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1
Plammootil, Suma Mary. "Herstellung und Etablierung von 4-Hydroxytamoxifen aktivierbaren PKC[alpha]- [PKC alpha]-, PKC[beta]1- [PKC beta1] und PKCd--Fusionsproteinen [PKC delta-Fusionsproteinen]." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971703302.
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Queirolo, Valeria <1981>. "Caratterizzazione e ruolo di PKCε e PKCδ in modelli di differenziamento megacariocitario normale e patologico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6757/1/Queirolo_Valeria_tesi.pdf.
Full textAbstract:
La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno.
In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica.
Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina.
Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo.
È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine.
I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.Protein kinases C (PKC) are known to be ubiquitously distributed and to have pleiotropic effects. Isoforms epsilon (PKCε) and delta (PKCδ) are involved in the regulation of cell growth, survival and differentiation; in particular, they have been also investigated for their role in the hematopoiesis and in aberrant processes of differentiation along the erythroid and megakaryocytic lineages. In this PhD thesis, the results of an in vitro study about the role of these two kinases in models of megakaryocytic (MK) differentiation, both normal and pathological, are presented. The observations about PKCε and PKCδ kinetics show how these proteins have a specific modulation during the MK differentiation that results in an opposite pattern of expression and, in the murine model if compared with the human model, also a reciprocal one. In particular, in human megakaryocytopoiesis, PKCε results down-modulated, whereas in mouse its levels increase. Instead, PKCδ shows a high and steady expression in maturing CD34+ MK committed, but it is strongly down-modulated during the latest phases of platelet maturation in the murine model. The study also elucidates the different pathways PKCε and PKCδ work through, being an inhibitory action of PKCε on RhoA during proplatelets (ppt) formation in the mouse model while, in the human MK differentiation, platelets production is regulated by PKCδ through Bcl-xL. In this dissertation it is also demonstrated how in an aberrant megakaryocytopoiesis, as in the pathologic model of primary myeloproliferative neoplasm (PMF), PKCε is strongly deregulated and it results in an altered Bcl-xL expression. A forced down-modulation of this kinase restores a normal MK differentiation and ppt maturation. Therefore, the data presented show that PKCε and PKCδ play a key role in proper megakaryocyte maturation and that PKCε could be a potential new therapeutic target for PMF.
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Queirolo, Valeria <1981>. "Caratterizzazione e ruolo di PKCε e PKCδ in modelli di differenziamento megacariocitario normale e patologico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6757/.
Full textAbstract:
La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno.
In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica.
Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina.
Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo.
È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine.
I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.Protein kinases C (PKC) are known to be ubiquitously distributed and to have pleiotropic effects. Isoforms epsilon (PKCε) and delta (PKCδ) are involved in the regulation of cell growth, survival and differentiation; in particular, they have been also investigated for their role in the hematopoiesis and in aberrant processes of differentiation along the erythroid and megakaryocytic lineages. In this PhD thesis, the results of an in vitro study about the role of these two kinases in models of megakaryocytic (MK) differentiation, both normal and pathological, are presented. The observations about PKCε and PKCδ kinetics show how these proteins have a specific modulation during the MK differentiation that results in an opposite pattern of expression and, in the murine model if compared with the human model, also a reciprocal one. In particular, in human megakaryocytopoiesis, PKCε results down-modulated, whereas in mouse its levels increase. Instead, PKCδ shows a high and steady expression in maturing CD34+ MK committed, but it is strongly down-modulated during the latest phases of platelet maturation in the murine model. The study also elucidates the different pathways PKCε and PKCδ work through, being an inhibitory action of PKCε on RhoA during proplatelets (ppt) formation in the mouse model while, in the human MK differentiation, platelets production is regulated by PKCδ through Bcl-xL. In this dissertation it is also demonstrated how in an aberrant megakaryocytopoiesis, as in the pathologic model of primary myeloproliferative neoplasm (PMF), PKCε is strongly deregulated and it results in an altered Bcl-xL expression. A forced down-modulation of this kinase restores a normal MK differentiation and ppt maturation. Therefore, the data presented show that PKCε and PKCδ play a key role in proper megakaryocyte maturation and that PKCε could be a potential new therapeutic target for PMF.
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BONALUME, VERONICA. "GABAA RECEPTOR AS A NOVEL REGULATOR OF PERIPHERAL PAIN SENSITIVITY AND LOCAL NEURON-GLIA INTERACTION." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/699522.
