLys) was tackled with the establishment of a chemoselective synthesis to obtain site-selectively phosphorylated lysine peptides and the development of mass spectrometric protocols to unambiguously identify modification sites. Nonetheless, no endogenous pLys site has been described or in-depth investigations of interacting enzymes have been conducted. In this thesis, several approaches to enhance the knowledge about pLys are introduced. These include the design of an alternative synthesis route to pLys peptides and the development as well as evaluation of two stable analogues as building blocks for peptide synthesis. Furthermore, the protonation of the phosphoramidate-nitrogen was investigated. In systematic phosphoramidate hydrolase and phosphatase activity assays, the interactions between phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) and various phospho-substrates were examined. Thereby, distinct selectivity for pLys, high sequence dependence of LHPP activity and a distinct binding motif were revealed. In addition to that, proteomic methods were evaluated regarding their suitability for pLys peptides. Over the course of this investigation, several pLys immunogens for the generation of monoclonal anti-pLys antibodies and a workflow for histone separation and analysis were developed. Furthermore, chelation-enhanced fluorescence of labeled phospho-peptides was studied as a tool for determining the degree of phosphorylation in enzyme activity or stability assays.
34
Landais, Jean-Christophe. "La Phosphorylation du collagène de l'os". Paris 6, 1991. http://www.theses.fr/1991PA066190.
Testo completoAbstract (sommario):
La mise en évidence de résidus d'acide gamma-phosphoglutamique (P-GLU) liés spécifiquement ou situés dans la chapine alpha 2 du collagène de type i de tissus minéralisés a permis de penser que ces résidus pouvaient être des sites de fixation du calcium et par suite d'initiation de la minéralisation observée dans des sites spécifiques des fibres de collagène. Nous avons essaye de localiser le p-glu dans le collagène extrait de l'os et dans le collagène synthétise par des ostéoblastes en culture de cellules. Les résultats obtenus sont contradictoires. Dans certains cas, nous observons une localisation très spécifique et dans d'autres cas, le p-glu est distribue tout le long des 2 chaines de collagène. Nous en avons aussi trouve dans une fraction peptidique soluble, de faible poids moléculaire et de caractère anionique. Nous en avons conclu que le p-glu pourrait être dans un petit peptide ayant une forte affinité pour des sites spécifiques de la chaine 2. Dans certaines conditions expérimentales, d'autres interactions non spécifiques pourraient se produire entre ce peptide et les 2 chaines de collagène
35
Dreier, Megan Renee. "The Regulation of Sororin by Phosphorylation". University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1333736093.
Testo completo
36
DELATTRE, DELPHINE. "Phosphorylation des proteines chez bacillus subtilis". Paris 7, 2000. http://www.theses.fr/2000PA077059.
Testo completoAbstract (sommario):
Les etudes concernant les phenomenes de phosphorylation des proteines sur les residus ser/thr ou tyr ont montre l'importance de ce mode de regulation chez les eucaryotes. L'objectif de cette these etait d'etudier la phosphorylation de proteines sur ces residus chez bacillus subtilis. Tout d'abord, une centaine de proteines phosphorylees ont ete detectees apres marquage in vivo et separation par electrophorese 2-d ( 3 2p). Certaines d'entre elles ont ete identifiees et les genes correspondants clones. Les proteines, fusionnees a un histag, ont ete surproduites et purifiees. Parmi elles, l'alkyl-hydroperoxyde-reductase, ahpc, impliquee dans la reponse au stress oxydant, a attire notre attention. Cette proteine fait partie des peroxyredoxines a 2 cysteines, son activite enzymatique requiert la formation de ponts disulfures intermoleculaires. Nous montrons ici pour la premiere fois que cette proteine est phosphorylee in vivo. En effet, le spot marque est reconnu par les anticorps anti-ahpc et est absent de la souche portant le gene interrompu (ahpc :: tn10). La proteine 6his-ahpc est phosphorylee in vitro en presence d'extraits proteiques de b. Subtilis et a ensuite ete purifiee. Une analyse de la stabilite de la liaison phosphate a differents agents a permis d'exclure une phosphorylation sur un residu ser, thr, arg, his, lys, asp ou glu. Nos resultats indiquent que les cysteines jouent un role important dans la phosphorylation de ahpc. En effet, la liaison phosphate est labile en presence d'agents reducteurs. Chez b. Subtilis, ahpc comporte 3 cysteines, c37, c47 et c166. L'analyse par phosphorylation in vitro des proteines mutees montre que la cysteine c37 n'intervient pas dans la phosphorylation de 6his-ahpc. Par contre, c47
37
Oliveira, Joana Machado de. "Abnormal protein phosphorylation in Alzheimer's disease". Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7388.
Testo completoAbstract (sommario):
Mestrado em Biomedicina MolecularAD is a neurodegenerative disorder neuropathologically characterized by the presence of senile plaques, neurofibrillary tangles and synaptic loss. The Aβ peptide, the major constituent of senile plaques, is a key player in AD pathology since increased Aβ production and aggregation was associated with neurotoxicity, activation of inflammatory response, and apoptotic cascades. These processes are associated with neuronal death, neurodegeneration and consequently gradual cognitive decline. Altered signal transduction is also thought to be one of the key aspects in AD pathology. Protein phosphorylation is recognized as a fundamental mechanism by which the regulation of key intracellular events is achieved. Several studies have reported abnormal protein kinase and protein phosphatase activities in AD brains as well as abnormal phosphorylation levels of APP and Tau proteins. Further, phosphorylation is one of the mechanisms that regulates APP function and processing. APP is a phospho-specific protein also described as being hyperphosphorylated in AD brains. Due to the key role played by A and abnormal phosphorylation in AD pathology, the aim of this study was to analyze de Aβ effects on APP phosphorylation at Thr668 and Tyr682 as well on protein phosphorylation in general. In this work we could observe an increase in the phosphorylation level of APP at these specific residues upon Aβ exposure at low concentrations. The phosphatase involved in dephosphorylating the above mentioned residue (Thr668) was found to be protein phosphatase 1. Additionally, it was also observed that both Aβ and APP phosphorylation at Thr668 can regulate APP interactions. The phosphoproteome was in fact altered in response to Aβ exposure, and it was shown that proteins involved in fundamental cellular processes such as gene transcription and intracellular levels of protein kinases and phosphatases are increased upon Aβ treatment. Taken together these findings suggest that Aβ plays a role in abnormal protein phosphorylation, potentially leading to abnormal signaling cascades, and consequently contributing to AD pathology.
