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Hasan, Diya [Verfasser]. "Hypoxic regulation and selective silencing of pyruvate kinase isoforms PKM1 and PKM2 by siRNA / Diya Hasan." Gießen : Universitätsbibliothek, 2012. http://d-nb.info/1064024580/34.
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Delobelle, Quentin. "Simulations de dynamique moléculaire à haute résolution des isoformes 1 et 2 de la pyruvate kinase musculaire." Electronic Thesis or Diss., Sorbonne université, 2024. http://www.theses.fr/2024SORUS067.
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Le métabolisme glycolytique joue un rôle essentiel dans les processus physiologiques normaux et dans la croissance des cellules cancéreuses. Les isoformes 1 et 2 de la pyruvate kinase musculaire, appelées PKM1/2, sont des protéines cruciales qui régulent ce métabolisme. La caractérisation de la dynamique structurelle de ces biomolécules est donc importante pour développer de nouveaux médicaments dirigés contre les formes exprimées dans les cellules tumorales. Pour ce faire, nous avons réalisé des simulations de dynamique moléculaire (DM) de ces deux protéines à haute résolution, en nous appuyant sur des techniques d'échantillonnage adaptatif couplées au champ de forces polarisable AMOEBA afin de comprendre la flexibilité conformationnelle de l'enzyme dans ses différents états d'oligomérisation. Nous proposons une analyse structurale à haute résolution pour PKM2 et apportons de nouvelles données structurales pour PKM1. En effet, nous étudions le processus d'oligomérisation de l'enzyme PKM2 (du monomère au tétramère) et nous sommes notamment attachés à analyser la structuration de sites structuraux clés, notamment le site actif et la poche de fixation du Fructose Biphosphate (FBP), un inducteur conformationnel physiologique. Nous montrons également que la fixation du TEPP-46, un activateur pharmacologique de PKM2, induit des modifications structurales très proches de celles induites par la fixation du FBP, bien que les domaines d'interaction soient distincts. Enfin, nous décrivons la première simulation DM à haute résolution de la forme active de PKM1, qui présente de fortes similitudes structurelles avec la forme tétramérique de PKM2 liée au FBP. Ces résultats fournissent de nouvelles informations sur la dynamique structurelle et la structuration des sites clés pendant l'oligomérisation de PKM2, qui pourraient s'avérer cruciales pour la découverte de nouvelles molécules activesIn both normal physiological processes and cancer cell growth, glycolytic metabolism plays a pivotal role. The Pyruvate Kinase Muscle isoforms 1 and 2, denoted PKM1/2, are crucial proteins that regulate this metabolism. Characterizing the structural dynamics of these biomolecules is thus imperative in order to develop new drugs targeting PMK enzymes in tumor cells. To address this, we performed extensive molecular dynamics (MD) simulations of these two proteins at high-resolution. To do so, we employed adaptive sampling techniques coupled with the polarizable AMOEBA force field to understand the conformational flexibility of the enzyme. We propose a high-resolution structural analysis for PKM2 and introduce structural insights for PKM1. We are studying the oligomerization process of the PKM2 enzyme (from monomer to tetramer) while focusing on the structuring of key structural sites, in particular the active site and the binding pocket for Fructose Biphosphate (FBP), a physiological conformational inducer. We also provide data explaining the impact of TEPP-46, a pharmacological activator, on PKM2 structure and their similarity with PKM2 bound to FBP in conformity to biological results. Finally, we propose the first high-resolution MD simulation of the biological active PKM1 and reveal important structural information in link with strong structural similarities with PKM2 when linked to FBP. These findings provide new insights concerning the structural dynamics and key sites structuration during PKM2 oligomerization that could be critical in drug discovery
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Dayton, Talya Lucia. "Examining the roles of the pyruvate kinase isoforms, PKMI1 and PKM2, in systemic metabolism and tumor development." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104176.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase, and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. PKM1 expression, on the other hand, is restricted mostly to skeletal muscle, heart, and brain. To interrogate the functional requirement for PKM1 or PKM2 during development and tissue homeostasis, we generated germline PKM1 (Pkm1-/-) or PKM2 null mice (Pkm2-/-). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2' mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. We found that Pkm1-/- mice are also viable and fertile. Thus, neither PKM isoform is required for embryonic or postnatal development. In Pkm1-/- mice, loss of PKM1 leads to compensatory expression of PKM2 in the tissues that normally express PKM1. Aside from distinct changes in the plasma metabolite profiles of Pkm1-/- mice compared to wild-type (WT) mice, germline loss of PKMI appears to be of little phenotypic consequence. In contrast, PKM2 loss leads to a striking phenotype: spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism. To interrogate the cell-autonomous functional requirement for PKM2 during tumor initiation, we used a conditional Pkm2 allele (Pkm2f/) to abolish PKM2 expression in the context of two Kras driven mouse models - for lung adenocarcinoma and soft-tissue sarcoma (STS). In the sarcoma model, where the presumed tumor cell-of-origin expresses PKM1, deletion of PKM2 led to delayed tumor formation. In contrast, in the lung cancer model the presumed tumor cell-of-origin expresses PKM2 and deletion of PKM2 had no effect on tumor latency or tumor area. PKM2-null sarcomas expressed PKM1 and contained a high number of infiltrating PKM2+ stromal cells. Metabolite analysis of sarcoma cell lines generated from PKM2-null and wild-type tumors revealed metabolic changes in the PKM2-null tumors. These results argue that the consequences of PKM2 loss during tumor initiation depend on the tumor type and that the requirement for PKM2 expression can be overcome through metabolic adaptation. Taken together, the work presented in this thesis provides key insights into the pleiotropic roles played by PKM1 and PKM2 in the contexts of normal and malignant proliferation and in tumor and systemic metabolism. Our findings contribute to a more complete understanding of the distinct cell-intrinsic and cell-extrinsic roles of PKM isoform expression in the context of cancer, and may potentially inform strategies that target metabolism for the treatment of cancer.
by Talya Lucia Dayton.
Ph. D.
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Torbett, Neil Edward. "Cellular roles of PKN1." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406323.
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Kuntz, Ashley L. "The Role of PKS1 in Root Phototropism." Miami University Honors Theses / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=muhonors1177531764.
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Kappe, Eva Christina. "Molekularbiologische Untersuchungen am PKD1-Gen der Katze." Giessen : VVB Laufersweiler, 2008. http://d-nb.info/990148246/04.
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Damasceno, Luis Eduardo Alves. "Envolvimento da enzima Piruvato Quinase M2 (PKM2) na diferenciação de linfócitos Th17 e patogênese da encefalomielite autoimune experimental." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17147/tde-23042018-150809/.
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Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17.Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.
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Kunze, Markus. "Abbau von Chlorbenzol in Pseudomonas putida GJ31: das Plasmid pKW1." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=967443148.
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Hughes, Jim. "Comparative analysis of the PKD1 gene and protein, polycystin-1." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300891.
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Sneddon, Tam Paterson. "The characterisation of genes (HG) homologous to the PKD1 locus." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365319.
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Charavin, Marine. "Synthèse d'agonistes non-peptidiques du récepteur à la prokinéticine PKR1." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF047/document.
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Les récepteurs couplés aux protéines G représentent la plus grande famille de récepteurs membranaires. Parmi eux, nous avons choisi d’étudier deux récepteurs apparentés : les récepteurs de la prokinéticine 1 et 2. Ces deux récepteurs ont pour ligands des hormones de nature peptidique, divisées en deux sous-groupes : les prokinéticines 1 et 2. Ces deux prokinéticines sont impliquées dans plusieurs processus physiologiques en se liant à leurs récepteurs PKR1 et PKR2. Il a été récemment montré que la prokinéticine 2 pouvait stimuler la prolifération et la différenciation des cellules souches progénitrices cardiaques, via les récepteurs PKR1 et PKR2. Il a également été reporté que l’activation de PKR1 protège les cardiomyocytes et les cellules progénitrices cardiaques de l’apoptose. Afin d’étudier ces effets nous avons synthétisé des agonistes non-peptidiques du récepteur PKR1. Nous avons donc poursuivi les études de pharmacomodulation d’une première famille de composés et développé une seconde famille d’agonistes potentiels originaux, déterminée par des études de modélisation moléculaire. Une sonde fluorescente a été synthétisée afin d’évaluer la liaison de nouveaux composés. Au cours de ces travaux nous avons découvert une nouvelle réaction multi-composante permettant la synthèse d’un composé dihydropyrrole polyfonctionnel. Nous nous sommes alors intéressés à son mécanisme et à sa limitation chimique dans le but de former de nouveaux hétérocycles fonctionnalisésThe G protein-coupled receptors represent the largest familly of membrane receptors. Among them,we choose to study two related receptors: prokineticin receptors 1 and 2. These two receptors have peptidic hormone ligands, divided in two sub-groups: prokineticins 1 and 2. Both prokineticins are involved in many physiological processes by binding to their receptors PKR1 and PKR2. It has recently been shown that prokineticin 2 could stimulate proliferation and differentiation of cardiac progenitor cells. It was also reported that activation of PKR1 protects cardiomyocytes and cardiac progenitor cells from apoptosis. To investigate these effects we synthesized non-peptidic receptor PKR1. We continued pharmacodulation studies of a first familly of compounds and developped a second familly of original potential agonists, determined by molecular modeling studies. A fluorescent probe was synthesized to access the binding of novel compounds. During this work we discovered a new multi-component reaction for the synthesis of a polyfunctional dihydrpyrrol compound. We then interested in the mechanism and its chemical limitation in order to form new functionalized heterocycles
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Asiedu, Marina N. "Spinal Sensitization Mechanisms Promoting Pain: Gabaergic Disinhibition and Pkmζ-Mediated Plasticity." Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/232496.
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As a major public health problem affecting more that 76.5 million Americans, chronic pain is one main reason why people seek medical attention. It is a pathological nervous system disorder that persists for months or years. Sensitization of nociceptive neurons in the dorsal horn of the spinal cord is crucial in the development of allodynia and hyperalgesia. The work presented in this thesis will focus on spinal protein kinase M zeta (PKMζ)-mediated plasticity and GABAergic disinhibition as spinal amplification mechanisms that orchestrate persistent changes in the dorsal horn of the spinal cord. As a result of central sensitization following peripheral nerve injruy, GABAergic disinhibition occurs due to an alteration in Cl- homeostasis via reduced KCC2 expression and function. Intrathecal administration of acetazolamide (ACT), a carbonic anhydrase inhibitor, attenuated neuropathic allodynia and spinal co-adminitation of ACT and midazolam (MZL), an allosteric modulator of the benzodiazepine class of GABAA receptors, synergistically inhibited neuropathic allodynia. Further studies concerning the impact of altered Cl-homeostasis via reduced KCC2-mediated Cl-extrusion capacity on the analgesic efficacy and potency of GABAA receptor agonist and allosteric modulators revealed that there is a differential regulation of the agonists and allosteric modulators at the GABAA receptor complex when Cl-homeostasis is altered. Another spinal amplification mechanism leading to central sensitization is PKMζ-mediated spinal LTP. In model of persistent nociceptive sensitization, allodynia induced by IL-6 injection or plantar incision was abolished by both the inhibition of protein translation machinery and PKMζ inhibitor, ZIP. However, only PKMζ inhibition prevented the enhanced pain hypersensitivity precipitated by a subsequent stimulus after the initial hypersensitivity had resolved, asserting that spinal PKMζ underlies the maintenance mechanisms of persistent nociceptive sensitization. Also, these results confirmed that the initiation mechanisms of persistent sensitization parallel LTP initiation mechanisms and the maintenance mechanisms of persistent sensitization parallel LTP maintenance mechanisms. Taken together, these results indicate that these amplification mechanisms drive a chronic persistent state in these models such that inhibition of these spinal amplication mechanisms will serve as an effective approach in the quenching chronic pain hypersensitivity in chronic pain models.