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The pathogenesis of neuropathic pain and its chronicization process are still not fully elucidated. For this reason, therapies currently ongoing in the clinic are aimed to treat mostly the symptoms, considering poorly the target mechanisms causing those pathologies. The approach that we choose to investigate new pharmacological targets for the therapy of chronic pain, was to firstly investigate the mechanisms of nociception in physiological condition, finding new molecular pathways responsible for nociception modulation. Secondly, we were aimed to study the contribution of these targets in the pathological condition. In particular, in this PhD thesis, I focused on the characterization of the GABAergic system, primarily the GABAA receptors (GABAAR), along peripheral nociceptors axons (forming C-fibers), evaluating their putative role in the modulation of pain peripheral conduction, during different physio-pathological conditions. GABAAR mediate fast synaptic inhibition in the dendrites, soma and axons of mature neurons that maintain low intracellular Cl- concentrations. In immature neurons as well as mature somatosensory neurons, the intracellular Cl- concentration is physiologically elevated and this gives rise to depolarizing GABAAR currents. We here report that, in somatosensory unmyelinated C-fibre axons, GABAAR mediates depolarizing currents and that the magnitude and time course of GABAAR responses are determined by NKCC1 activity. GABA (1 µM-1 mM) depolarizing responses were mediated specifically by axonal GABAAR, indeed they were mimicked by muscimol (>1 µM) and gaboxadol (THIP; >1 µM) and blocked by bicuculline (50 µM). Depolarizing axonal responses to GABA were completely absent in mice lacking β3 GABAAR subunit (β3,fl/fl), in either all sensory neurons (AdvillinCRE) or in nociceptor neurons expressing NaV1.8 (snsCRE). qRT-PCR analyses suggested that the most common GABAAR composition in somatosensory DRG neurons was a2, b3 and g2. To examine the physiological role of axonal GABAAR, C-fibres were subjected to a sustained frequency challenge (3 min at 2.5 Hz) and three main effects were found. First, the amplitude of axonal GABA responses was increased, and this effect was deemed secondary to an NKCC1 mediated shift in E Cl-. Second, GABAAR activation increased the axonal conduction velocity of C-fibres. Third, axonal GABAAR was activated by endogenous ligands. Our results indicate that C-fibres sustained firing increases NKCC1 activity, shifting ECl- toward more positive values. In this condition, constitutive GABAAR currents maintain nociceptor conductance during sustained firing. Established the capability of peripheral GABAergic currents to stabilize nociceptor conductance during sustained activity, we investigated the GABAergic modulation in pathological conditions characterized by hypersensitivity of nociceptors. In this regard, we studied in vivo model of inflammatory pain on snsCRE;b3-/- mice. The outcomes indicated that GABAAR activity increment mechanical allodynia and prevent the insurgence of hyperalgesia. In addition, GABAAR activity prolonged the recovery time, maintaining the hypersensitive phenotype for a longer period of time (up to 4 weeks). This finding corroborated the hypothesis that GABAergic transmission within peripheral fibers is able to stabilize the physiological conduction of pain, although it appeared dangerous in pathological condition, promoting nociceptor hypersensitivity and chronicization. We found that ALLO, a neuroactive steroid able to activate GABAAR and endogenously synthesized in PNS, induces the release of the growth factor BDNF from SCs, which is able to target trkB receptors on axons, in turn inducing PKCe upregulation and activation. PKCe is a typical protein kinase known to be involved in the process of pain chronicization. Overall, these set of data suggested that GABAAR is involved in a complex paracrine mechanism mediated by SCs, which activate the GABAAR and subsequently modulate its hyperactivity by the BDNF release. In conclusion, the results presented in this PhD thesis highlight the novel role of peripheral GABAAR in the modulation of nociceptor conduction in different physio-pathological conditions affecting the peripheral nervous system. Moreover, our findings stressed the role of local neuron-glia interaction in such mechanisms. GABAAR is able to dynamically stabilize nociceptor conduction of action potential during sustained activity, preventing excessive C-fiber slowing. On the other hand, GABAAR promotes pain hypersensitive state after neuronal inflammation and prolong the symptoms, likely leading to the pain chronicization. SCs play a fundamental role in the regulation of GABAergic signalling along C-fibers, although further studies are needed to unveil this process. in this direction might leads to the individuation of new pharmacological targets, exploiting endogenous pathways to obtain selective peripheral treatments. Hopefully, the complete comprehension of all the mechanisms would lay the basis for future identification of novel, possibly local, therapeutic strategies for the treatment of peripheral neuropathies and associated chronic pain.
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Le, Good Jessie Ann. "Regulation of atypical PKCs." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313738.
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Kerai, Preeti. "Structural and functional characterisation of PKCI." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325029.
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Crossland, V. M. "Cell cycle specific recruitment of PKCε." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1352790/.