A Doença de Alzheimer é uma patologia neurodegenerativa caracterizada pela presença de placas senis, emaranhados neurofibrilhares e perda sináptica. Aβ, o principal componente das placas senis, tem um papel fundamental na patologia, uma vez que, o aumento da sua produção e agregação está associada a neurotoxicidade, activação da resposta inflamatória e cascatas apoptóticas. Estes processos estão associados à morte neuronal, neurodegeneração e, consequentemente, com o declínio cognitivo gradual. Considera-se também alterações em vias de sinalização celular como um dos aspectos fundamentais da patologia. A fosforilação de proteínas é reconhecida como um mecanismo fundamental capaz de regular eventos intracelulares. Vários estudos têm descrito uma actividade anormal de proteínas cinases e fosfatases em cérebros de doentes, bem como níveis anormais de fosforilação da PPA (Proteína Precursora de Amilóide de Alzheimer), Tau, entre outras proteínas. A fosforilação é um dos mecanismos que regula as funções e processamento da PPA sendo esta uma proteína fosfo-específica descrita como hiperfosforilada nos cérebros de doentes de Alzheimer. Devido à importância do Aβ e aos níveis de fosforilação anormal descritos na DA, os objectivos deste estudo eram a análise dos efeitos do Aβ na fosforilação da PPA, em específico nos resíduos Thr668 e Tyr682, bem como nos níveis de fosforilação geral. Neste trabalho observaram-se níveis aumentados de fosforilação nos resíduos especificados após a exposição ao Aβ sugerindo que este possa estar relacionado com a fosforilação anormal da PPA e, consequentemente, com o processamento desta. O trabalho desenvolvido sugere ainda o envolvimento da proteína fosfatase 1 na desfosforilação da PPA no resíduo Thr668. Adicionalmente, tanto o Aβ como a fosforilação no resíduo Thr668 regulam as interacções da PPA. Análise do fosfoproteoma revelou alterações em resposta ao tratamento com Aβ, estando proteínas envolvidas na transcrição de genes, cinases e fosfatases, aumentadas após o tratamento com Aβ. Todos estes resultados sugerem que o Aβ pode estar relacionado com a fosforilação anormal de proteínas conduzindo a uma sinalização intracelular anormal e consequentemente, contribuindo para a DA.
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Bastos, Maria de Fátima Camões Sobral de. "Aluminium neurotoxicity and neuronal phosphorylation systems". Doctoral thesis, Universidade de Aveiro, 2007. http://hdl.handle.net/10773/938.
Testo completoAbstract (sommario):
Doutoramento em BiologiaO alumínio é o terceiro elemento mais abundante na Terra. Uma vez que se encontra distribuído ubiquamente pelo meio ambiente e é utilizado em vários produtos e processos, a população humana está inevitavelmente exposta diariamente a este metal. De facto, o alumínio tem sido relacionado com diversas doenças neurodegenerativas como: a esclerose lateral amiotrófica, a demência de Parkinson (DP), a doença de Alzheimer (DA) e a encefalopatia relacionada com a diálise. A fosforilação de proteínas é um dos principais mecanismos reguladores intracelulares da maior parte das vias de sinalização nas células eucarióticas. Este processo dinâmico regula o estado de fosforilação e/ou a actividade das proteínas através de um balanço entre as proteínas cinases, que fosforilam, e as proteínas fosfatases (PP) que desfosforilam as proteínas. A proteína fosfatase 1 (PP1) é uma fosfatase específica para serina/treonina que está envolvida em importantes mecanismos celulares tais como o ciclo celular, contracção muscular e apoptose, entre outros. A PP1 tem três isoformas conhecidas, denominadas PP1α, PP1β e PP1γ,. O gene que codifica para a isoforma gama pode sofrer splicing alternativo originando a isoforma ubíqua PP1γ1 e a isoforma enriquecida no testículo PP1γ2. A fosforilação anormal de proteínas tem sido associada a várias patologias, incluindo cancro, diabetes e várias doenças neurodegenerativas (DP, doença de Huntington e DA). Uma das proteínas que se encontram anormalmente fosforiladas na DA são, por exemplo, os neurofilamentos (NF). Neste contexto, avaliou-se o efeito do alumínio na expressão de proteínas (PP1, NF) e realizou-se um estudo comparativo entre duas linhas celulares com características diferentes, as células PC12 e COS-1. Observou-se que o alumínio induziu um decréscimo na viabilidade celular, assim como na expressão e actividade de ambas as isoformas da PP1 em ambas as linhas celulares. Verificou-se que este efeito podia ser revertido retirando-se o alumínio. Um estudo semelhante foi ainda realizado num sistema neuronal utilizando culturas primárias de neurónios corticais. A expressão de ambas as isoformas da PP1 permaneceu inalterada após a exposição ao alumínio. No entanto, o alumínio induziu um decréscimo da expressão dos NF e da sinaptofisina, uma proteína que marca os terminais sinápticos. Por fim, estudou-se o efeito do alumínio na expressão de outras proteínas em sistemas in vitro e in vivo utilizando a tecnologia SELDI-TOF MS. Esta tecnologia permitiu detectar várias proteínas cuja expressão foi alterada devido ao alumínio. Com este trabalho pretendeu-se contribuir para um melhor conhecimento da neurotoxicidade do alumínio.