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Ryu, Kimoon. "The expression and purification of the regulatory domain of PKG1 alpha." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12609.
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Thesis (M.A.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Cyclic guanosine dependent protein kinases play key roles in many functions throughout the body. Three different types of these kinases exist in humans: type one alpha, one beta, and two. The different kinases exist in different locations within the body and also have different substrates that they phosphorylate. Obtaining a pure sample of these kinases has always been challenging due to cyclic nucleotides that co-purify and contaminate the final samples. If cAMP free samples of the kinase were able to be purified, then figuring out the conformation of the enzyme with and without cyclic nucleotides as well as its activation and binding kinetics of the enzymes can be determined. This information will help us to investigate the detailed mechanism of activation of the enzyme and to develop drugs to target these kinases better. Purification of the enzyme is started with growing them in an adenyl ate cyclase deficient Escherichia coli cell line, TP2000, to avoid cyclic nucleotide contaminations. With use of immobilized-metal affinity, ion exchange, and size exclusion chromatography, I successfully purified the regulatory domain of type 1 alpha kinase yielding 490 µL of the protein at a concentration of 2.2 mg/ml that is completely free of cAMP for the first time. The resulting samples will help us to not only measure its affinity to various cyclic nucleotide ligands, but also initiate co-crystal screen with its native ligand, cGMP.
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Hatfield, Spencer. "Establishing Methods For Protein Purification And Activity Analysis For Pkn1 And Pkn5 From Chlamydia Trachomatis." OpenSIUC, 2016. https://opensiuc.lib.siu.edu/theses/1903.
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Chlamydia are Gram negative, obligate intracellular bacterial pathogens responsible for diseases that affect both animals and humans. Only two vaccines have been developed for Chlamydia, targeting C. felis and C. abortus, and development of antibiotic resistance and/or persistent infection forms has been documented for multiple species. Consequently, identification of new therapeutic targets is critical for prevention and treatment of chlamydial infections. These bacterial pathogens have a unique biphasic developmental cycle beginning with the infectious and environmentally stable elementary body, which enters a host cell and envelopes itself in the host membrane forming an inclusion. While residing in the inclusion, the EB transitions into the metabolically active and replicative reticulate body. The RB divides by binary fission before converting back into the EB and exiting the host cell by inclusion extrusion or cell lysis. The signals that initiate morphogenesis and the mechanism(s) mediating the transition between EB and RB forms are poorly understood. Eukaryote-like serine/threonine kinases (Hank’s type kinases) have recently been described to play major roles in cellular development and pathogenicity in prokaryotes. Chlamydia encode three Hank’s type kinase; Pkn1, PknD, and Pkn5. We hypothesize that these kinases control bacterial differentiation and metabolism by regulating protein activity via phosphorylation, making them potential targets for anti-chlamydial therapeutics. To aid in future efforts to elucidate the roles of these Hank’s type kinases in the physiology of C. trachomatis, my thesis developed protocols for the affinity purification of recombinant Pkn1 and Pkn5 and assessed the efficacy of a high-throughput kinase assay for Pkn1.
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Hestermann, Jörg-Oliver. "Beitrag zur allgemeinen Konzeption von parallelkinematischen Maschinen (PKM) /." Stuttgart : IfW, 2007. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=016575131&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Kappe, Eva Christina [Verfasser]. "Molekularbiologische Untersuchungen am PKD1-Gen der Katze / eingereicht von Eva Christina Kappe." Giessen : VVB Laufersweiler, 2008. http://d-nb.info/990226506/34.
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Rowles, Thomas Andrew. "Investigation into the phosphorylation of PICK1 by the atypical protein kinase PKMζ." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572674.
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Phosphorylation is a post-translational modification that is known to regulate many aspects of cellular function, including the mechanisms which underlie synaptic plasticity. PICK1 is a neuronal protein known to be involved in AMPA receptor trafficking, and which has been shown to interact with PKCa and act as a substrate for this conventional PKC isoform. This study further investigates the phosphorylation of PICK1. Phosphorylated regions of PICK1 are first identified through the use of a radioactive phosphorylation assay and serine-412 in the C- terminus of PICK1 is identified as a phosphorylated residue. The kinases involved in PICK1 phosphorylation are then investigated and it is shown that the atypical PKC isoform PKMζ is likely to be the kinase responsible for phosphorylating PICK1 at serine-412. Corroboration of this comes from the finding that PICK1 interacts with PKMζ, and that treatment with the PKMζ inhibitory peptide ZIP reduces the signal obtained from a phosphospecific antibody targeted against PICK1 phosphorylated at serine-412. Finally, functions and signals associated with PICK1 phosphorylation are investigated. It is shown that PICK1 strengthens the known interaction of PICK1 with the actin polymerising Arp2/3 complex, and that NMDA treatment decreases the observed level of PICK1 phosphorylation.
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Moeke, Cassidy Hameora. "Detection and modification of pyruvate kinase isoforms by myeloperoxidase-derived oxidants HOCl and HOSCN." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16903.
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Pyruvate kinase M2 is a glycolytic rate-limiting enzyme that has been shown to induce apoptosis through a caspase-independent pathway in response to oxidative damage by H2O2. Recent studies examining the potential redox regulation of PKM2 showed this form of programmed cell death was linked to the oxidation of a single cysteine amino acid Cys358. Myeloperoxidase (MPO), a leukocyte-derived heme enzyme, and the oxidants it generates, play a key role in killing invading pathogens at sites of inflammation, but can also generate host tissue damage, including within the inflamed artery wall in the context of cardiovascular disease and atherosclerosis. Two oxidants produced by this enzyme, hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), react with several biomolecules including amino acids such as Cys, Met, cystine, His, Lys, Trp and Tyr in the case of HOCl, while HOSCN is considered to be a relatively specific oxidant of cysteine residues. In the current study we have examined whether and how oxidation of PKM1 and PKM2 are induced by myeloperoxidase-derived oxidants formed at sites of inflammation. These studies indicate that HOCl and HOSCN rapidly inhibit the catalytic activity of pyruvate kinase in a dose-dependent manner for isolated PKM1 and PKM2 (1.25-80 μM HOSCN and 2-16 μM HOCl, P < 0.01) and for HCAEC (150-400 μM HOSCN and HOCl, P < 0.01) and HCASMC cellular systems (25-400 μM HOSCN and 100-400 μM HOCl, P < 0.01), with this loss of activity associated with oxidation of Cys residues as determined by Thio-Glo assay and the formation of reducible protein aggregates. Peptide mass mapping experiments provided confirmation of Cys oxidation through the derivatization of sulfenic acid / sulfenyl thiocyanate residues with dimedone, where the amino acids Cys 49,152, 165, 326, 358 and 474 were found to contain the 138 Da dimedone adduct. This research also presents the first comprehensive evidence identifying the presence of PKM2 in human coronary endothelial and smooth muscle cells, mammary artery clinical specimens and carotid or abdominal atherosclerotic lesions using a combination of activity measurements, western blot and mass spectrometry techniques.
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Courtellemont, Thibault. "Septin regulation by the Protein Kinase C in the budding yeast, Saccharomyces cerevisiae." Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20007/document.
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La cytokinèse est un processus fondamental prenant place à la fin de la mitose et permettant la séparation des deux cellules filles. Un défaut de cytokinèse peut mener à une ségrégation anormale des chromosomes et engendrer des phénomènes de cancer. Dans beaucoup d'organismes eucaryotes, la cytokinèse nécessite l'assemblage et la contraction d'un anneau d'actomyosine permettant la formation d'un sillon et la réorganisation de la membrane cellulaire au site de clivage. Dans la plupart de ces organismes, des protéines du cytosquelette appelées septines participent à la cytokinèse. Chez la levure bourgeonnante, Saccharomyces cerevisiae, cinq septines sont exprimées durant la mitose (Cdc3, Cdc10, Cdc11, Cdc12 et Shs1). Ces protéines ont la capacité de s'assembler en un anneau au niveau du site de bourgeonnement, lieu de séparation entre la cellule mère et la cellule fille. Cet anneau de septines permet la fixation et le recrutement de nombreuses protéines intervenant dans la cytokinèse. La dynamique des septines change durant le cycle cellulaire, ce qui a une importance dans la régulation de la cytokinèse. La stabilisation de cet anneau est accompagnée d'un changement du niveau de phosphorylation des septines, mais les kinases responsables de ces modifications restent inconnues. Les travaux de l'équipe de Simonetta Piatti ont mis en évidence un nouveau rôle de la GTPase Rho1 et de sa cible, la protéine kinase C (Pkc1), dans la régulation de la dynamique des septines. Le but de ce travail de thèse était de déterminer les voies moléculaires par lesquelles la protéine Pkc1 intervient dans le recrutement et la stabilisation de l'anneau de septines. Pour se faire nous avons purifié le complexe de septines chez la levure bourgeonnante en présence ou en absence de la protéine Pkc1 et nous l'avons analysé par spectrométrie de masse. Cette analyse nous a permis d'observer que le niveau de phosphorylation d'un cluster (îlot) de 5 sérines était diminué sur Shs1. L'alignement de séquence nous a permis de constater que ce domaine était conservé dans la septine Cdc11. Par ailleurs ces deux protéines sont connues pour jouer un rôle dans l'assemblage des filaments et la formation de l'anneau de septines. Il a déjà été observé qu'un mutant phosphomimétique du cluster de sérine de la septine Shs1 empêche la formation des filaments in-vitro. Nous avons voulu caractériser le rôle de ce cluster dans la protéine Cdc11 en créant un mutant non-phosphorylable (CDC11-9A) et un mutant phosphomimétique (CDC11-9D). De manière très évidente, le mutant phosphomimétique provoque des problèmes de cytokinèse dans les cellules dont le gène codant la protéine Shs1 a été supprimé. A l'inverse le mutant non-phosphorylable améliore le phénotype des cellules ne comportant pas Shs1. Ces résultats sont en parfait accord avec l'observation selon laquelle les protéines Shs1 et Cdc11 pourraient avoir des fonctions très similaires, et mettent en avant le rôle important du cluster de sérines phosphorylées de Cdc11 lors de la cytokinèse. Nous avons constaté que Pkc1 ne phosphoryle pas directement les septines, mais agit par l'intermédiaire de kinases et de phosphatases impliquées dans la régulation des septines. Nous avons pu montrer que Pkc1 régule l'interaction de Gin4 avec les septines, cette kinase étant connue pour sa capacité à phosphoryler Shs1. De plus, nous avons observé que Pkc1 impacte sur le niveau de phosphorylation des deux autres kinases de la même famille, Hsl1 et Kcc4. Par ailleurs, la délétion de PKC1 diminue drastiquement la quantité de protéines Kcc4 dans la cellule.L'absence de Pkc1 augmente également l'interaction entre les septines et Bni4, une sous-unité régulatrice de la phosphatase PP1. Nous avons également observé que Bni4-PP1 peut déphosphoryler Cdc11, expliquant la diminution de son niveau de phosphorylation en cas d'absence de la protéine Pkc1.Ces travaux mettent en évidence que Pkc1 est un nouveau régulateur majeur des septines dans la levureCytokinesis is the last step of mitosis and is the fundamental process leading to the physical separation of two daughter cells. Defects in cytokinesis generate polyploid cells that are prone to chromosome missegregation and cancer development. In animal cells and fungi, cytokinesis requires the formation and contraction of an actomyosin ring that drives ingression of the cleavage furrow. Additional cytoskeletal proteins called septins contribute to cytokinesis. In the budding yeast Saccharomyces cerevisiae, five different