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Protein kinase C (PKC) comprises a family of serine/threonine kinases which play central roles in intracellular signal transduction typically triggered by recruitment to membraneous compartments. The epsilon isoform of PKC (PKCå) has been shown to localize to cell-cell contacts and to the cytokinetic furrow/midbody, indicative of a role in the cell cycle. Both recruitment patterns can be visualized under conditions of PKCå inhibition, which is selectively achieved using a gatekeeper mutant (PKCå-M486A) and the inhibitor NaPP1. Initial studies indicated that interphase and mitotic cells were not distinguished in their capacity for PKCå-M486A recruitment as evidenced by optical trapping experiments. I therefore assessed whether recruitment to the furrow/midbody is a general property reflecting the juxtaposition of two membranes and a cell-cell contact response. I have successfully used fluorescence recovery after photobleaching (FRAP) to distinguish between the localization at the furrow/midbody from that at cell-cell contacts by measuring PKCå-M486A turnover at these two compartments. It is demonstrated that PKCå-M486A has a slower turnover at the furrow/midbody. The distinct kinetic behaviour of PKCå M486A at the furrow/midbody is indicative of other factors contributing to recruitment and/or retention. Sites and domains within PKCå-M486A were therefore assessed for their involvement in this process using a combination of mutagenesis and confocal microscopy. Through these studies I have identified a short motif in the regulatory domain of PKCå-M486A, the inter C1 domain (IC1D), that is in part required for the accumulation of PKCå-M486A at the furrow/midbody. The deletion of this domain (PKCå-ÄIC1D-M486A) prevents the kinase being recruited to the furrow/midbody despite, the recruitment and furrow/midbody localization of the co-expressed PKCå-M486A. Given that the IC1D was previously identified as an actin-binding region, I have assessed the relationship between actin and PKCå-M486A recruitment by manipulating actin polymerization. Using latrunculinA, an inhibitor of F-actin assembly, I have shown that PKCå-M86A and RhoA colocalize and are stabilized in the same compartment in conditions where F-actin is depolymerized. Importantly, the behaviour is observed for both active and inactive PKCε-M486A. This condition may be analogous to a stage in midbody biogenesis and may be evidence of the requirement of F-actin for normal PKCε and RhoA behaviour in cytokinesis. These data show some progress towards understanding the unique behaviour of PKCε at the furrow/midbody and indicate a complex relationship between PKCε, actin and RhoA.
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Pena, Darlene Aparecida. "Anticorpos conformacionais para PKCs clássicas e suas aplicações." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-17082016-074719/.
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A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCβI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínasThe protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCβI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins.
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McCarthy, Joy. "PKCε and cardioprotection : an exploration of putative mechanisms." Doctoral thesis, University of Cape Town, 2006. http://hdl.handle.net/11427/3429.
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Includes bibliographical references.Recent studies have investigated the underlying regulatory mechanisms that may explain the cardioprotective role of PKCε. Sub-proteome analysis has identified interactions between activated PKCε and various mitochondrial proteins, which orchestrate mitochondrial homeostasis, including proteins governing mitochondrial oxidative phosphorylation, electron transfer, ion transport and control of mitochondrial permeability transition (MPT). MPT disruption is regarded as a key step in the initiation of an apoptotic cascade. However, brief pore opening may be beneficial in triggering the generation of small amounts of protective reactive oxygen species (ROS) and restoring calcium homeostasis. PKCε also interacts with adenine nucleotide translocases (ANTs), inner mitochondrial membrane proteins essential for ATP production and an integral component of the permeability transition pore. An augmented capacity to generate ATP would fundamentally enhance resilience to ischemia.
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Snider, Adam K. "PKC gamma regulates connexin 57." Thesis, Manhattan, Kan. : Kansas State University, 2010. http://hdl.handle.net/2097/4128.
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Bahte, Svenja-Katharina Paula. "Identifikation neuer Bindungspartner von PKC- d [PKC-delta] mit Hilfe des Yeast-two-hybrid-Screens." [S.l.] : [s.n.], 2004. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=013081490&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Quittau-Prévostel, Corinne. "Mutant D294G de la PKC[alpha] tumorigénèse humaine : la PKC[alpha], un nouveau suppresseur de tumeur." Montpellier 1, 1997. http://www.theses.fr/1997MON1T005.
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Gobbi, Giuliana <1971>. "Il ruolo della PKCε nel differenziamento eritroide e megacariocitario." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/313/1/TESI_GOBBI_.pdf.
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Gobbi, Giuliana <1971>. "Il ruolo della PKCε nel differenziamento eritroide e megacariocitario." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/313/.
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Gaboardi, Gian Carlo <1978>. "Ruolo della PLCγ1 e della PKCε nel differenziamento miogenico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/1076/1/Tesi_Gaboardi_Gian_Carlo.pdf.
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Phospholipase C (PLC) has been known to be a key effector protein in signal transduction pathway
for cell proliferation and differentiation.
Studies on signalling through the insulin/IGF-1 receptors in muscle differentiation have revealed
that PLCγ1 is involved during this process and that both mRNA and protein levels were increased
during myogenesis. Based on increasing signal transduction pathways that required both PLCγ1 and
PKCε, we investigated its role in insulin stimulation of skeletal muscle differentiation. The precise
effects of insulin on specific PKC isoforms are as yet unknown. Insulin stimulation produced a
gradual increase in PKCε expression and activation of PKCε through skeletal muscle
differentiation. By immunoprecipitation we have demonstrated that endogenous PLCγ1 and PKCε
belong to the same immunocomplex that increase during through myogenic differentiation.