Aluminium is the third most abundant element on Earth. Aluminium is ubiquitous in the environment and is used in a variety of products and processes, thus, daily exposure of the general population to this metal is unavoidable. Indeed, aluminium has been implicated with various neurodegenerative disorders like: amyotrophic lateral sclerosis (ALS)/Parkinson’s dementia (PD) complex of Guam, Alzheimer’s disease (AD) and dialysis encephalopathy. Aluminium is known to interfere with several mechanisms of the nervous system, including alteration of cytoskeletal proteins, behavioural abnormalities, neurotransmission systems, oxidative damage, energy metabolism, second messengers, and also to induce neuronal apoptosis. Protein phosphorylation is a major intracellular regulatory mechanism of all signalling pathways in the eukaryotic cell. This dynamic process regulates the net phosphorylation state and the activity of proteins by a balance between protein kinases, which phosphorylate, and protein phosphatases (PP), which dephosphorylate proteins. Protein phosphatase 1 (PP1) is a serine/threonine specific phosphatase which is involved in the control of important cellular mechanisms such as the cell cycle, muscle contraction and apoptosis, among others. PP1 has three known isoforms termed PP1α, PP1β and PP1γ. The gene for PP1γ produces by alternative splicing a ubiquitously expressed PP1γ1 and a testis-specific PP1γ2 isoform. Abnormal protein phosphorylation has been associated with various disorders, including cancer, diabetes, and several neurodegenerative disorders (PD, Huntington’s disease and AD). Besides tau other proteins that are abnormally phosphorylated in AD are the neurofilaments (NF). In this context, the aluminium effect on the expression of proteins (PP1, NF) was evaluated. A comparative study was performed using two cell lines with different characteristics, PC12 and COS-1 cells. It was observed that aluminium induced a decrease in the cellular viability, as well as in the expression and activity of both PP1 isoforms in both cell lines. This effect was reverted following aluminium withdrawal. A similar study was also performed in a neuronal system, primary cortical neuron culture. The expression of both PP1 isoforms remained unchanged after aluminium exposure. However, aluminium induced a decrease in the expression of NF and of synaptophysin, a protein marker for synaptic terminals. Finally, the effect of aluminium on the expression of other proteins in in vitro and in vivo systems was evaluated using SELDI-TOF MS technology. In this study, several proteins with altered expression due to aluminium were detected. This work aimed to contribute to the better understanding of aluminium neurotoxicity.
39
Panayiotou, R. "Phosphorylation mediated regulation of MRTF-A". Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1462479/.
Testo completoAbstract (sommario):
The transcription factor SRF (Serum Response Factor) regulates expression of target genes in response to changes in actin dynamics, by virtue of association with the Myocardin Related Transcription Factor (MRTF) family of transcription cofactors. MRTF-A senses changes in actin dynamics via direct G-actin binding to its RPEL domain. While bound to actin MRTF-A continuously shuttles between the nucleus and cytoplasm, but localises to the cytoplasm due to a high export rate. Because MRTF-A is exported by Crm1 in an actin dependent manner, dissociation from actin leads to nuclear accumulation and SRF activation. Concomitantly, MRTF-A is phosphorylated on multiple residues. The aim of this thesis was to determine the role of phosphorylation in MRTF-A function. An MRTF-A derivative lacking all 26 identified phosphorylation sites was generated. Evidence is presented that phosphorylation of MRTF-A is required for its full capacity to activate SRF. One phosphorylation site, S98, is located within the RPEL domain. Phosphorylation of S98 attenuates actin binding to the RPEL domain and promotes nuclear accumulation of MRTF-A. I found that S98 is phosphorylated by ERK, which relies on an ERK binding motif just N-terminal of S98. Thus S98 represents a means by which MAP kinase signalling can impinge on MRTF-A regulation. In contrast, S33 phosphorylation promotes export of MRTF-A conferred by a Crm1 NES that was identified within its N-terminus. I have shown that this NES can act as an autonomous NES, but cooperates with the RPEL domain to confer actin dependent Crm1 mediated export. MRTF-A phosphorylation, can therefore fine-tune MRTF-A regulation by affecting both localisation and activity.
40
Fagerholm, Susanna. "Bidirectional signalling and phosphorylation of CD11 /". Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/fagerholm/.
Testo completo
41
Al, Jabry Tariq Saud Hamad. "Characterising the phosphorylation of YB-1". Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/15336.