septins are expressed during the mitotic cell cycle (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1). All septins, except for Shs1, are essential for cell viability. Yeast septins form filaments that in turn organize into a ring at the bud neck, which is the constriction between the mother and the future daughter cell where cytokinesis takes place. The septin ring then expands into a rigid septin collar that acts as scaffold for cytokinesis by recruiting most cytokinetic proteins to the bud neck. Cell cycle-regulated changes in septin ring dynamics are thought to be important for its cytokinetic functions and formation of the rigid septin collar is accompanied by septin phosphorylation. However, the kinases responsible for these modifications have not been fully characterized. Unpublished data from our laboratory indicate that the Rho1 GTPase, which is essential for actomyosin ring formation and contraction, and its target protein kinase C (Pkc1) contribute to deposition and stabilization of the septin ring. Here, we have addressed how Pkc1 regulates septin ring deposition and/or stability. To this end, septin complexes were purified from yeast and analyzed by mass spectrometry, comparing wild type and pkc1Δ mutant cells. This mass spectrometry analysis clearly showed that phosphorylation of a cluster of residues in Shs1 decreased in the absence of Pkc1. Interestingly, we found that this cluster is conserved in the septin Cdc11, which together with Shs1 is known to play an important role in the assembly of high-order structures like filaments and rings. Phosphomimetic mutations of the phosphorylatable cluster in Shs1 have been previously shown to disrupt filament formation in-vitro. We therefore proceeded to mutagenise the same cluster in Cdc11, generating a phosphomimetic (CDC11-9D) and in a non-phosphorylatable mutant (CDC11-9A). Strikingly, the phosphomimetic CDC11-9D caused cytokinesis defects in cells lacking Shs1, whereas the non-phosphorylatable CDC11-9A allele partially rescued the sickness of shs1∆ mutant cells. These observations are in agreement with the notion that Cdc11 and Shs1 share overlapping functions and highlight an important role of the phosphorylatable cluster of Cdc11 for cytokinesis. We also found that Pkc1 does not phosphorylate septins directly, but rather regulates the activity of septin kinases and phosphatases. Consistently, we show that Pkc1 affects the interaction between septins and the bud neck kinase Gin4, which is known to interact with septins and to phosphorylate them. In addition, Pkc1 impacts on the phosphorylation of two additional bud neck kinases, Hsl1 and Kcc4, which are part of the same family of Nim1-related kinases as Gin4. In addition, PKC1 deletion leads to a dramatic decrease in the levels of Kcc4 , so that it is barely detected at the bud neck.Deletion of PKC1 affects also the interaction between septins and the Bni4 protein, which is a regulatory subunit for the PP1 phosphatase at the bud neck. In turn, we found that Bni4-PP1 modulates Cdc11 phosphorylation, thereby explaining how the latter is decreased in the absence of Pkc1. Altogether, our data strongly suggest that Pkc1 is a novel major regulator of septins in yeast
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Newby, Linda Jane. "Identification of ligands to the PKD1 protein, polycistin-1, using genetically modified cells." Thesis, University of Sheffield, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425192.
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Parker, Sara Shannon. "Crossed Wires: PKMζ Antagonizes Apkc And The Par Complex To Regulate Morphological Polarity." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/594959.
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A cell's composition is not uniform, but is comprised of many molecular gradients to compartmentalize functions into specialized subcellular domains. This organization is called polarity–the asymmetry of morphology and composition. Though it's a feature of nearly all prokaryotic and eukaryotic cells, polarity is plastic and highly dynamic, and is continuously instructed by the crosstalk between extracellular cues and internal effector pathways. One of the master regulators of polarity is the Par complex, canonically comprised of Cdc42, Par6, Par3 and atypical protein kinase C (aPKC). The Par complex defines the apical domain of epithelia and the neuronal axon, directs cell migration and the assembly of cell junctions, and restricts other polarity complexes to their respective domains. We have identified a novel polarity protein that counteracts the activities of the Par complex in cells. PKMζ, a truncated isoform of aPKC normally found in neurons, competes with full-length aPKC for substrate interactions. This competition results in the disruption of the canonical Par complex and its instruction of cell polarity, manifesting as a block in axon specification in developing neurons, or as a loss of the apical-basal axis of epithelial polarity. By eliminating PKMζ's ability to compete with aPKC for interaction with Par3, the effect on polarity is mitigated, while RNAi-mediated reduction of Par3 levels similarly rescues PKMζ-associated defects. We further report that PKMζ is aberrantly transcribed in certain epithelial cancers, and its expression correlates with grade. Malignant epithelial phenotypes are driven by PKMζ's Par3-dependent disruption of polarity, and its Par3- independent promotion of anoikis resistance. We demonstrate that PKMζ, as the catalytic fragment of aPKC, is surprisingly competent to influence polarity independently of its kinase activity, while other aPKC isoforms require their catalytic function to permit apical development. Together, this body of work presents PKMζ as an endogenous inhibitor of Par complex function, whose presence provides bistability to the dynamics of symmetry-breaking.
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Sousa, Mauri Félix de. ""Efeitos renais da haploinsuficiência do gene Pkd1 (Polycystic kidney disease 1) em camundongos"." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/5/5148/tde-21122005-163447/.
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Vários estudos mostram que na doença renal policística autossômica dominante os cistos surgem a partir de um mecanismo de "dois-golpes". A patogênese das manifestações não-císticas, contudo, é pouco compreendida. Neste estudo usamos uma linhagem de camundongos endogâmica com uma mutação nula em Pkd1, onde animais heterozigotos apresentam formação cística renal mínima até 40 semanas de idade. O clearance de inulina e o número de glomérulos foram menores em machos Pkd1+/- que Pkd1+/+, enquanto o volume glomerular médio foi maior em heterozigotos. A excreção urinária de NO2/NO3 não diferiu significantemente entre os dois grupos. Avaliamos a osmolalidade urinária máxima em machos e fêmeas Pkd1+/- and Pkd1+/+, porém não foi detectada diferença significante entre os grupos heterozigoto e selvagem. Nossos resultados oferecem evidência direta de que a haploinsuficiência de Pkd1 resulta em anormalidades anatômicas e funcionais renais e sugerem que o estado haploinsuficiente de Pkd1 possa resultar na redução do número de néfrons por diminuir a ramificação tubular renal durante a nefrogêneseSeveral studies show that in autosomal dominant polycystic kidney disease cysts arise through a "two-hit" mechanism. The pathogenesis of non-cystic features, however, is poorly understood. In this study we used an inbred mouse line with a null mutation of Pkd1, where heterozygotes had minimal renal cyst formation up to 40 weeks of age. Inulin clearance and the number of glomeruli were lower in Pkd1+/- than in Pkd1+/+ males, while a higher average glomerular volume was observed in heterozygotes. The urinary excretion of NO2/NO3 did not significantly differ between the two groups. Maximal urinary osmolality was evaluated in Pkd1+/- and Pkd1+/+ males and females, but no significant difference was detected between the heterozygous and the wild type groups. Our results provide direct evidence that haploinsufficiency for Pkd1 results in anatomic and functional abnormalities of the kidney and suggest that Pkd1 haploinsufficiency may result in a reduced number of nephrons by diminishing renal tubule branching during nephrogenesis
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Riscal, Romain. "L'oncogène Mdm2 : nouvelles fonctions et implications dans le métabolisme des cellules cancéreuses." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONT3512/document.
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L'oncoprotéine MDM2 est reconnue comme un régulateur négatif majeur du suppresseur de tumeur p53, mais plus de preuves indiquent que ses activités oncogéniques vont au-delà de p53. Ici, nous montrons que MDM2 est recruté à la chromatine indépendamment de p53 pour réguler un programme transcriptionnel complexe impliqué dans le métabolisme des acides aminés et l'homéostasie redox. L'identification des gènes cibles de MDM2 au niveau du génome entier met en évidence un rôle important pour les facteurs de transcription ATF3/4 dans le recrutement de MDM2 à la chromatine. Ce recrutement de MDM2 à la chromatine est un processus étroitement régulé qui se produit lors d'un stress oxydatif et lors d'une déprivation en serine/glycine et est modulé par la pyruvate kinase M2 (PKM2) qui est une enzyme métabolique. La déplétion de la protéine MDM2 endogène dans des cellules déficientes en p53 altère le métabolisme sérine/glycine, le rapport NAD+/NADH et le recyclage de la glutathion (GSH), important leurs état redox et leurs potentiel tumorigènique. Nos données illustrent une fonction précédemment insoupçonnée de MDM2 à la chromatine impliquée dans le métabolisme des cellules cancéreusesThe mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD+/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatinbound MDM2 in cancer cell metabolism
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Merzoug, Messaouda. "La protéine kinase D1, PKD1, un acteur essentiel de la physiologie du mélanome et une cible de perturbateurs endocriniens dans les tumeurs du sein." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS045.
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La protéine kinase D1, PKD1, est une sérine/thréonine kinase activée par de nombreux facteurs mitogènes. Les études, menées jusqu’à présent sur les fonctions de PKD1, ont montré qu’elle semble jouer un rôle dans la régulation de plusieurs processus biologiques fondamentaux impliqués dans le développement des tumeurs. Cependant, le rôle précis et les cibles de PKD1 restent largement méconnus. Au cours de ce travail, nous avons tout d’abord démontré que l’inhibition de PKD1 dans les cellules de mélanome inhibe la croissance et la motilité cellulaire, induit l’expression de la E-cadhérine et une diminution de la N-cadhérine. D’autre part, nous nous sommes intéressés au rôle des perturbateurs endocriniens dans les cellules tumorales mammaires et avons démontré que PKD1 est une cible des perturbateurs endocriniens (PE). Les PE, tels que le bisphénol A (BPA) et les phtalates, sont des produits chimiques ubiquitaires de notre environnement. Leur rôle dans la croissance tumorale mammaire est bien documenté. Néanmoins, les mécanismes moléculaires précis par lesquels ces molécules agissent demeurent encore inconnus. Au cours de notre travail, nous avons démontré que ces PE induisent de façon dose-dépendante la prolifération des cellules MCF-7 (cellules d’adénocarcinome mammaire) et que ce processus biologique est dépendant de l’expression de PKD1. Ainsi, l’ensemble de ce travail fait apparaître, d’une part, que PKD1 pourrait être une nouvelle cible thérapeutique anti-tumorale potentielle dans le mélanome et que, d'autre part, PKD1est une cible moléculaire de certains PE dans le cancer du seinProtein kinase D1, PKD1, is a serine/threonine kinase which can be activated by mitogens and that regulates various functions involved in the development of tumors. However, its precise role and targets are still unclear. Our study demonstrates that PKD1 inhibition in melanoma cells decreased cell growth and motility, and reversed the E- to N-cadherin switch. On the other hand, we examined the role of endocrine disruptors (EDs) in breast cancer cells and identified PKD1 as a target of these compounds. EDs, such as bisphenol A (BPA) and phthalates, have been found molecular mechanisms are still ubiquitously throughout our environment. Their role in breast tumor growth has been well documented. However, their precisemolecular mechanisms are still unknown. Our study demonstrates that EDs induce dose-dependently MCF-7 cell growth and that this biological process is dependent upon PKD1expression. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target in melanoma and identifies PKD1 as a new molecular target of some EDs in breast cancer cells
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Rietscher, Katrin [Verfasser]. "Regulation of PKP1´s function in intercellular adhesion, proliferation and barrier formation / Katrin Rietscher." Halle, 2018. http://d-nb.info/1161729615/34.