Furthermore, the SH domain of PLCγ1 is involved in the protein complex and that its confine to the
Golgi membrane. PLCγ1 has been involved in cyclin D3 up-regulation. By overexpression and
silencing approach we have evidenced that PKCε modulate the espression of cyclin D3; the kinase
dead form of PKCε doesn’t maintain the same ability. Using a reporter hGH vector we proved that
PKCε acts at transcriptional level by affecting the -37 region of cyclin D3 promoter, as has been
described previous for PLCγ1. In summary this data proved the involvement of PKCε in the
regulation of cyclin D3 expression, together with PLCγ1.
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Gaboardi, Gian Carlo <1978>. "Ruolo della PLCγ1 e della PKCε nel differenziamento miogenico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/1076/.
Full textAbstract:
Phospholipase C (PLC) has been known to be a key effector protein in signal transduction pathway
for cell proliferation and differentiation.
Studies on signalling through the insulin/IGF-1 receptors in muscle differentiation have revealed
that PLCγ1 is involved during this process and that both mRNA and protein levels were increased
during myogenesis. Based on increasing signal transduction pathways that required both PLCγ1 and
PKCε, we investigated its role in insulin stimulation of skeletal muscle differentiation. The precise
effects of insulin on specific PKC isoforms are as yet unknown. Insulin stimulation produced a
gradual increase in PKCε expression and activation of PKCε through skeletal muscle
differentiation. By immunoprecipitation we have demonstrated that endogenous PLCγ1 and PKCε
belong to the same immunocomplex that increase during through myogenic differentiation.
Furthermore, the SH domain of PLCγ1 is involved in the protein complex and that its confine to the
Golgi membrane. PLCγ1 has been involved in cyclin D3 up-regulation. By overexpression and
silencing approach we have evidenced that PKCε modulate the espression of cyclin D3; the kinase
dead form of PKCε doesn’t maintain the same ability. Using a reporter hGH vector we proved that
PKCε acts at transcriptional level by affecting the -37 region of cyclin D3 promoter, as has been
described previous for PLCγ1. In summary this data proved the involvement of PKCε in the
regulation of cyclin D3 expression, together with PLCγ1.
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ZINI, SILVIA. "PKC: Paffard Keatinge-Clay architetto itinerante." Doctoral thesis, Università IUAV di Venezia, 2015. http://hdl.handle.net/11578/278352.
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Burguière, Eric. "Rôle de la DLT cérébelleuse hétérosynaptique des fibres parallèles dans la navigation." Paris 6, 2006. https://tel.archives-ouvertes.fr/tel-00129346.
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Récemment, il a été proposé que le cervelet participe à l’acquisition de fonctions cognitives telle que la navigation. Un des mécanismes de plasticité synaptique du cervelet, la Dépression synaptique à Long Terme hétérosynaptique des fibres parallèles (DLT), est déjà connu pour être impliqué dans les apprentissages moteurs. L’objectif de ce travail de thèse était de déterminer si cette DLT participe également à l’acquisition d’une tâche de navigation. A l’aide de tests de navigation développés dans l’équipe, j’ai étudié les performances de souris transgéniques L7-PKCI dont ce mécanisme de DLT est altéré. Dans ces tests, les souris L7-PKCI étaient déficientes dans la capacité à élaborer une trajectoire efficace pour rejoindre leur but. Ces résultats suggèrent qu’un rôle essentiel du cervelet dans la navigation, et plus particulièrement de la DLT, est d’adapter en permanence la sortie motrice afin d’effectuer une trajectoire optimale.
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Gresham, Rebecca. "The role of PKCι in cell polarity, mitosis and cancer." Thesis, University of Bath, 2010. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.545318.
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Worthmann, Kirstin [Verfasser]. "Die Rolle der atypischen Protein Kinase C Isoformen PKC[lambda]/[iota] und PKC[zeta] in Podozyten / Kirstin Worthmann." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover, 2011. http://d-nb.info/1012625508/34.
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Cabrerizo, Benito Yolanda. "Studies on a PKC-PLD-MAPK pathway." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399767.
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Kostelecky, B. D. "An investigation of PKC isoform functional specificity." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18705/.
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Protein kinase C (PKC) isozymes are vital signalling proteins in many intracellular processes including cell survival, proliferation and migration. As such, changes in their expression levels have been linked to many types of cancer. The various PKC family members provoke differential responses in cancer highlighting the need for study of individual isoforms. This investigation of PKC has aimed to determine how kinase domain structure, regulatory region interactions and binding partners confer functional specificity to individual PKC isoforms. X-ray crystallographic, biochemical and biophysical studies have been employed to explore the architecture of these PKC interactions. A panel of recombinant PKC kinase domains has been cloned, expressed and purified to characterise their maturation, activities and structures. Kinetic constants have been determined for several PKC kinase domains in various phosphorylation states. Additionally, inhibition by novel low molecular weight inhibitors provided by collaborators at Cancer Research Technologies (CRT) has been probed. The PKCζ kinase domain has been crystallised with one of the CRT inhibitors and the structure determined at 2.8Å resolution. A panel of PKC isoform regulatory regions has also been expressed and purified. The results presented here show it is possible to reconstitute an intact PKC holoenzyme complex after expression of the domains as individual polypeptides. The protocols and materials developed during this thesis project will be further used in the laboratory with the aim of crystallising a PKC holoenzyme complex. This thesis also presents the crystal structure of PKCε-binding partner 14-3-3 bound to an asymmetric PKCε di-phosphorylated peptide determined at 2.2Å resolution. The PKCε di-phosphorylated peptide in the crystal structure was derived from the PKCε V3 variable region containing one consensus 14-3-3-phospho-binding motif and one divergent 14-3-3-binding motif. A thermodynamic analysis of the interaction between 14-3-3 and the PKCεV3 di-phosphopeptide reveals an increased affinity more than two orders of magnitude greater than the singly phosphorylated species. Together, the results of this study provide a multifaceted examination of PKC functional specificity by isoform-specific low molecular weight inhibitors, regulatory domains and binding partner interactions and provide a solid platform for exploring further aspects of PKC regulation.