Testo completoAbstract (sommario):
The Y-Box-binding protein-1 (YB-1) is a ubiquitous and multifunctional protein involved in several cellular processes such as transcription, mRNA splicing, and translation. YB-1 has been shown to be of significance to the area of cancer research, where it has been observed to be expressed at elevated levels in~40% of invasive breast carcinomas and a number of other malignancies. Within the nucleus, it has been reported to induce the transcription of a variety of genes, including oncogenes that upregulate cell proliferation, migration, and invasion. The phosphorylation of YB-1 at serine 102 has been reported to be necessary for its nuclear translocation and transcriptional activities.In most cases, the majority of the cell’s YB-1 is present within the cytoplasm, where it is involved in processes such as mRNA translation. YB-1 translationally upregulates several EMT associated and promoting genes, potentially contributing to metastasis. Its phosphorylation at serine 102 was also reported to allow the translation of latent oncogenic transcripts that upregulate cell proliferation.«br /» «br /» The general goal of this study was to functionally characterise YB-1 phosphorylation and determine its significance on tumour malignancy. However, the specific aims of this project were: (1) To purify and characterise the phosphorylation status of cytoplasmic and nuclear YB-1 by mass spectrometry; (2) To characterise their phosphorylation profiles under normal conditions, and after treatment with a variety of genotoxic agents; (3) To determine the significance of phosphorylation on YB-1 localisation and YB-1 driven cell proliferation and migration (hallmarks of cancer), using phosphorylation-defective mutants. As detailed in this thesis, a total of 7 distinct YB-1 phosphorylation residues have now been identified from A549 lung cancer cell lysate. The contribution of these phosphorylation sites to the regulation of YB-1 localisation and functions remain unknown. Nuclear and cytoplasmic YB-1 were both individually purified and identified by mass spectrometry, and a total of 7 phosphorylation sites were identified under normal conditions, and 6 under genotoxic stress (doxorubicin, cisplatin). Experiments with the phosphorylation-defective mutants suggest that dephosphorylation in general increases YB-1 nuclear localisation in colorectal and lung cancers, with the most nuclear localisation observed with NLS-proximal phosphorylation mutants. The data also suggests that phosphorylation at the NLS proximal serine 167, 174, and 176 sites are necessary for lung cancer cells to proliferate at an accelerated rate. On the other hand, phosphorylation-defective serine 165 and serine 174 & 176 double-mutants appear to act as oncoproteins that upregulate proliferation. Migration speed was also observed to increase with the serine 174-176 double and the threonine 271 phosphorylation-defective mutants, suggesting dephosphorylation at those residues increase bone cancer cell migration. Phosphorylation-defective mutants were also found to generally increase migration persistence (i.e. towards a gradient) in bone cancer cells. These studies implicate additional YB-1 phosphorylation residues in the regulation of two major hallmarks of cancer, proliferation and metastasis, and can potentially be of valuable prognostic value in future therapeutic endeavours in cancer research.
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Willder, Jennifer Mary. "Androgen receptor phosphorylation in prostate diseases". Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5797/.
Testo completoAbstract (sommario):
Prostatic diseases are common; benign prostate hyperplasia (BPH) is almost ubiquitous in elderly men and 899,000 men were diagnosed with prostate cancer worldwide in 2008. The incidence of both is increasing and expected to continue to rise. Therefore, prostatic diseases represent a considerable economic burden, but there are currently no reliable markers available to accurately differentiate indolent from aggressive disease nor to predict who will benefit from treatment for either BPH or prostate cancer. This results in over and under-treatment of both diseases with consequent patient related morbidity and mortality. The molecular mechanisms underlying the natural history of prostatic diseases remain elusive. It is accepted that prostate cell growth and survival are exquisitely dependent upon activation of the androgen receptor (AR) by androgens. Following ligand binding, AR undergoes further phosphorylation at serine residues, which inhibit proteolytic degradation, stabilise AR and influence AR transactivation. It is therefore plausible that alterations in AR phosphorylation may drive prostatic disease progression. However, few studies have explored the significance of AR phosphorylation, or the kinases driving AR serine phosphorylation in the clinical setting. The over-riding objective of this study was to establish the clinical relevance of AR serine phosphorylation status in prostate tissue in both BPH and prostate cancer. The specific aims of the current study were: • To firstly establish and validate a panel of AR phosphospecific antibodies. • To evaluate site specific AR serine phosphorylation expression levels in prostate cancer and BPH patient cohorts, with full clinical data and follow-up. • To investigate the expression of candidate kinases mediating such phosphorylation. This involved establishing tissue banks with linked comprehensive clinical databases, and utilising this tissue to establish AR phosphorylation expression profiles for each patient. Six AR phosphospecific antibodies (pARS81, pARS94, pARS213, pARS515, pARS578, pARS650) were verified using peptide competition assays and western blotting. Cdk1, ERK1/2, Akt and PKC were identified as putative kinases mediating AR phosphorylation using the online kinase search tool Scansite 2.0. Immunohistochemistry was performed on hormone naïve diagnostic prostate cancer tissue relating to 90 patients. High expression levels of AR phosphorylation at serine sites 81, 515 and 578 were each associated with a poorer clinical outcome. Following cox regression analysis, cytoplasmic pARS515 expression (p=0.038, HR 4.5 (95% CI 1.1–20.6)) and pARS81 nuclear expression (p=0.030, HR 0.033 95% CI 0.002-0.721) were independently associated with shorter time to biochemical relapse and shorter disease specific survival respectively. Cdk1 and/or pCdk1161 were significantly associated with pARS81 and pARS515 as predicted by Scansite 2.0. Similarly, nuclear PKC expression was significantly associated with pARS578 expression both in the cytoplasm and the nucleus. In patients with PSA at diagnosis ≤20ng/ml, high cytoplasmic pARS515 expression was associated