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Bennehar, Moussab. "Some contributions to nonlinear adaptive control of PKMs : from design to real-time experiments." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS033/document.
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La popularité des robots parallèles s’est considérablement accrue lors des dernières décennies. Cette popularité a été stimulée par les nombreux avantages qu’offrent les robots parallèles par rapport à leurs homologues traditionnels sériels concernant certaines applications industrielles nécessitant de fortes accélérations et une bonne précision. Toutefois, afin d'exploiter pleinement leur potentiel et de tirer le meilleur de leurs capacités, un long chemin reste encore à parcourir. En plus de la conception mécanique, l'étalonnage et l'optimisation de la structure, le développement d’une commande efficace joue un rôle primordial dans l’amélioration de la performance globale des robots parallèles. Cependant, ces derniers sont connus par leur dynamique fortement non linéaire qui s’accroît considérablement lorsque de fortes accélérations sont sollicitées conduisant à des vibrations mécaniques. En outre, les incertitudes sont abondantes dans ces systèmes en raison des hypothèses simplificatrices de modélisation, l'usure des composants du robot et les variations de l'environnement. De plus, leur dynamique couplée et la redondance d'actionnement dans certains mécanismes donnent lieu à des problèmes de commande complexes et difficiles à gérer. Par conséquent, les stratégies de commande développées pour les robots parallèles devraient tenir compte de tous les enjeux et défis mentionnées précédemment. L'objectif principal de cette thèse réside dans la proposition de nouvelles stratégies de commande adaptatives pour les robots parallèles tenant compte de leurs caractéristiques et particularités afin d'améliorer leurs performances de suivi de trajectoires. En outre, les stratégies de commande développées devraient être validées d'abord en simulation, puis à travers des expérimentations temps-réel sur les robots parallèles à notre disposition. Dans ce contexte, trois contributions majeures sont proposées dans le cadre de cette thèse. Tout d'abord, une nouvelle classe de contrôleurs adaptatifs avec des gains de retour non linéaires temps-variant est proposée. La deuxième contribution réside dans le développement d’une version adaptative de la commande robuste RISE. Pour la troisième contribution, la stratégie de commande adaptative L1, récemment développée, est appliquée pour la première fois sur un robot parallèle, suivie de deux nouvelles extensions basées-modèle. Des simulations numériques ainsi que des expérimentations temps-réel sur différents prototypes de robots parallèles sont présentées et discutées. Tous les contrôleurs proposés sont validés pour différents scénarios permettant ainsi de montrer leur pertinence et efficacitéParallel Kinematic Manipulators (PKMs) have gained an increased popularity in the last few decades. This interest has been stimulated by the significant advantages of PKMs compared to their traditional serial counterparts, with respect to some specific industrial tasks requiring high accelerations and accuracy. However, to fully exploit their potential and to get the most of their capabilities, a long path is still to be covered. In addition to mechanical design, calibration and optimization of the structure, efficient control development plays an essential role in improving the overall performance of PKMs. However, PKMs are known for their highly nonlinear dynamics which increases considerably when operating at high accelerations leading to mechanical vibrations. Moreover, uncertainties are abundant in such systems due to model simplifications, the wear of the components of the robot and the variations of the environment. Furthermore, their coupled dynamics and actuation redundancy in some mechanisms give rise to complex and challenging control issues. Consequently, the developed control schemes should take into account all the previously mentioned issues and challenges. The main goal of this thesis lies in the proposal of new adaptive control schemes for PKMs while considering their characteristics and particularities in order to improve their tracking capabilities. Moreover, the developed control strategies should be first validated through numerical simulations, then through real-time experiments on available PKMs. Within this context, three main contributions are proposed in this thesis. First, a new class of adaptive controllers with nonlinear time-varying feedback gains is proposed. The second contribution lies in an adaptive-based extended version of RISE robust feedback control strategy. For the third contribution, the recently developed L1 adaptive control strategy is applied for the first time on a PKM, followed by two novel model-based extensions. Numerical simulations as well as real-time experiments on various PKMs prototypes are provided and discussed. All the proposed controllers are validated for different operating conditions in order to show their relevance and efficiency
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Kesiry, Riad. "GRP78/BiP is Involved in Ouabain-induced Endocytosis of the Na/K-ATPase in LLC-PK1 Cells." University of Toledo Health Science Campus / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1096302498.
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Stewart, Mark Steven. "Mpt5 is a novel positive regulator of the Pkc1-mediated integrity pathway of Saccharomyces cerevisiae." Thesis, University of Glasgow, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252537.
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Balbo, Bruno Eduardo Pedroso. "Camundongos com deficiência em Pkd1 apresentam disfunção cardíaca, fenótipo atenuado por knockout de galectina-3." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5148/tde-09122014-123524/.
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Anormalidades miocárdicas destacam-se entre as manifestações cardiovasculares da doença renal policística autossômica dominante (DRPAD). Para investigar a patogênese dessas manifestações, analisamos o fenótipo cardíaco em camundongos com diferentes perfis de deficiência de Pkd1. Avaliamos o modelo Pkd1cond/cond:Nestincre (CI), com cistos renais e hipertensão, na idade de 20-24 semanas, e heterozigotos para mutação nula em Pkd1 (Pkd1+/-; HT) entre 10-13 semanas, representando um modelo não cístico de haploinsuficiência gênica. Animais Pkd1cond/cond (não cístico; NC) e Pkd1+/+ (selvagem, SV) foram usados como controles. Análises ecocardiográficas de camundongos CI e HT revelaram diminuição da fração de ejeção do ventrículo esquerdo, indicando disfunção sistólica. A relação E/A e o tempo de desaceleração foram consistentes com disfunção diastólica em animais CI. Ecocardiografia por speckle-tracking mostrou redução na deformidade cardíaca (strain) nos modelos CI e HT. Os corações de ambos os grupos apresentaram índices de apoptose maiores e fibrose discreta. Neste cenário, investigamos galectina-3 (Gal-3) como modificador potencial do fenótipo cardíaco na DRPAD. Duplos-mutantes Pkd1cond/cond:Nestincre;Lgals3-/- (CIG-) e Pkd1+/- ;Lgals3-/- (HTG-) cursaram com melhora da função sistólica e de strain comparados a CIs e HTs, não diferindo de NCs e SVs. Animais HTG- apresentaram melhora parcial da função diastólica. Apoptose e fibrose cardíaca mostraram-se reduzidas em CIG-s e HTG-s, alcançando valores similares a NCs e SVs. Análises de western blot revelaram expressão de Gal-3 maior em corações CIs que NCs, porém o mesmo não ocorreu entre HTs e SVs. Os duplos-mutantes não apresentaram diferença na ureia sérica quando comparados a CIs e HTs, assim como nas frações de excreção de Na+, Cl- e K+. Por fim, empregamos um modelo renal cístico grave, homozigoto para um alelo que impede a clivagem da policistina-1 no sítio GPS (Pkd1V/V; VV), e mostramos que a ausência de galectina-3 aumentou a sobrevida em animais Pkd1V/V;Lgals3-/- (VVG-). Nossos resultados demonstram disfunção e alterações de deformidade miocárdica em diferentes modelos de deficiência de Pkd1, à semelhança da DRPAD humana, e revelam que knockout de Gal-3 resgata significativamente este fenótipoMyocardial abnormalities stand out among ADPKD cardiovascular manifestations. To elucidate their pathogenesis, we analyzed the cardiac phenotype in distinct models of Pkd1-deficiency. We evaluated Pkd1cond/cond:Nestincre (CY) cystic, hypertensive mice at 20-24 weeks of age, and Pkd1+/- (HT) noncystic mice at 10-13 weeks, a model of gene haploinsufficiency. Pkd1cond/cond (noncystic; NC) and Pkd1+/+ (wild type, WT) animals were used as controls. Echocardiographic analyses in CY and HT mice revealed decreased left ventricle ejection fraction (LVEF), indicating systolic dysfunction, as well as E/A ratios and deceleration times consistent with diastolic dysfunction in CY animals. Speckle-tracking echocardiography showed reduced cardiac deformability in both models. CY and HT hearts presented higher apoptotic rates and mild fibrosis. In this scenario, we investigated galectin-3 (Gal-3) as a potential modifier of the ADPKD cardiac phenotype. Double mutants Pkd1cond/cond:Nestincre;Lgals3-/- (CYG-) and Pkd1+/-;Lgals3-/- (HTG-) displayed improved systolic and deformability parameters compared to single mutants, while such values did not differ from NCs and WTs. HTG-s presented a partial improvement in diastolic function. CYG- and HTG- hearts showed decreased apoptosis and fibrosis, reaching NC and WT baselines. Western blot analyses revealed higher Gal-3 expression in CY than NC hearts but no difference between HT and WT mice. CYG- and HTG- animals showed no difference in BUN and in the fractional excretion of Na+, Cl- and K+ compared to CYs and HTs. We also employed a more severe renal cystic model, homozygous for an allele that hinders polycystin-1 cleavage at the GPS site (Pkd1V/V; VV), and showed that Pkd1V/V;Lgals3-/- mice present longer survival than VVs. Our findings demonstrate myocardial dysfunction and abnormal deformability in different Pkd1-deficient models, reproducing human ADPKD, and reveals that Gal-3 knockout significantly rescues this phenotype
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Huang, Qi. "Development of a PKM control system : by design, modelling, simulation and integration /." Stockholm, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3138.
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Chihabi, Kutibh. "Protein Kinase Mzeta (PKM-ζ) Regulates Kv1.2 Dependent Cerebellar Eyeblink Classical Conditioning." ScholarWorks @ UVM, 2017. http://scholarworks.uvm.edu/graddis/719.
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Learning and memory has been a topic that has captured the attention of the scientific and public communities since the dawn of scientific discovery. Without the faculty of memory, mammals cannot experience nor function in the world; among homosapiens specifically, language, relationships, and personal identity cannot be developed (Eysenck, 2012). After all, some philosophers such as John Locke argued we are nothing but a collection of past memories in which we have developed and improved upon (Nimbalkar, 2011).