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Roberts, Sarah Anne. "An investigation of protein kinase C in Swiss 3T3 cells using phorbol esters." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369190.
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Dovas, Athanassios. "Regulation of RhoA by PKCa during syndecan-4-mediated cell adhesion." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434916.
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Lamy, Carole. "Synthèse de fluoroalcènes et disaccharides comme peptidomimétiques visant l'interaction PKCε-RACK." Lyon 1, 2008. http://www.theses.fr/2008LYO10049.
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La famille des protéines kinase C est impliquée dans de la transduction d’un grand nombre de signaux cellulaires entre le récepteur et l’appareillage intracellulaire qui effectuera la fonction désirée. Son inhibition fait l’étude de nombreuses recherches, notamment dans la recherche d’inhibiteurs visant une seule des isozymes de la PKC. Une nouvelle stratégie consiste à viser non pas l’inhibition de l’enzyme elle-même, mais plutôt d’empêcher sa localisation sur la membrane cytoplasmique par l’inhibition de l’interaction avec son récepteur, RACK-2. L’objectif de cette thèse est la synthèse de fluoroalcènes et de disaccharides comme peptidomimétiques de l’octapeptide EAVSLKPT, un inhibiteur de l’interaction de PKCε-RACK décrit par Mochly-Rosen et al. , afin de mieux comprendre le rôle de cette interaction dans la transduction du signal. La synthèse de peptidomimétiques fluoroalcènes portant des chaînes latérales fonctionnalisées est inspirée de la voie développée par Waelchli pour la préparation de dipeptides non fonctionnalisés, comme analogues de l’hormone parathyroïde. Une deuxième famille de peptidomimetiques a été conçue sur la base de disaccharides. Ce squelette confère à la molécule une semi-rigidité, car en effet, la liaison aglyconique offre différentes orientations bien définies de la molécule dans l’espace. Les dix positions fonctionnalisables permettent l’accès à un grand nombre de molécules, tandis que sa taille permet de mimer une molécule qui est assez étendue dans l’espace. La modélisation et l’analyse conformationnelle ont permis de trouver une molécule qui se juxtapose au modèle pharmacophore: un D-Glucose-β(1-3)-L-Glucose substitué en position 4 par un tétrahydropyrane et en position 3’ par une chaîne portant un acide carboxyliqueThe protein kinase C family has been implicated in the transduction of a large variety of cellular signals between the receptor and the intracellular machinery that will be responsible for the desired action. This has led to a considerable interest in the inhibition of PKC, in particular in the inhibition of single isoforms of PKC. A novel approach consists of targeting the localization of PKC on the cytoplasmic membrane, rather than the enzyme itself, by inhibiting the interaction of PKC with its receptor, RACK-2. The objective of this work is the design and synthesis of fluoroalkene and disaccharide-based peptidomimetics of the octapeptide EAVSLKPT, an inhibitor of the PKCε-RACK interaction reported by Mochly Rosen et al. The fluoroalkene peptidomimetics were synthesized by a route inspired by Waelchli’s synthesis of unfunctionalized peptidomimetics of the parathyroïd hormone. The second family of peptidomimetics was designed based on a disaccharide scaffold. This skeleton offers a semi-rigid backbone with several well defined conformations around the aglyconic bond, ten functionaliseable positions, and a sufficient size to mimic a relatively linear peptide chain. Pharmacophore modeling and conformational analysis allowed us to identify a particular saccharide, a D-glucose β(1-3) L-Glucose substituted at the 4 position with a tetrahydropyran and the 3’ position with a carboxy-bearing chain, which matched the peptide pharmacophores in a favorable solution conformation
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ZECCHINI, Erika. "Oxidative stress in health and disease: role of PKCζ and p66Shc." Doctoral thesis, Università degli studi di Ferrara, 2010. http://hdl.handle.net/11392/2389327.