with significantly shorter time to biochemical relapse (p=0.019). This translated into significantly shorter disease-specific survival (p<0.001, 10y survival 38.1% vs 100%). Prostate cancer patients with a low serum PSA level at diagnosis may be suitable for delayed radical treatment via active surveillance. An investigation was therefore undertaken in 51 prostate cancer patients treated by active surveillance. Active surveillance is a deferred radical treatment approach which provides a potential solution to the problem of over treatment as a result of over-diagnosis. However some patients harbour occult aggressive disease and delay in treatment may result in disease progression and failure of radical therapy. Although none of the individual AR serine phosphorylation sites were associated with clinical outcome measures on univariate analysis, high expression of total AR in the cytoplasm (p=0.021, HR 4.6 (95% CI 1.3-16.8)) and presence of perineural invasion in the tumour specimen (p=0.003, HR 8.6 (95% CI 2.1-35.7)) were deemed independent with regards to shorter time to treatment intervention in a cox regression analysis. Validation of the results seen in the first active surveillance prostate cancer cohort was undertaken in a second prospectively collected cohort consisting of 84 active surveillance patients. The results in the first cohort were not replicated in the second. Although cytoplasmic pARS81 was associated with time to intervention (p=0.032) and pARS515 expression trended towards an association (p=0.072), an increase in patient numbers in both cohorts may have provided more reliable results. However even with the numbers available in contrast to the first active surveillance cohort, but in line with the pilot prostate cancer cohort, Cdk1 was associated with pARS515 expression, and pCdk1161 trended towards an association. BPH is also an androgen driven disease dependent upon the AR. Previous research into predictive and prognostic markers in BPH is scant. Therefore a comprehensive analysis of clinical and novel pathological factors, including markers of inflammation, was performed in 336 BPH patients. Following this a complete panel of AR serine phosphorylation sites, and associated kinases, was analysed with reference to clinical outcome measures in the BPH cohort. Low expression levels of total AR and AR phosphorylated at Ser-81, 515 and 650 were associated with poorer clinical outcomes. Low expression of smooth muscle pARS515 (p=0.029, HR 0.31 (95% CI 0.10-0.94)) and older age (p=0.004, HR 5.13 (95% CI 1.43-18.41)) were deemed independent on cox regression analysis with regards to shorter time to postoperative acute urinary retention (AUR). Furthermore, low expression of pARS515 in the smooth muscle was associated with increased incidence of postoperative AUR in patients over 70 years old (25.1% vs 2.8% at 10 years following transurethral resection of prostate (TUR)), (p=0.002, HR 0.20 (95% CI 0.06-0.62)). This may have important clinical implications in postoperative counselling of these patients. In addition it may influence the decision to commence early postoperative medical treatment (with 5-alpha-reductase inhibitors and/or alpha blockers) on a prophylactic basis in these patients. Cytoplasmic pARS650 expression (p=0.010, HR 0.50 (95% CI 0.29-0.86)) and PSA at diagnosis (p=0.018, HR 1.89 (95% CI 1.11-3.16)) were independently associated with time to failure of surgical intervention. Furthermore, low expression of pARS650 in the cytoplasm was associated with increased failure of surgical intervention in patients with PSA ≥4ng/ml at diagnosis (45.5% vs 13% at 5 years post TUR), (p=0.026, HR 0.52 (95% CI 0.29-0.93)). This comprehensive study on immunohistochemical expression of site specific AR serine phosphorylation and associated kinases fills a gap in the current literature. It has demonstrated the clinical significance of AR serine phosphorylation in prostate cancer and BPH and uncovered potentially exciting new avenues for future investigation. Site specific serine phosphorylation of the AR may serve as a prognostic and predictive biomarker in prostatic disease and has potential as a future target for therapeutic intervention.
43
Schubert, Alexander Fabian. "Mechanism of PINK1-mediated ubiquitin phosphorylation". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277271.
Testo completoAbstract (sommario):
Ubiquitin phosphorylation by PINK1 (PTEN-induced Putative Kinase 1) is crucial for mitochondrial quality control and loss or mutation of PINK1 can lead to autosomal recessive juvenile parkinsonism (AR-JP). PINK1 is an unusual kinase, as it is characterised by three unique insertions in its kinase N lobe and a C-terminal region after the kinase domain. Despite great effort, a structure of PINK1 could not be determined and the molecular mechanism of ubiquitin phosphorylation and the effect of the PINK1 AR-JP patient mutations remained elusive. The versatile modifier ubiquitin (Ub) is also an unusual kinase substrate, as its phosphorylation site (Ser65) is not exposed, but protected by the Ub fold. Hence, it was not clear how a kinase would be able to target Ser65 of Ub. This work shows that Ub needs to adopt a previously described conformation in order to be efficiently phosphorylated by PINK1. NMR experiments revealed that in a small population of Ub the last β-strand is retracted, resulting in a more accessible Ser65 loop. It could be shown that PINK1 binds the Ser65 loop in this C-terminally retracted conformation (Ub-CR), but not in the ‘common’ conformation. In addition, it could be shown that Ub trapped in the Ub-CR conformation by point mutations (Ub TVLN) is phosphorylated significantly faster than Ub wt, which only adopts the Ub-CR conformation at very low frequency. To further elucidate how PINK1 binds and phosphorylates Ub, the kinase domain of Pediculus humanus corporis (Ph)PINK1 was crystallised in complex with Ub TVLN stabilised by a nanobody. The structure revealed many peculiarities of PINK1, such as the architecture of the unique insertions and the C-terminal region. Together with NMR and mass spectrometry studies, the structure explains how PINK1 interacts with ubiquitin via insertion-3 and its activation segment, and how PINK1 utilises the Ub- CR conformation for efficient Ser65 phosphorylation. In addition, the structure shows that two autophosphorylation sites in the N lobe regulate PINK1, by stabilising the functionally important insertions. The structure helped our understanding of the molecular basis of over 40 AR-JP patient mutations and may guide the design of ARJP therapeutics in the future.
44
Goveas, Liesel Mary. "LRRK2 phosphorylation - phosphophenotypes, biomarkers and targeting". Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS015.