Understanding the cellular mechanisms behind learning, and the subsequent formation of memory, has been a topic that has garnered scientific interest for many decades. One particular kinase that has been at the center of attention in the last decade is the serine/threonine kinase PKM-ζ, an N-terminal truncated form of PKC-ζ that renders it constitutively active (Hernandez et al., 2003). PKM-ζ has long been implicated in a cellular correlate of learning, long-term potentiation (LTP). Inhibition of PKM-ζ with Zeta-inhibitory peptide (ZIP) has been shown in many brain structures to disrupt maintenance of AMPA receptors, irreversibly disrupting numerous forms of learning and memory that have been maintained for weeks.
The voltage-gated potassium channel Kv1.2 is a critical modulator of neuronal physiology, including dendritic excitability, action potential propagation, and
neurotransmitter release. While expressed in various mammalian tissues, Kv1.2 is most prevalent in the cerebellum where it modulates both dendritic excitability of Purkinje cells (PCs) and basket cell (BC) inhibitory input to PCs. Because PCs are the main computational unit of the cerebellar cortex and provide its sole output (Napper et al., 1988; Harvey et al., 1991), regulation of synaptic Kv1.2 is predicted to have a major role in cerebellar function. Pharmacological inhibition of Kv1.2 in cerebellar PC dendrites increases excitability (Khavandgar et al., 2005), while its inhibition in BC axon terminals increases inhibition to PCs (Southan & Robertson, 1998). Interestingly, two prior studies have demonstrated that PKC-ζ, an atypical Protein Kinase C, is able to phosphorylate and bind cerebellar Kvβ2, a Kv1.2 auxiliary subunit. (Gong et al., 1999; Croci et al., 2003).
Delay eyeblink conditioning (EBC) is an established model for the assessment of cerebellar learning. Despite being highly expressed in the cerebellum, no studies have examined how regulation of cerebellar PKM-ζ may affect cerebellar-dependent learning and memory nor have they examined the possible effect PKM-ζ may have on Kv1.2. The goal of this dissertation was to determine whether PKM-ζ could modulate EBC in a Kv1.2 dependent manner. Through the use of microscopy techniques we have shown that PKM-ζ is highly expressed in the cerebellar cortex, primarily in the PC, and by the use of pharmacological manipulations, it was found that PKM-ζ has an important role in regulating the acquisition of EBC. Through the use of biotinylation, flow cytometry, and behavioral manipulations, it was determined that PKM-ζ regulates Kv1.2 during eyeblink conditioning. These studies provided the first evidence that PKM-ζ has a role for learning and memory in the cerebellum, and the first evidence of PKM-ζ regulating a voltage-gated ion channel rather than a ligand-gated ion channel such as AMPA receptors.
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Chen, Xin Jie. "Étude du plasmide pKD1 et développement de systèmes d'expression de gènes chez la levure Kluyveromyces lactis." Paris 11, 1987. http://www.theses.fr/1987PA112299.
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Le nouveau plasmide circulaire, pKD1, isolé chez Kluvveromvces drosophilarum présente une organisation de son génome similaire à celle du plasmide 2 micron chez Saccharomyces cerevisiae, cependant ils ont très peu d'homologie au niveau de la séquence nucléotidique. L'analyse de la séquence nucléotidique de pKD1 montre qu'il a une taille de 4757 paires de bases, qu'il possède trois phases de lecture ouvertes (A, B et C) et une paire de répétitions inversées de 346 paires de bases. La molécule de pKD1 existe sous deux formes isomériques, ceci étant dû à une recombinaison intramoléculaire au niveau des répétitions inversées. La séquence protéique de la phase de lecture ouverte A a une homologie significative avec la protéine recombinase FLP du plasmide 2 micron. Le plasmide pKD1 a été transféré chez Kluyveromyces lactis où il peut être maintenu stablement sans pression de sélection. Nous avons développé un système de transformation efficace chez K. Lactis en utilisant des plasmides dérivés de pKD1. Cela nous a permis aussi de localiser les fonctions de pKD1 (origine de réplication, FLP, REP). De nombreux vecteurs (vecteurs de clonage, vecteurs d'expression, vecteurs pour la détection de promoteur et vecteurs de sécrétion) ont été construits en utilisant des séquences de pKD1 et différents marqueurs génétiques: le gène URA3 de S. Cerevisiae, le gène de résistance à la kanamycine de Tn903 et le gène lacZ d'E. Coli). Nous avons également développé un nouveau système de fusion de gènes à partir du gène de résistance à la kanamycine de Tn903, ce qui nous a permis d'analyser plusieurs promoteurs de S. Cerevisiae et K. LactisThe new circular plasmid, pKD1 (or 1. 6µ), isolated from Kluyveromyces drosophlilarum, has a genome organization analogous to that of the 2 micron plasmid from Saccharomyces cerevisiae, although these plasmids share little sequence homology. Nucleotide sequence analysis of pKD1 revealed that this 4757 base-pair long plasmid contained three major open reading frames, A, B, and C, and a pair of inverted repeats of 346 base-pairs. The molecule exists in two isomeric forms generated by internal recombination at these repeats. The amino acids sequence encoded by open reading frame A has significant homology with the FLP recombinase of the 2 micron plasmid. The plasmid pKD1 can be transferred to Kluyveromyces lactis where it can be stably maintained. Using pKD1-derived recombinant plasmids, we developed an efficient transformation system for K. Lactis. This also allowed us to map the functions of pKD1 (replication origin, REP, FLP). A series of useful cloning vectors, expression vectors, promoter-probe vectors and secretion vectors were constructed using the pKD1 sequence and three different genetic markers: URA3 gene of S. Cerevisiae, kanamycin resistance gene of Tn903, and LACZ gene of E. Coli. A new gene fusion system was developed using the kanamycin resistance determinant of Tn903 as the indicator gene; in this way several promoters from S. Cerevisiae and K. Lactis were analysed
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Chen, Xin Jie. "Etude du plasmide pKD1 et développement de systèmes d'expression de gènes chez la levure Kluyveromyces lactis." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37603889w.
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Lu, Yiyang. "Exploring Rapamycin-induced Pro-survival Pathways in Tuberous Sclerosis Complex and the Development of Alternative Therapies." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1613752713277464.
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Perrichot, Régine. "Analyse moléculaire du gène PKD1 impliqué dans la transmission de la polykystose rénale autosomique dominante (doctorat : sciences de la vie et de la santé)." Brest, 2000. http://www.theses.fr/2000BRES3101.
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Sen, Satarupa. "Regulation of cellular glucose metabolism by HIV-1 infection." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/280025.
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BiologyPh.D.
Regulation of Glucose metabolism is known to play an important role in pathogenesis of many diseases. Primarily because deregulation of this metabolic pathway can lead to either apoptosis or extended life span of the cells involved. Viruses are parasitic in nature, they utilize the host cellular pathways to support their own progeny; hence it is expected that viruses would regulate the central glucose metabolism of infected host cells. Human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome, and it uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+ T cells induces G2 arrest and cell death within 2-3 days, HIV-1 infection of macrophages results in longer survival of infected cells and low constitutive viral production, generating viral reservoirs. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of infected cells by altering the enzymatic activity and protein expression of various glycolytic components. The data suggests that the two HIV-1 target cell types exhibit very different metabolic outcomes. During viral replication in monocyte/macrophage lineage cells we observe increase in glycolytic protein expression and the same proteins show no modulation in T-cell lines post viral replication. Similar differential regulation is observed in case of enzymatic activity of glycolytic enzymes as well. We also conducted proteomic studies in collaboration with the proteomics core. HIV-1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Vpr is known to cause cell cycle block in infected cell and bring about cell death. However, macrophages are resistant to cell death and are viral reservoir, even Vpr over expression does not cause apoptosis in these cell types. The goal of the study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. We observed that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. We then focused on infected monocyte macrophages to identify if glycolytic components such as HK and G6PD were regulated by HIV-1 infection/replication. We report that Hexokinase-1 (HK-1) enzyme expression increases post infection of PBMCs where as the enzymatic activity of HK decreases. Similar effect is seen with HIV-1 replication in latently infected monocyte cell lines U1. The G6PD enzyme activity and expression both increases in infected PBMCs and in U1 cells post induction of viral replication with PMA. We also found that HK-1 translocate to the mitochondria of U1 cells post induction of HIV-1. It is known that the product of HK activity, Glucose 6-phosphate (G6P) releases HKI from the outer leaflet of mitochondria. Hence we conclude that the viral infection decreases HK activity to have less G6P produced in cell and increases G6PD enzyme activity ensuring the remaining G6P is quickly used up, supporting the adherence of outer mitochondrial membrane bound HK1. This sequence of cellular events ensures longer survival of infected cells supporting the viral progeny to propagate in the cell. We further show that suppressing the Pentose phosphate pathway (PPP) by blocking G6PD activity is not only detrimental to the survival of the infected cells it also suppresses viral replication and promoter level transactivation of the viral LTR. Next we sought to identify if glycolytic enzyme PKM2, that is also known to play a nonmetabolic dual role as a protein kinase regulating gene transcription has any effect on the transcription of HIV-LTR. Our study demonstrates upregulation of pyruvate kinase isoform M2 (PKM2) expression in whole cell extracts and nuclear extracts of HIV-1JRFL infected PBMCs and during reactivation of HIV-1 in chronically infected U1 cells. We then focused on understanding the potential role of PKM2 on HIV-1 LTR transactivation. Our studies demonstrate that over expression of PKM2 leads to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -120 bp to -80 bp to be essential for PKM2 mediated transactivation. This region contains the NFKB DNA binding site and mutation of NFKB binding site attenuated PKM2 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis confirmed interaction of PKM2 with HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional co-activator of HIV-1 LTR. Hence it opens up another possible target to curb HIV-1 replication at transcriptional level. This study sheds light on the regulation of glycolytic pathway of host cells by HIV-1 infection and its consequences for the virus, opening up new avenues to target viral replication and identify glycolytic markers of HIV-1 pathogenesis.
Temple University--Theses
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Prades, Julien. "Dynamique linéarisée totale : Application aux robots parallèles." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTS106/document.