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Reactive oxygen species (ROS) are short-living and highly reactive molecules formed by
incomplete one-electron reduction of oxygen. Mitochondria produce low levels of ROS as an
inevitable consequence of oxidative metabolism. Low levels of ROS are normally reduced by nonenzymatic
and enzymatic oxidizing agents, such as glutathione, thioredoxin, superoxide dismutase
(SOD), catalase and peroxidases. Oxidative stress results from exposure to high levels of ROS,
which are not detoxified by cellular antioxidizing agents, and produces cellular damage due to the
oxidation of cellular constituents. ROS, however play not only a damaging role: recent studies have
demonstrated that they also actively participate in a diverse array of biological processes, including
normal cell growth, induction and maintenance of the transformed state, and cellular senescence.
Moreover, ROS can also provide a signaling function, by acting as an intracellular messenger
involved in the transduction of the signaling of cytokines such as TNF-α and IL-1β. We focused
our attention on the effect of ROS in the activities of two proteins: PKCζ and p66Shc. On the one
hand, PKCζ is a serine-threonine kinase belonging to the atypical subfamily of PKC proteins. We
showed that in an in vitro model redox stress induces PKCζ translocation from the cytosol, where it
is located in resting conditions, to the nucleus. Nuclear PKCζ protects cells from apoptotic stimuli
such as H2O2 or ceramide and this protective effect is reverted by the selective inhibition of the
nuclear pool of the protein. The protective effect of nuclear PKCζ is involved in the
chemoresistance of tumor cells to chemotherapic drugs, as demonstrated by the fact that the
selective inhibitor of nuclear PKCζ can revert the chemoresistance, suggesting nuclear PKCζ as a
suitable target in anticancer therapies. On the other hand p66Shc is a Shc protein involved in stress
responses, in particular it is activated by redox stress and it produces ROS itself. Our purpose is to
investigate a possible involvement of p66Shc in two different phenomena: autophagy and adipogenic
transdifferentiation of skeletal muscle cells by comparing wt mice with p66Shc KO mice.
Autophagy is a general term referring to pathways for the degradation of cellular constituents
(cytosol and organelles) by the autophagolysosome; it is activated mainly by nutrient starvation and
it plays a dual role: it is primarly a surviving mechanism, but it also leads to cell death (called type
II cell death) thus possibly acting as an alternative to apoptosis. Starting from this notion, and from
the fact that p66Shc KO cells are protected from apoptosis, we investigated a possible role of p66shc
as a key element in the switch from apoptosis to autophagy. We observed that, while in wt cells
redox stress induces apoptosis, cells lacking p66Shc in the same condition activate the autophagic
pathway. We are now trying to investigate the biological effect of these observation. The second
aspect of our work is the investigation of a putative role of p66Shc in the adipogenic
transdifferentiation of skeletal muscle precursor cells. To this purpose we used an in vivo model and
we observed that mice lacking p66Shc exposed to muscle damage (e.g. freeze injury or redox stress)
show lower adipocyte accumulation than wt mice, thus suggesting a role of p66 in the activation of
adipogenic differentiation pathway. The key purpose of this work is the evaluation of many
different effects of ROS production, looking for possible molecular targets to fight pathological
processes such as chemoresistance and adipogenic degeneration of skeletal muscle.
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Peel, N. R. "Dissecting compartmentalised atypical PKC controls in cell migration." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1419046/.
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Atypical Protein Kinase C (aPKC) isoforms are essential regulators of polarised cell behaviour and in migrating NRK cells translocate to the leading edge in a complex with the exocyst and KIBRA. Engineered delivery of upstream signals to the plasma membrane places leading edge ERK activation downstream of aPKC and demonstrates partial sufficiency in regulating cell migration and adhesion. This model system provides the opportunity to probe the leading edge to better understand events downstream of aPKC. Multiple screening approaches have identified cytoskeletal and translation processes as putative targets of this pathway. Based on in silico candidate screening it is shown that multi-site phosphorylation of Parvin alpha is important for focal adhesion maturation. These phosphorylation events are triggered following acute focal adhesion turnover, which can be blocked by aPKC and MEK inhibition. Based upon proteomic approaches, a novel role for the putative aPKC/ERK substrate Cdc42 effector protein 1 (Cdc42ep1) has been identified. siRNA knockdown of Cdc42ep1 phenocopies aPKC loss; focal adhesions enlarge and turnover less efficiently. This impacts on polarized cell motility as knockdown prevents cell orientation and efficient wound closure. Finally, a novel role for aPKC is reported in relation to leading edge translation. Active translation at the leading edge is reduced following aPKC and MEK inhibition and compartmentalised distribution of translation factors is modulated following pathway intervention. This includes the eukaryotic translation initiation factor 3A (eIF3A), one hit identified by proteomic screening. eIF3A interacts with the exocyst and localises to the leading edge in an aPKC-dependent fashion. In addition, eIF3A is shown to regulate polarised migration and adhesion maturation. The data presented in this thesis illustrate combined screening and validation to delineate compartmentalised signalling events. Localised aPKC/exocyst/ERK activity is necessary for cytoskeletal controls and the polarized delivery and activation of translation machinery at the leading edge.
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Gumpert, Nicolas Maximilian. "Funktionale Bedeutung der Protein Kinase C - d [C - delta] (PKC - d) [(PKC - delta)] in der Reorganisation der zytoskelettären Architektur humaner Keratinozyten." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=964885824.