Testo completoAbstract (sommario):
Les mutations de la Leucine-Rich Repeat Kinase 2 (LRRK2) sont liées à la maladie de Parkinson (MP). Le gène LRRK2 code pour une protéine multiphosphorylée comportant plusieurs domaines fonctionnels impliqués dans l'interaction protéine-protéine, la GTPase et l'activité kinase. Le ciblage de l'activité kinase de LRRK2 fait l'objet de nombreuses recherches, et plusieurs composés sont en cours d'évaluation en test clinique. D'autres fonctions de LRRK2 sont également étudiées pour leurs liens avec les pathomécanismes de la MP. Des études suggèrent que la phosphorégulation de LRRK2 est essentielle à son fonctionnement pathologique et physiologique. LRRK2 comprend deux types de sites de phosphorylation, à savoir les sites hétérologues et d'autophosphorylation. Un groupe de phosphosites hétérologues (S910, S935, S955, S973) présente une phosphorylation réduite dans les cerveaux atteints de la MP sporadique, dans les mutants de LRRK2 et après l'inhibition pharmacologique de l'activité kinase de LRRK2. L'état de phosphorylation de LRRK2 influence sa localisation subcellulaire. Ces résultats ont conduit à l'identification de PP1 et PP2A comme phosphatases déphosphorylant les sites hétérologues de LRRK2. Notre équipe s'intéresse à l'étude des conséquences de la déphosphorylation de LRRK2 et du rôle de ses phosphatases, avec un intérêt particulier pour la modulation de cette interaction comme nouvelle approche thérapeutique pour la MP. Par conséquent, la présente thèse de doctorat vise à déterminer les phénotypes de LRRK2 dans différents états de phosphorylation, et à réaliser des études d'interaction physique et fonctionnelle entre LRRK2 et PPP.Les résultats obtenus dans ce projet de thèse démontrent qu'il n'y a pas d'altération significative de la stabilité des phosphomutants purifiés de LRRK2 (S860/910/935/955/973/976 6x S>D/A) par rapport au WT. Nous confirmons l'hyperactivation de l'autophosphorylation induite par RAB29 sur un phosphomutant mimant la déphosphorylation (6xS>A). Il est intéressant de noter que l'expression de RAB29 affecte les niveaux totaux de LRRK2 de certains phosphomutants et du mutant R1441G. Le modèle de Drosophila melanogaster exprimant la forme 6xS>D du LRRK2 humain présente une sensibilité accrue au stress lysosomal induit par la chloroquine, par rapport aux mouches 6xS>A et WT. En outre, un lien fonctionnel entre LRRK2 et la sous-unité catalytique de PP1 (Ppp1CA) a été mis en évidence par une analyse protéomique et immunoblot des vésicules extracellulaires urinaires (uEV) de rats ; la présence de Ppp1CA est multipliée par trois dans les uEV de rats LRRK2 KO par rapport aux rats WT. Concernant l'interaction physique entre LRRK2 et ses phosphatases, une interaction in vivo entre LRRK2 endogène et Ppp1CA et Ppp2CA est confirmée dans le cerveau de rats (avec LRRK2 KO comme témoin). De plus, des coIPs ont été réalisées pour cartographier l'interaction entre LRRK2 et PPP2CA, avec des points de contact pour PPP2CA dans les domaines Ankyrine, Kinase et WD40 de LRRK2. Enfin, deux séquences candidates à la liaison avec LRRK2, dérivées d'une expérience de PEP-Scan avec PPP2CA, présentent une affinité de liaison de l'ordre du nanomolaire avec LRRK2, ce qui suggère que ces deux peptides pourraient potentiellement moduler l'interaction LRRK2:PPP2CA.Dans l'ensemble, la vérification de l'hypothèse que la promotion de la phosphorylation de LRRK2 est thérapeutique a conduit à la découverte de phénotypes de phosphorylation de LRRK2, justifiant la poursuite des travaux pour vérifier cette hypothèse. Les résultats confirment de manière robuste l'interaction physique entre LRRK2 et PPP, et identifient de courtes séquences peptidiques ayant une forte affinité de liaison pour LRRK2, qui peuvent servir de modulateurs candidats des complexes LRRK2:PPP. La présente thèse de doctorat ouvre donc la voie aux futurs tests biologiques des modulateurs de LRRK2:PPP à l'aide de résultats de nos tests de phosphophénotypageMutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are linked to autosomal dominant Parkinson's disease (PD). The LRRK2 gene encodes a multiphosphorylated protein with several functional domains involved in protein-protein interaction, GTPase and kinase activity. Mutations in the catalytic core of LRRK2 that segregate with disease alter its kinase activity. Targeting LRRK2 kinase activity is widely investigated with several compounds in late preclinical and early clinical stages. Other LRRK2 functions are also under investigation for their links to PD pathomechanisms, which could serve as potential targets and disease biomarkers. Studies suggest LRRK2 phosphoregulation is pivotal to its pathological and physiological functioning. LRRK2 comprises two distinct phosphorylation sites, namely the heterologous and autophosphorylation sites. A cluster of heterologous phosphosites (S910, S935, S955, S973) show reduced phosphorylation in sporadic PD brains, in disease mutants of LRRK2 and pharmacological inhibition of LRRK2 kinase activity. The phosphorylation status of LRRK2 influences its subcellular localization. These results spurred the identification of PP1 and PP2A as the phosphatases dephosphorylating the LRRK2 heterologous sites. Our team has been focused on studying the consequences of LRRK2 dephosphorylation and the role of its phosphatases, with particular interest of modulating this interaction as a novel therapeutic approach for PD. Therefore, the present doctoral thesis aims to determine the phenotypes of LRRK2 in varying states of phosphorylation, and carry out physical and functional interaction studies between LRRK2:PPP.The results obtained in this thesis project demonstrate there is no significant alteration in the stability of the purified LRRK2 phosphomutants (S860/910/935/955/973/976 6x S>D/A) when compared to the WT. We confirm RAB29 induced hyperactivation of autophosphorylation of the phosphodead hexamutant (6xS>A). Interestingly, RAB29 expression affects total LRRK2 levels of some phosphomutants and R1441G mutant. The Drosophila melanogaster model expressing the 6xS>D form of human LRRK2 shows increased sensitivity to chloroquine induced lysosomal stress as compared to the 6xS>A and WT flies. Furthermore, a functional link