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Les travaux de recherche de ce manuscrit se concentrent sur l’analyse des fréquences de vibrations des robots. Nos applications concernent plus particulièrement les architectures à cinématique parallèle. Dans un premier temps nous avons considéré les robots parallèles redondants en actionnement pour lesquels nous envisageons d’augmenter la fréquence de leurs oscillations en utilisant les efforts internes intrinsèques à ce type de structure. L’objectif est d’utiliser leur actionnement pour mettre en tension leur structure, et par conséquent, par analogie avec une corde vibrante, augmenter la fréquence de leurs oscillations. Nous avons étudié plusieurs robots plans redondants et nous montrons que dans le cadre de robots typiquement conçus pour être rigides,l’influence des efforts internes rajoutés n’a que peu d’importance. La suite de nos travaux soutient la proposition suivante : "les trajectoires très dynamiques influencent les fréquences des oscillations de la plateforme mobile". En effet, les robots parallèles quand ils sont conçus pour être légers, peuvent atteindre de grandes accélérations. Nous avons choisi de nous intéresser à l’étude de l’impact que peut avoir les effets dynamiques sur la fréquence des oscillations de la plateforme mobile de ces robots. Les robots considérés pour nos développements sont des robots parallèles plans, redondants en actionnement ou non. Nous proposons d’étudier cette influence en nous basant sur un développement au premier ordre du modèle dynamique. Cette linéarisation du modèle dynamique se veut plus complète que celles proposées dans la littérature. Nous expliquons et vérifions la validité de notre approche par une étude sur le lien entre accélération et vitesse et la fréquence d’oscillation pour les robots série PR (pendule sur glissière verticale) et RR (double pendule en rotation horizontale). Ensuite, nous généralisons notre modélisation au premier ordre et l’appliquons aux quatre robots PRR-2 PRR-3, PRR-4 et Dual-V pour voir si nous sommes capable d’en dégager une tendance concernant l’évolution des fréquences d’oscillation. Nous constatons que, en fonction des trajectoires, la dynamique a une influence faible mais visible, souvent positive sur l’augmentation des fréquences d’oscillation de la plateforme mobile. Cependant, les trajectoires et les lois horaires étant imposées, nous ne pouvons que subir cette influenceThe research work of this thesis manuscript focus on the analysis of the frequency of robots’ vibrations. Our applications mainly revolve around architectures with parallel kinematics. First we examined parallel robots which are redundant in actuation and for which we are considering an increase of their oscillations’ frequency using the internal forces inherent to this type of structure. The aim is to use their actuation is the tensioning of their structure, and consequently, by analogy with a vibrating-wire, to enhance theiroscillation frequency. We have studied several redundancy planar robots and we demonstrate that in the case of robots which are typically designed to be stiff, the impact of added internal forces is of low relevance. The continuation of our research supports the following proposal: “High dynamics trajectories have an impact on the oscillation frequency of the mobile platform.” Indeed parallel robots, when designed to be light, can reach greater accelerations. We chose to concentrate on the study of the impact that dynamic effects canhave on the oscillation frequency of those robots’ mobile platform. The robots examined for our developments are planar parallel robots whether they have redundant actuation or not. We offer to study this impact based on a prime order development of the dynamic model. This linearisation of the dynamic model is intended to be more complete than those suggested by literature. We explain and verify the validity of our approach with a study on the link between speed and oscillation frequency on PR robots (pendulum on a vertical sliding guide) and RR robots ( double pendulum rotating horizontally). Then we will generalize our first order model and apply it to the four robots ( PRR-2 PRR-3, PRR-4, and Dual-V) to see if we are able to identify a pattern regarding the evolution ofoscillation frequencies. We observe that, depending on the trajectories, the dynamics have a low but noticeable, and often positive, impact on the increase of oscillation frequency of the mobile platform. However, since the trajectories and speed input laws are imposed, we have no choice but to be subjected to this impact
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Weston, Benjamin Saul. "Autosomal dominant polycystic kidney disease : biochemical studies of polycystin-1, the product of the human PKD1-gene." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313530.
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Osko, Wioletta Anna [Verfasser], Albert [Gutachter] Brühl, and Sandra [Gutachter] Bensch. "Fachliche Differenzierung im Kontrast zu Klassifikationsergebnissen im Pflegekomplexmaßnahmenscore (PKMS) / Wioletta Anna Osko ; Gutachter: Albert Brühl, Sandra Bensch." Vallendar : Philosophisch-Theologische Hochschule Vallendar, 2021. http://d-nb.info/1231711175/34.
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Milani, Vagner. "Análise mutacional da região 3' do gene PKD1 em pacientes com rins policísticos no sul do Brasil." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2006. http://hdl.handle.net/10183/8896.
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Elattar, Afaf Ibrahim Mohammed Behery. "The role of hematopoietic stem/progenitor cells (HSPCs) in the development of inflammation in non-alcoholic steatohepatitis (NASH)." Thesis, The University of Sydney, 2020. https://hdl.handle.net/2123/22138.
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Non-alcoholic fatty liver disease (NAFLD) is a common chronic hepatic disease that affects about a quarter of the global population. Between 5 and 10% of patients with NAFLD develop non-alcoholic steatohepatitis (NASH), the inflammatory and progressive form that is characterized by liver cell injury/death and inflammation. NASH patients are at higher risks for developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. Indeed NAFLD/NASH is the third leading indication for liver transplantation. NAFLD is also a systemic disease which is able to disrupt metabolic homeostasis and is an independent risk factor for cardiovascular disease and diabetes. Unfortunately, to date, there is no approved drug available for the treatment of patients with NAFLD or NASH. Hence, there is an urgent unmet need to understand the underlying mechanisms of disease in order to identify novel therapeutic targets. Many experimental and clinical data indicated that inflammatory circulating monocytes and monocyte-derived macrophages play a central role in the progression of both NASH and cardiovascular disease (CVD). Indeed, it is well established that the presence of NASH is an independent risk factor of CVD, though the mechanism underlying this effect is unknown. Importantly, multiple clinical trials have been initiated with the liver as the target organ, however, some of them have been terminated due to concerns of an increase in the risk of CVD. Hence, both the US Food and Drug Administration (FDA) and the American Association for the Study of Liver Disease (AASLD) has stated that novel NAFLD/NASH therapies should be at least neutral from a cardiovascular risk perspective and ideally also reduce the risk of CVD in NASH. Hence uncovering the link between these diseases can potentially provide new orthogonal therapeutic targets for NASH and possibly CVD. Orthogonal therapies by definition aim to fine-tune critical nodes involved in multiple related conditions, thus functioning as a rheostat. Bone marrow hematopoietic stem/progenitor cells (BM-HSPCs) are the primary source of myeloid cell production. The proliferation of HSPCs and myeloid-biased hematopoietic stem cells (clonal haematopoiesis) leads to greater myelopoiesis that is causally linked to the development of CVD and atherosclerotic plaque formation. Cholesterol accumulation in HPSCs can also stimulate HSPC proliferation and myelopoiesis. However, no study to date has reported on the relationship between liver pathology and the rate of bone marrow stem cell haematopoiesis during liver injury in NASH. The aim of this thesis was to elucidate the link between NASH progression, innate immunity and the hematopoietic system. In novel data, we were able to demonstrate cross-talk between the liver and the bone marrow hematopoietic system. Indeed, in multiple murine models, the induction of inflammation within the liver in NASH accelerated the rate of myeloid cell production (myelopoiesis) within the bone marrow. Feeding mice a cholesterol-rich (ChR) diet led to the infiltration of immune cells to the liver, the formation of inflammatory foci and higher levels of inflammatory cytokines and chemokines. Mass cytometry (Cytof) and flow cytometric analysis of the liver demonstrated broad infiltration of immune cells especially bone marrow-derived myeloid cells to the liver of this ChR diet-fed mice compared to those fed normal chow (NC), confirming the induction of a systemic inflammatory response. Subsequently, we investigated the profile of immune cells in primary sites such as the bone marrow (BM). Cytof on bone marrow confirmed that the direction of inflammatory changes that happens in the liver is replicated in the immune profile of BM with over-production of myeloid-derived cells at this site. Importantly, we detected the emergence of a population of myeloid cells in ChR diet fed mice which express markers of myeloid-derived cells that infiltrate liver. This enhanced production of immune cells in BM (myelopoiesis) has not been reported in NASH. We next examined the effects of the ChR diet on HSPC homeostasis. Interestingly, this demonstrated that the level of the HSPC population associated positively with the level of liver inflammation. The fact that the ChR diet-induced haematopoiesis might not be surprising, as cholesterol-related pathways in stem cells have been shown to be involved in haematopoiesis. To investigate whether cholesterol is the causal link between myelopoiesis and liver inflammation, we undertook studies using a methionine choline-deficient (MCD) diet and a diet supplemented with 0.1% 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC). In both models, liver injury and inflammation were independent of cholesterol and mirrored many of the morphological features of NASH. In these experiments, we demonstrated that the mere presence of liver injury and an initial hepatic inflammatory response was sufficient to stimulate bone marrow stem cell proliferation and myelopoiesis. These findings demonstrate the existence of cross-talk between liver and bone marrow (BM) that could be a link to explain the simultaneous occurrence of NASH and CVD risk and CVD disease. To find an appropriate target that can reduce liver inflammation and restore hematopoietic homeostasis in BM (to treat NASH and its CVD complications), we first performed RNA-Seq on liver samples and built a biological network using weight gene co-expression network analysis (WGCNA). Biological networks represent a mathematical model of biological systems and their functions. Biological networks are modular which means a group of genes (modules) are involved in similar biological functions. Not surprisingly, we noticed upregulation of a module of genes related to inflammation and the immune response in ChR diet-fed mice. Screening of genes in this network indicated co-expression of genes related to the non-canonical NF-kB pathway and the enzyme pyruvate kinase M2 (PKM2). The latter is a rate-limiting enzyme of the glycolytic pathway. In addition, mining the literature in silico, we noticed that PKM2 plays a role in BM hematopoietic stem cell homeostasis and is hence a promising target. We confirmed the association of RelB of the non-canonical NF-kB pathway with PKM2 in publicly available human and mice datasets. It has been suggested that co-expressed genes in a module are usually co-regulated and are functionally relevant. Thus we investigated the co-regulation of these pathways in myeloid cells in vitro and in vivo in three models of liver injury in mice. To test the effect of modulation of PKM2 activity, we treated mouse bone marrow-derived macrophages (mBMDMs) and human monocytes derived macrophages (hMDMs) with TEPP-46 (which keeps PKM2 in the tetramer form with high glycolytic enzyme activity) and then stimulated the cells with LPS and palmitate as an inflammatory stimulus. Using qPCR, western blotting, ELISA and Seahorse we demonstrated the inhibition of inflammation in the cells treated with TEPP-46. Finally, we performed an intervention in three murine NASH models by oral gavage with TEPP-46 daily for 2 weeks. In these studies, we observed that in vivo modulation of PKM2 alleviates liver injury and restores homeostasis in the bone marrow and reduces inflammation in the liver. In conclusion, our results explore a novel concept of how NASH independently increases cardiometabolic risk. This link we believe at least partly is explained by: 1. Crosstalk between the liver and the bone marrow hematopoietic system that is initiated by the development of hepatic inflammation. 2. This hepatic inflammation and its persistence/progression are associated with alterations of the homeostatic balance in stem cell production, and differentiation of HSPCs towards the myeloid lineage (myelopoiesis) in the bone marrow. This we believe leads to greater production and delivery of inflammatory immune cells to peripheral organs such as the liver and the cardiovascular system, thereby perpetuating inflammatory injury in these tissues. 3. A metabolic regulator, the enzyme PKM2 is a unique modulator of this balance, creating a new class of orthogonal drugs to treat both NASH and cardiovascular disease. 4. Modulation of PKM2 activity can lessen inflammation both locally and can restore homeostasis of BM- hematopoietic stem cells. The following graphical abstract summarizes our findings: See legend on next page Graphical abstract 1: Interplay between the liver, the bone marrow hematopoietic system and immune cells during NASH progression. In response to injurious stimuli that result in NASH (such as a highly processed western diet), hepatic myeloid cells (especially Kupffer cells) become activated and switch their metabolism to glycolysis to yield lactate in the presence of dimeric PKM2 (a glycolytic enzyme). Dimeric PKM2 can translocate to the nucleus, induce the expression of RelB, and activate the non-canonical NF-kB pathway. As a result, there is a greater secretion of inflammatory cytokines (TNF-a, IL-1β, CCL2). This initial liver inflammation signals to the bone marrow to stimulate myeloid-biased hematopoietic stem cells (HSCs), increases myelopoiesis, and ultimately results in exacerbated liver inflammation. Bone marrow myelopoiesis is a critical link to CVD (atherosclerosis) due to the higher flux of myeloid immune cells to plaque. The enzyme PKM2 that is involved in glycolysis and gene transcription was identified as a key regulator of myelopoiesis Targeting PKM2 (by using TEPP-46) can dampen inflammation both locally and can restore normal bone marrow homeostasis of HSCs. Hence, PKM2 could be a potent orthogonal therapeutic target to treat NASH. Abbreviations: HSCs: Hematopoietic stem cells; HSPC: Hematopoietic stem and progenitor cell; GMP: granulocyte monocyte progenitor, PKM2: Pyruvate kinase M2, NF-kB: Nuclear factor kappa-light-chain-enhancer of activated B cells, CVD: Cardiovascular disease, TNF-a: Tumor necrosis factor-alpha, IL-1β: interleukin 1 beta, CCL2: Chemokine (C-C motif) ligand 2, RelB: avian Reticuloendotheliosis viral oncogene homolog B, TEPP-46: Thienopyrrolopyridazinone.