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Martín, Flix Marta. "Inestabilitat cromosòmica i radiosensibilitat en cèl·lules defectives en ATM i DNA-PKcs." Doctoral thesis, Universitat Autònoma de Barcelona, 2008. http://hdl.handle.net/10803/3817.
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ATM (Ataxia-Telangiectasia Mutated) i DNA-PKcs (DNA-dependent Protein Kinase, catalytic subunit) són dues proteïnes que pertanyen a la família de les PIKKs (PhosphatidylInositol 3-Kinase-related Kinase) i ambdues desenvolupen funcions importants en la via de resposta al dany en el DNA (DDR). ATM i DNA-PKcs són activades en resposta als trencaments de doble cadena del DNA (DSBs) que es produeixen a conseqüència de l'actuació de múltiples factors, entre ells les radiacions ionitzants. Un cop activades, les dues cinases realitzen funcions diferents -però significativament complementàries- en aquesta via: ATM és capaç de: (1) aturar el cicle cel·lular en qualsevol fase; (2) activar diverses proteïnes implicades en la via de reparació homòloga (HR) del DNA i (3) induir l'activació de la via de l'apoptosi cel·lular en cas que el dany sigui massiu i/o irreparable. Per la seva part DNA-PKcs és una proteïna mestra de la via de reparació no homòloga del DNA (NHEJ), on activa i regula a la resta de factors participants d'aquesta via. Finalment DNA-PKcs també pot induir la via de l'apoptosi en resposta a un dany massiu. L'absència d'alguna d'aquestes dues cinases comporta, en els individus afectes, l'aparició de síndromes d'inestabilitat cromosòmica caracteritzats per una major probabilitat de desenvolupar processos cancerígens. Les cèl·lules derivades dels individus afectes són extremadament radiosensibles i acumulen aberracions cromosòmiques. L'objectiu d'aquesta tesi doctoral és determinar quins factors contribueixen de manera específica a la radiosensibilitat i a la inestabilitat cromosòmica de les cèl·lules deficients en ATM i en DNA-PKcs. Per assolir aquest objectiu es va analitzar: (1) l'espectre d'aberracions radio-induïdes en ambdós tipus cel·lulars; (2) l'evolució i resolució d'aquestes aberracions al llarg del temps; (3) la possible implicació del metabolisme telomèric en aquestes aberracions i (4) la cinètica de reunió de DSBs de les cèl·lules deficients en ATM i la de les cèl·lules deficients en DNA-PKcs.
Després de ser irradiats, ambdós tipus cel·lulars acumulen un nombre significativament elevat d'aberracions cromosòmiques. El metabolisme telomèric només contribueix de manera marginal a la inestabilitat cromosòmica en cèl·lules deficients en DNA-PKcs i no té repercussions en la línia cel·lular deficient en ATM emprada en aquest estudi. En analitzar la cinètica de reunió dels DSBs radio-induïts es va fer palès que la deficiència en DNA-PKcs indueix un alentiment de la mateixa. Una reparació més lenta de les lesions en el DNA afavoreix (1) l'acumulació de fragments cromosòmics i, alhora, (2) la reunió il·legítima dels mateixos. La cinètica alentida explica l'ampli espectre d'aberracions radio-induïdes obtingut en aquestes cèl·lules així com la seva persistència en el temps, esdevenint el principal factor responsable de la radiosensibilitat i inestabilitat cromosòmica en cèl·lules deficients en DNA-PKcs. En canvi les cèl·lules deficients en ATM reparen la majoria dels DSBs radio-induïts amb una cinètica comparable a la de les cèl·lules normals, però una fracció dels trencaments roman sense reparar inclús a llargs temps post-irradiació. Per tant, l'acumulació de trencaments pendents de ser reparats a llargs temps post-irradiació (48 i 72 h) apareix com el principal factor responsable de la radiosensibilitat i inestabilitat cromosòmica d'aquestes cèl·lules. Però com s'explica la persistència d'aquests trencaments durant vàries divisions post-irradiació? Un cop descartat un alentiment de la cinètica de reparació ens varem plantejar la possibilitat de que l'absència d'ATM impedís la correcta detecció d'aquests DSBs. Per tal d'avaluar aquesta hipòtesi es va realitzar un anàlisi de la presència de γH2AX i Mre11 en els extrems cromosòmics trencats. La majoria de les delecions cromosòmiques presents en les cèl·lules deficients en ATM presenten marcatge amb les dues proteïnes en el punt de trencament, però una fracció considerable de les mateixes (25%) no presenta cap tipus de marcatge. El resultat obtingut suggereix que els trencaments no senyalitzats no estan essent correctament detectats i que la maquinària de reparació no és activa en ells. Proposem que l'acumulació de trencaments pendents de reparar són una característica de les cèl·lules deficients en ATM, i que aquesta acumulació contribueix de manera important a la seva radiosensibilitat i inestabilitat cromosòmica. Mentre que la fracció de trencaments correctament senyalitzats per γH2AX i Mre11 podrà ser reparada al llarg del temps, la fracció de trencaments sense senyalitzar podria romandre sense reparar durant temps indefinit, contribuint especialment a la inestabilitat cromosòmica d'aquestes cèl·lules.