between LRRK2 and PP1 catalytic subunit alpha (Ppp1CA) is shown by proteomic and immunoblot analysis of rat urinary extracellular vesicles (uEVs); a 3-fold increase of Ppp1CA is shown in uEVs of LRRK2 KO compared to WT rats. Concerning the physical interaction between LRRK2 and its phosphatases, an in vivo interaction between endogenous LRRK2 and Ppp1CA and Ppp2CA is confirmed in rat brains (using LRRK2 KO control). In addition, coIPs were performed to map the interaction between LRRK2 and PPP2CA, with contact points for PPP2CA in the Ankyrin, Kinase and WD40 domains of LRRK2. Finally, 2 candidate LRRK2 binding sequences derived from a peptide scanning experiment with PPP2CA, display a nanomolar range binding affinity with LRRK2, suggesting these 2 peptides could potentially modulate the LRRK2:PPP2CA interaction.Altogether, testing the hypothesis that promoting LRRK2 phosphorylation may be therapeutic led to the discovery of some phenotypes of LRRK2 phosphorylation, additional work is required to verify the hypothesis. The results of this doctoral thesis robustly confirm the physical interaction between LRRK2:PPP, and identify short peptide sequences with a high binding affinity for LRRK2, which may serve as candidate modulators of LRRK2:PPPcomplexes. Hence, the present doctoral thesis paves the way for the future biological testing of LRRK2:PPP modulators using readouts obtained in our phosphophenotype testing
45
Hébert, Chatelain Etienne. "Impact des phosphorylations sur tyrosine sur le métabolisme mitochondrial : régulation et impacts fonctionnels des phosphorylations induites par la Src kinase". Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21830/document.
Testo completoAbstract (sommario):
La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellulesMitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells
46
Faure, Camille. "Régulation de la localisation et de l'activation de la tyrosine kinase Pyk2 (Proline-rich tyrosine kinase 2)". Paris 6, 2010. http://www.theses.fr/2009PA066736.
Testo completoAbstract (sommario):
Proline-rich tyrosine kinase 2 (Pyk2) est une tyrosine kinase non récépteur de 110-kDa deappartenant à la famille de focal adhesion kinase (FAK). Elle a été initialement caractérisée comme une enzyme cytoplasmique. Elle est, régulée par autophosphorylation (sur la tyrosine 402) en réponse à l’des augmentations de la concentration de Ca2+calcium libre cytosolique. L’autophosphorylation de Pyk2 est cruciale pour son activation puisqu’elle permet le recrutement des membres de la famille de Src, conduisant à l’ac’tivation de nombreuses voies de signalisation. Pyk2 existe sous deux isoformes produites par épissage alternatif, une isoforme enrichie dans le système nerveux contenant l’exon alternatif et une forme spécifique des cellules hématopoïétiques, dans laquelle cet exon est absent. Le laboratoire s’intéresse à l’isoforme de Pyk2 abondante dans les neurones qui joue un rôle important dans l’induction de la plasticité synaptique dans l’hippocampe. Au cours de ma thèseN, nous nous sommes intéressés à la régulation de l’activation et de la localisation de Pyk2 en réponse à la dépolarisation membranaire. Nous avons montré observé dans différents modèles, que des augmentations de Ca2+ induisent indépendamment l’activation et la relocalisation de Pyk2 dans le noyau en impliquant la calcineurine, probablement en amont de ces évènements. Nous avons la dépolarisation de la membrane induisait une accumulation nucléaire rapide et transitoire de Pyk2. Nous avons montré que cette relocalisation de Pyk2 dans le noyau était calcium-dépendante, mais indépendante de son autophosphorylation, de son activité kinase et du recrutement des kinases de la famille de Src. En revanche, nous avons démontré qu’en réponse à la dépolarisation, l’autophosphorylation et la localisation subcellulaire de Pyk2 sont régulées par une protéine sérine/thréonine phosphatase, la calcineurine. Afin de caractériser le mécanisme moléculaire permettant la régulation de la localisation de Pyk2, nous avons recherché les séquences responsables de son trafic nucléo/cytoplasmique. Nous avons utilisé une stratégie de délétion qui nous a permis d’isoléer une région de Pyk2, localisée entre les domaines kinase et FAT, nécessaire à lsa localisation cytoplasmique de Pyk2 contenantdans les cellules non stimulées. Nous avons démontré la présence d’ une séquence d’export nucléaire qui dans cette région, comprenant les résidus hydrophobes Leu-735, Phe-739, Val-741. Au cours de ces expériences, nous avons observé que cette région mimait en partie la régulation de la localisation de Pyk2 induite par la dépolarisation membranaire suggérant que l’accessibilité de ces séquences d’export et/ou d’import nucléaire est régulée au cours de la stimulation pour permettre la translocation nucléaire de Pyk2. De manière intéressante, la séquence d’export nucléaire que nous avons caractérisée appartient en partie à l’exon alternatif et n’est pas fonctionnelle dans l’isoforme spécifique des cellules hématopoïétiques révélant. Ces données suggèrent que cet épissage alternatif traduirait une spécificité tissulaire de lala régulation de sla localisation de Pyk2 mise en évidence par la dépolarisation membranaire. Nous avons également pu observéer une accumulation nucléaire de Pyk2 dans d’autres modèles, ainsi qu’ in vivo, suggérant l’existence des fonctions nucléaires de Pyk2 différentes de ses fonctions cytoplasmiques connues. L’ensemble de ces résultats permet de révéler que des augmentations de calcium induites par la dépolarisation membranaire induisent indépendamment l’activation de Pyk2 et sa re-localisation dans le noyau. Ces deux phénomènes impliquent la calcineurine, qui agit probablement en amont de ces évènements. D’autre part, la localisation cytoplasmique de l’isoforme neuronale de Pyk2 est assurée par une séquence d’export nucléaire située au niveau du site d’épissage alternatif, permettant d’appréhender les bases d’une spécificité neuronale Tyrosine kinase
47
Blanquart, Christophe. "Etude des modifications post-traductionnelles du Peroxisome Proliferator Activated Receptor Alpha (PPAR Alpha) et de leurs conséquences sur les fonctions biologiques de ce récepteur nucléaire". Lille 2, 2003. http://www.theses.fr/2003LIL2P015.