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Pasquier, Adrien. "Lysosomal degradation of insulin granules promotes β-cell failure in type 2 diabetes." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ083/document.
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Notre équipe a récemment découvert l’importance du ciblage des granules d’insuline aux lysosomes lors d’une mise à jeun chez les cellules pancréatiques β. Le diabète de type 2 (TD2) est caractérisé par la résistance à l’insuline couplé au dysfonctionnement des cellules β-et à leur perte. Je souhaitais évaluer le ciblage des granules d’insuline aux lysosomes dans le contexte diabétique. Grâce à un modèle murin, nous avons trouvé que le nombre des lysosomes contenant des granules d’insuline était augmenté chez les cellules β-provenant de souris diabétiques en comparaison aux contrôles. Ceci était accompagné par l’augmentation des niveaux de la protéine lysosomale CD63. Parce que PKD1 contrôle le ciblage des granules d’insuline aux lysosomes lors d’une mise à jeun, nous nous sommes demandé si PKD1 était importante lors d’un diabète de type 2. Dans nos modèles, les niveaux de PKD1 étaient diminués en conditions diabétiques en comparaison aux contrôles. De plus, l’inhibition de PKD1 entrainait l’augmentation du ciblage des granules d’insuline aux lysosomes et accélérait l’apparition du diabète dans notre modèle murin. Nous souhaitions ensuite savoir si l’activation de PKD1 dans les cellules pancréatiques β-pouvait être avantageuse dans un contexte diabétique. De fait, grâce à l’utilisation d’un composé spécifique, nous avons pu montrer que l’activation de PKD1 menait à l’augmentation des niveaux d’insuline sur des ilots pancréatiques humains et ralentissait l’apparition du diabète dans notre modèle murin. Pour conclure, j’ai aussi débuté la caractérisation des lysosomes sur d’autres types cellulaires des ilots pancréatiques. Nous avons observé que LIMP2, une autre protéine lysosomale, était fortement exprimée chez les cellules pancréatiques αOur team recently uncovered the importance of the targeting of insulin granules to the lysosomal compartments in pancreatic β-cells during fasting. Type 2 Diabetes (T2D) is characterised by insulin resistance coupled with pancreatic β-cell failure which account for both β-cells dysfunction and β-cells death. I wanted to assess the targeting of insulin granule to the lysosomes in the context of T2D. Using murine diabetic model, we found that the number Granule-containing Lysosomes was enhanced in diabetic β-cells in comparison to controls. This was accompanied by an increase in the level of the lysosomal protein CD63. Because PKD1 controls the targeting of insulin granule to the lysosomes during fasting, I wondered if PKD1 was important during T2D. PKD1 levels were decreased in our diabetic models in comparison to controls. Moreover inhibition of PKD1 led to enhanced targeting of the insulin granules to the lysosomes and accelerated apparition of diabetes in our murine model. I also tested if activation of PKD1 in pancreatic β-cells could be beneficial in the context of diabetes. Indeed using a specific compound, we showed that PKD1 activation led to an increase in insulin levels and delayed onset of diabetes in our murine model. My work thus uncovered mechanisms underlying a fundamentally new process in β-cells with potential implications for novel therapeutic directions in T2D. Finally, I started to assess lysosomes in another pancreatic islets cell type. I found that LIMP2, another lysosomal membrane protein, was specifically highly expressed in the pancreatic α-cells
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Hackmann, Karl. "Untersuchungen zur Expression der murinen Gene Pkd1 und Pkd2, den orthologen Genen der Autosomal-dominanten-polyzystischen Nierenerkrankung (ADPKD)." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975570323.
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Liu, Xuefeng. "Role of PKMz in morphological and synaptic development of optic tectal neurons in Xenopus laevis tadpoles in vivo." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/28820.
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PKMz (Protein Kinase M zeta) is a recently identified isoform of Protein Kinase C. It is persistently active upon synthesis because its sequence resembles the catalytic domain of PKC zeta but lacks the auto-inhibitory regulatory domain. Previous studies found that PKMz is critical for LTP maintenance, as well as learning and memory in the adult rat brain. However, it is not known whether and how it functions in developing neural systems. I have identified endogenous PKMz in Xenopus laevis tadpoles brain and found that its expression pattern is temporally and spatially correlated with synaptogenesis and dendritogenesis within tadpole retino-tectal system. By in vivo rapid time-lapse imaging and three-dimensional analysis of dynamic dendritic growth, I find that exogenous expression of PKMz within single neurons stabilizes dendritic filopodia by increasing dendritic filopodial lifetimes and decreasing filopodial additions, eliminations, and motility, whereas long-term in vivo imaging demonstrates restricted expansion of the dendritic arbor. Alternatively, blocking endogenous PKMz activity in individual growing tectal neurons with ZIP (zeta-inhibitory peptide) destabilizes dendritic filopodia and over long periods promotes excessive arbor expansion. Consistent with its established roles in regulating adult glutamatergic synaptic transmission, I also examined role of PKMz in regulating developing synapses, using both immunohistochemistry and in vivo patch clamp recording. Specifically, I find that knocking down endogenous PKMz using a morpholino impairs both transmission and maturation of glutamatergic synapses, and consistently induces promoted dendritic expansion as seen in ZIP treated neurons. The model that PKMz regulates dendritogenesis by regulating glutamatergic synaptic transmission was further investigated using a novel seizure model based on Xenopus tadpoles. I find that PTZ induced seizure activity increases normalized expression level of brain PKMz, which is required for over-stabilization of dendritic filopodia dynamics induced by seizure activity. Based on these findings, together with previous results from other related studies, I have constructed a discreet and stochastic computational model to simulate synaptotropic dendritic growth mechanism. I show that as formation of nascent synapses promotes dendritic expansion into region of synaptic partners by promoting maintenance of dendritic filopodia, synapse maturation drives further dendritic refinement and stabilization of appropriate dendritic structure.
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Seiler, Nicole [Verfasser], and Heinz [Akademischer Betreuer] Schmidt. "Pflegekomplexmaßnahmen-Score Erwachsene – PKMS-E : Ein Spannungsfeld zwischen betriebswirtschaftlichen, pflegerischen und ethischen Aspekten / Nicole Seiler ; Betreuer: Heinz Schmidt." Heidelberg : Universitätsbibliothek Heidelberg, 2017. http://d-nb.info/1177690551/34.
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Bastos, Ana Paula Almeida. "A haploinsuficiência de Pkd1 aumenta a lesão renal e induz formação de microcistos após isquemia/reperfusão em camundongos." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/5/5148/tde-25082010-112042/.
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A maior parte dos casos de doença renal policística autossômica dominante (DRPAD) é causada por mutações no gene PKD1 (Polycystic Kidney Disease 1). O insulto por isquemia/reperfusão (IR) constitui-se em uma causa freqüente de lesão renal aguda, incluindo a população de pacientes com DRPAD, mas a relação entre policistina-1 e IR é essencialmente desconhecida. Uma vez que a policistina-1 modula proliferação, diferenciação celular e apoptose em sistemas de cultura de células, sua menor atividade biológica na DRPAD poderia favorecer um maior grau de lesão renal. Utilizamos uma linhagem endogâmica de camundongos 129Sv com uma mutação nula em Pkd1 para testar esta hipótese. Camundongos Pkd1+/- não apresentam cistos renais até 12 semanas de vida, constituindo-se em um modelo puro de haploinsuficiência para este gene. Um insulto IR bilateral de 32 min foi induzido em camundongos machos de 10-12 semanas de idade, heterozigotos e selvagens, por meio do clampeamento reversível de ambos os pedículos renais. Os animais foram analisados 48 h, 7 dias (d) e 14 d após o insulto. Camundongos Pkd1+/- apresentaram FENa, FEK e SCr mais elevadas que animais Pkd1+/+ 48 h após IR. O dano cortical residual foi mais severo em heterozigotos que em selvagens em todos os tempos avaliados. A marcação para PCNA também foi mais alta em camundongos Pkd1+/- que Pkd1+/+ 48 h e 7 d pós-IR, enquanto a taxa de apoptose e a infiltração inflamatória intersticial foram maiores em heterozigotos que em selvagens nos seguimentos de 48 h, 7 d e 14 d pós-IR. A expressão renal de p21 foi menor nos camundongos Pkd1+/- que Pkd1+/+ no tempo de 48 h pós-insulto, tanto no nível transcricional como traducional. Análises adicionais realizadas 6 semanas após o insulto IR revelaram dilatação tubular e formação de microcistos nos camundongos haploinsuficientes para Pkd1, assim como fibrose renal aumentada nesses animais, comparados aos camundongos selvagens. Por fim, um insulto de 35 min de isquemia/reperfusão acompanhou-se de uma mortalidade precoce substancialmente maior nos animais Pkd1+/-. Esses achados sugerem que isquemia/reperfusão induza uma lesão mais severa em rins de camundongos haploinsuficientes para Pkd1, um processo aparentemente dependente de uma deficiência relativa da atividade de p21, assim como dilatação tubular e formação de microcistos. Em conjunto, nossos resultados sugerem que a heterozigose para mutação nula em Pkd1 em camundongo (e talvez em humanos) esteja associada a um risco aumentado para lesão renal por isquemia/reperfusão e a um pior impacto desse insulto sobre a progressão da doença renal.The majority of autosomal dominant polycystic kidney disease (ADPKD) cases are caused by mutations in the PKD1 gene. Ischemia/reperfusion is a frequent cause of acute kidney injury, including the ADPKD patient population, but the relationship between polycystin-1 and ischemia/reperfusion is essentially unknown. Since polycystin-1 modulates cell proliferation, cell differentiation and apoptosis in cell culture systems, its lower biological activity in ADPKD might amplify the degree of renal injury. Using an inbred 129Sv mouse line with a Pkd1-null mutation, 32-min renal ischemia/reperfusion was induced in 10-12 week-old male non-cystic mice, heterozygotes and wild types. The animals were analyzed at 48h, 7 days (d) and 14d after the insult. Pkd1+/- mice showed higher FENa, FEK and SCr than Pkd1+/+ animals at 48h of follow-up. The residual cortical damage was more severe in heterozygotes than wild types at all evaluated time points. The PCNA staining was also higher in Pkd1+/- than Pkd1+/+ mice at 48h and 7d, while cell apoptotic rates and the interstitial inflammatory infiltration were higher in heterozygotes than wild types at 48h, 7d and 14d postischemia/ reperfusion. The expression of p21 was lower in Pkd1+/- than Pkd1+/+ kidneys at 48h, both at the transcriptional and translational levels. Additional analyses performed 6 weeks after the insult showed tubular dilatation and microcyst formation in the haploinsufficient mice, and increased renal fibrosis in these animals compared to wild types. Thirty-fivemin ischemia/reperfusion, at last, was accompanied by a substantially higher early mortality of Pkd1+/- animals. These findings suggest that ischemia/reperfusion induces a more severe injury in kidneys of Pkd1- haploinsufficient mice, a process that is apparently dependent on a relative deficiency of p21 activity, as well as tubular dilatation and microcyst formation. Altogether, our results suggest that mouse Pkd1-null heterozygosity (and maybe human) is associated with a higher risk for renal ischemia/reperfusion injury and with a worse impact of this insult upon renal disease progression.