Testo completoAbstract (sommario):
PPARα appartient à la superfamille des récepteurs nucléaires (RN). C'est un facteur de transcription activé par des ligands. PPARα est impliqué dans la régulation du métabolisme des lipides et des lipoprotéines ainsi que dans le contrôle de la réponse inflammatoire. Ces différentes fonctions lui confèrent un rôle protecteur contre l’athérosclérose. Le but de nos travaux a été de mettre en évidence diverses modifications post-traductionnelles de PPARα ainsi que de définir leurs conséquences sur les fonctions de ce RN. Ainsi, nous avons montré que la protéine PPARα est ubiquitinée, ce qui conduit à sa dégradation par le système ubiquitine-protéasome. Nous avons également mis en évidence que les ligands de PPARα diminuent son ubiquitination le protégeant ainsi de la dégradation. Enfin, nous avons montré que le système ubiquitine-protéasome est impliqué dans la régulation de l'expression des gènes cibles de PPARα dans des cellules hépatiques humaines en contrôlant le taux intracellulaire de ce RN. Comme il a été décrit dans la littérature, PPARα est une protéine phosphorylée. Nous avons alors étudié le rôle des protéines kinases C (PKC) dans la régulation de l'activité de PPARα. De manière originale, nos résultats montrent que l’activité des PKC est nécessaire à l'obtention d'une activation optimale des propriétés transactivatrices de PPARα par ses ligands dans les cellules hépatiques humaines. Nous avons également observé que les PKC diminuent les propriétés de transrépression de ce RN. Nos résultats mettent ainsi en évidence l'existence d'un mécanisme de contrôle des propriétés de transactivation et de transrepression de PPARα par les PKC. Enfin, nous avons montré que l'inhibition des PKC dans les cellules hépatiques humaines diminue la phosphorylation de PPARα et que ce RN est phosphorylé par les PKC classiques in vitro. En conclusion, nos travaux ont permis de mettre en évidence deux nouvelles voies de régulation de l'activité de PPARα par des modifications post-traductionnelles, le système ubiquitine-protéasome et la voie des PKCPPARa belong to the nuclear receptor (NR) superfamily. This NR is a transcription factor activated by ligands. PPARa is implicated in the regulation of the lipid and lipoprotein metabolism and in the control of the inflammatory response. These functions confer anti-atherogenic properties to PPARa. The aim of our work was to identified new post-translational modifications of PPARa and to determine their consequences on the functions of this NR. In a first study, we have shown that PPARa is ubiquitinated, which lead to the degradation of this NR by the ubiquitin-proteasome pathway. We shown too that the PPARa ligands protect this NR from degradation by decreasing its ubiquitination. Finally, we have demonstrated that the ubiquitin-proteasome pathway is implicated in the control of PPARa target genes expression by regulating its intracellular level. As it was already described in the literature, PPARa is a phosphoprotein. Then, in a second study, we have evaluated the role of the protein kinases C (PKC) in the regulation of the PPARa activities. Our results demonstrate that PKC activity is necessary to obtain an optimal induction of the PPARa transcriptional activity by its ligands. We have observed too that PKC decrease the transrepression properties of PPARa. Our results describe a new control mechanism of the PPARa transactivation and transrepression functions by the PKC. Finally, we have shown that PKC inhibition decreases the PPARa phosphorylation and that this nuclear receptor is phosphorylated by the classical PKC in vitro. In conclusion, our work describe two new regulation mechanisms of the PPARa functions by post-tranlational modifications, the ubiquitin-proteasome system and the PKC signalling pathway
48
Abukhalaf, Imad Kazem. "Application of Synthetic Peptides as Substrates for Reversible Phosphorylation". Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc277577/.
Testo completoAbstract (sommario):
Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.
49
Halbedel, Sven. "Regulation of HPr phosphorylation in Mycoplasma pneumoniae". Doctoral thesis, [S.l.] : [s.n.], 2006. http://webdoc.sub.gwdg.de/diss/2006/halbedel.
Testo completo
50
Sundstrom, Jeffrey Antonetti David A. "Identification and functional analysis of occludin phosphorylation". [University Park, Pa.] : Pennsylvania State University, 2008. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-2566/index.html.
Testo completo