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Neiva, Luciana Barros de Moura. "Toxicidade da polimixina B em células LLC-PK1 e a enzima heme oxigenase-1." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/7/7139/tde-07052009-112206/.
Full textAbstract:
Na lesão renal aguda, os mecanismos de defesa atuam como genes protetores, como a proteína heat shock 32 (HSP 32), também conhecida como heme oxigenase-1 (HO-1). A polimixina B (PmxB) é um antimicrobiano nefrotóxico. O objetivo deste estudo foi caracterizar a participação da enzima HO-1 na toxicidade da PmxB em células LLC-PK1. As células foram submetidas aos seguintes tratamentos: Controle (CTL- 0µM); Hemin (indutor de HO-1, 25µM); Hemin II (250M), Protoporfirina de zinco (ZnPP - inibidor de HO-1, 10M,); Nitro-L-arginina-metilester (L-NAME - inibidor de iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM de Hemin uma hora antes da PmxB); PmxB + ZnPP (10M de ZnPP uma hora antes da PmxB); PmxB + Hemin + L-NAME (25M de Hemin e 0,1mM de L-NAME uma hora antes da PmxB). Os grupos foram avaliados em 24 e 72 horas. Foram analisados os seguintes parâmetros: desidrogenase láctica (DHL), peroxidação lipídica (MDA), expressão gênica da HO-1 por RT-PCR, síntese protéica da HO-1 por imunofluorescência, óxido nítrico (NO) pelo método de Griess e expressão protéica da HO-1 e da iNOS por western blotting. Os resultados mostraram que a PmxB elevou o DHL com aumento dos níveis de MDA. O Hemin e a ZnPP elevaram as variáveis DHL, MDA e óxido nítrico (NO). O indutor de HO-1 incrementou a expressão protéica da HO-1 e da iNOS. A PmxB se confirmou como citotóxica e a HO-1 intensificou a lesão por mecanismos oxidativos. O efeito da HO-1 na lesão celular parece ser mediado pelo NOIn the acute kidney injury, the mechanisms of defense act as protector genes, as the protein heat shock 32 (HSP 32), also known as heme oxygenase-1 (HO-1). The polymyxin B (PmxB) is a nephrotoxic antimicrobial. The aim of this study was to distinguish the role of the HO-1 enzyme in the PmxB toxicity in LLC-PK1 cells. The cells were submitted to the following treatments: Control (CTL- 0µM); Hemin (inhibitor of HO-1, 25µM); Hemin II (250M), Zinc protoporphyrin (ZnPP - inhibitor of HO-1, 10M,); NG-nitro-L-arginine methyl ester (L-NAME - inhibitor of iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM of Hemin one hour before the PmxB); PmxB + ZnPP (10M of ZnPP one hour before the PmxB); PmxB + Hemin + L-NAME (25M of Hemin and 0,1mM of L-NAME one hour before the PmxB). All groups were evaluated in 24 and 72 hours. The following parameters were analysed: lactate dehydrogenase (LDH), lipid peroxidation (MDA), genic expression of HO-1 by RT-PCR, protein syntesis of HO-1 by immunofluorescence, nitric oxide (NO) by Griess method and protein expression of HO-1 and of iNOS by western blotting. The results showed that PmxB increased the LDH and the levels of MDA. Hemin and ZnPP also increased the LDH variables, MDA and nitric oxide (NO). The inducer of HO-1 improved the protein expression of HO-1 and of iNOS. The PmxB was confirmed as a cytotoxic and the HO-1 intensified the failure by oxidative mechanisms. The effect of HO-1 in the cell injury seemed to be mediated by NO
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Raju, Vanamala Bindinganavile. "The cardiotonic steroid Marinobufagenin (MBG) induces Epithelial-Mesenchymal Transition (EMT) in LLC-PK1 cells." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1211990253.
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Gluth, Markus. "Untersuchungen zum Einfluss von RhoA und der RhoA Effektorkinase PKN auf die TNF-induzierte Barrieredysfunktion in humanen intestinalen Epithelzellen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16529.
Full textAbstract:
Chronisch entzündliche Darmerkrankungen stellen eine Gruppe von chronischen, häufig in Schüben verlaufenden Erkrankungen mit rezidivierenden Entzündungen des Gastrointestinaltraktes dar. Es konnte gezeigt werden, dass eine gestörte Barrierefunktion einen wichtigen Schritt für die Pathogenese darstellt und dass das Zytokin Tumornekrosefaktor alpha (TNF) eine entscheidende Rolle dabei spielt. Die Rolle der kleinen GTPase RhoA bei der TNF-induzierten Barrieredysfunktion ist aufgrund der Komplexität der Signalwege nicht vollständig verstanden. Daher sollte der Einfluss von RhoA und der RhoA Effektorkinase PKN auf diese Prozesse in vitro mit Hilfe eines induzierbaren Expressionssystems untersucht werden, welches die kontrollierte Expression einer konstitutiv aktiven (KA) RhoA- und PKN-Mutante sowie einer dominant negativen (DN) PKN-Mutante ermöglichte. Die Induktion der KA RhoA Expression führte zu einer Störung der epithelialen Barriere. Eine simultane Interferon-gamma und TNF-Behandlung resultierte ebenfalls in einer gestörten Barrierefunktion, welche in KA RhoA Zellen weniger stark ausgeprägt war. Die TNF-Behandlung führte zu einer Aktivierung von PKN, weshalb dieses Protein ein Kandidat für die Vermittlung dieser Effekte darstellte. Inhibition von PKN mit Inhibitoren oder der Expression der DN Mutante führten zu einer Aggravierung der TNF-induzierten Barrieredysfunktion, welche durch eine Verringerung des transepithelialen elektrischen Widerstandes und eine erhöhte Ionenpermeabilität charakterisiert war. Diese Veränderungen wurden von einer Erhöhung des Myosin Leichtketten und NF-kappaB p65-Phosphorylierungsniveaus sowie von morphologischen Veränderungen begleitet. Im Gegensatz dazu konnten diese Veränderungen durch die Expression der KA PKN Variante abgeschwächt bzw. verhindert werden. Diese Ergebnisse liefern Hinweise auf eine potenzielle Rolle der RhoA Effektorkinase PKN bei der Modulation der TNF-induzierten Barrieredysfunktion in intestinalen Epithelzellen.Inflammatory bowel diseases are relapsing systemic inflammatory diseases of the gastrointestinal tract associated with high morbidity and costs. A plethora of studies demonstrated that impaired intestinal barrier function is a key step in the pathogenesis of inflammatory bowel diseases and that the cytokine tumor necrosis factor alphpa (TNF) is of pivotal importance for this effect. Although the small GTPase RhoA has been implicated in the control of tight junction function, its role in TNF induced barrier dysfunction is not entirely understood due to the complexity of its downstream signaling pathways. Therefore, the contribution of RhoA and its effector kinase PKN on TNF induced barrier dysfunction was investigated in vitro. An inducible expression system that allowed the doxycyline controlled expression of a constitutively active (CA) RhoA and PKN mutant as well as a dominant negative (DN) PKN mutant was generated. Induction of CA RhoA expression led to an impaired epithelial barrier. Simultaneous Interferon-gamma and TNF treatment also resulted in barrier perturbation, but this defect was attenuated when CA RhoA was expressed. As treatment with TNF resulted in activation of the RhoA effector kinase PKN, this protein constitutes a candidate molecule for the mediation of these effects. Inhibition of PKN by inhibitory compounds as well as expression of a dominant negative PKN mutant aggravated TNF-induced barrier dysfunction, characterized by a decline in transepithelial electrical resistance and increased ion permeability. These alterations were accompanied by an increase in myosin light-chain and NF-kappaB p65 subunit phosphorylation level as well as morphological changes of the tight junctions. Conversely, expression of a CA PKN mutant attenuated or prevented these changes. These results provide support for a potential role of the RhoA effector kinase PKN in modulating the barrier disrupting effects of TNF in the intestinal epithelium.
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Wang, Haizhen. "The Nucleocytoplasmic Shuttling Functions of P68 in Cancer Cell Migration and Proliferation." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/113.
Full textAbstract:
P68 RNA helicase (p68), as a DEAD family protein, is a typical RNA helicase protein. P68 functions in many other biological processes, which include the regulations of the gene transcription, cell proliferation and cell differentiation. In our group, Y593 phosphorylated p68 was found to have a function in the epithelial mesynchymal transition, which is an important process for cancer metastasis. In the present study, we found that p68 is a nucleocytoplasmic shuttling protein. The protein carries two functional nuclear exporting signal sequences and two nuclear localization signal sequences. Calmodulin, a calcium sensor protein, is well known to play roles in cell migration by regulating the activities of its target proteins at the leading edge. Calmodulin interacts with p68 at the IQ motif of p68. However, the biological function of this interaction is not known. In this study, we found that the p68/calmodulin protein complex functions as a microtubule motor in migrating cells. The shuttling function of p68 along with the motor function of p68/calmodulin causes the leading edge distribution of calmodulin in migrating cells. Disruption the interaction between p68 and calmodulin inhibits cancer cell metastasis in an established mouse model. On the other hand, Y593-Y595 double phosphorylated p68 were found to interact with PKM2, an important tumor isoform of pyruvate kinase. The shuttling function of p68 is reasoned to promote the dimer formation of PKM2 and transport the PKM2 to the cell nucleus. The nuclear PKM2 was found to function as a protein kinase to promote cell proliferation. In specific, the nuclear PKM2 phosphorylates and activates Stat3, an important transcription factor functions in cell proliferation. Overall, p68 is found to have functions in both cell migration and cell proliferation, and these two functions depend on the nucleocytoplasmic shuttling activity and the post-translational modification of p68.