ATM (Ataxia-Telangiectasia Mutated) and DNA-PKcs (DNA-dependent Protein Kinase, catalytic subunit) belong to the PIKKs (PhosphatidylInositol 3-Kinase-related Kinase) family, and both proteins develop important functions in the DNA damage response pathway (DDR). ATM and DNA-PKcs are activated by the presence of DNA double strand breaks (DSBs), which are produced by multiple factors, ionizing radiations among these. Once activated, both kinases display different -but significantly complementary- functions in the DDR: ATM is able to: (1) halt the cell cycle; (2) activate several proteins implicated in the homologous repair pathway (HR) and (3) if the cell harbours massive and/or irreparable damage, ATM can initiate the apoptosis pathway. Meanwhile, DNA-PKcs is a master protein belonging to the non homologous end joining repair pathway (NHEJ), where it activates and regulates the remaining factors implied in this repair pathway. Finally, if the cell carries extensive damage DNA-PKcs can also induce the apoptosis pathway. Individuals affected by the absence of either kinase develop chromosomal instability syndromes, which are characterized by a special cancer predisposition. Cells obtained from the affected individuals are extremely radiosensitive and accumulate chromosomal aberrations.
The main goal of this doctoral thesis is to determine which factors specifically contribute to the radiosensitivity and chromosomal instability of ATM and DNA-PKcs deficient cells. In order to attain this goal we analyzed: (1) the spectrum of radio-induced aberrations in both cell types; (2) the evolution and resolution of these aberrations over time; (3) the possible implication of telomeric metabolism in these aberrations and (4) the DSBs joining kinetics of ATM and DNA-PKcs deficient cells.
After irradiation both cellular types accumulate a significant number of chromosomal aberrations. While telomeric metabolism contributes -although only marginally- to the chromosomal instability in DNA-PKcs deficient cells, it has no influence on the ATM deficient cell line employed in this study. Analysis of the DSBs joining kinetics demonstrates that DNA-PKcs deficiency induces a delay in the repair kinetics of radio-induced lesions. Slower DNA repair favours the accumulation of chromosomal fragments as well as their illegitimate joining displayed by DNA-PKcs deficient cells. Thus, the slower repair kinetics explain the broad aberration spectrum obtained in these cells, as well as their persistence in time, revealed to be the main factor responsible for radiosensitivity and chromosomal instability in DNA-PKcs deficient cells. On the other hand, ATM deficient cells are able to repair the majority of the radio-induced DSBs with normal joining kinetics except for a fraction of breaks, which remain unrepaired even at long post-irradiation times. Therefore, the accumulation of unrepaired breaks at long post-irradiation times (48 and 72 hr) is revealed to be the main factor responsible for the radiosensitivity and chromosomal instability of AT cells. But how can the persistence of these breaks in an unrepaired state during several cell divisions be explained? After discarding a delayed DSBs joining kinetic, we reflected upon the possibility of the absence of ATM preventing proper detection of unrepaired DSBs. In order to evaluate this hypothesis we analysed the presence of γH2AX and Mre11 signalling in the broken chromosome ends scored in AT cells. The majority of the chromosome deletions displayed both γH2AX and Mre11 labelling at the break point, but a significant fraction (25%) was devoid of any labelling. The results obtained suggest that unlabelled breaks are not being correctly detected and that the cell repair machinery is not active on them. We propose that the accumulation of breaks waiting for efficient repair is a hallmark of ATM deficient cells, and that this accumulation makes a major contribution to their radiosensitivity and chromosomal instability. While the fraction of correctly γH2AX and Mre11-labelled breaks will eventually be repaired, the fraction of unlabelled breaks remains invisible to the DNA damage repair machinery, thus especially contributing to the chromosomal instability of these cells.
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Meng, Zhigang [Verfasser]. "Identification of PKCα as a CK1δ C-terminal targeting kinase / Zhigang Meng." Ulm : Universität Ulm, 2016. http://d-nb.info/1106329945/34.
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Qian, Yu. "Analyser le gène PKC-2 chez Caernorhabditis elegans et crible les mutants contre sérotonine chez le C. elegans souche pkc-2 (ok328)." Phd thesis, Université Claude Bernard - Lyon I, 2009. http://tel.archives-ouvertes.fr/tel-00712129.
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La myopathie de Duchenne est une maladie génétique qui se caractérise principalement par une dégénérescence progressive des muscles squelettiques dont la cause est l'absence de dystrophine fonctionnelle dans les muscles. A ce jour, il n'existe toujours pas de traitement efficace contre ces maladies. Comme le plus grand gène connu chez l'Homme, la dystrophine code pour une protéine de 427kDa. La protéine connecte l'actine avec le DAPC (Dystrophin Associated Protein Complex) dans les muscles striés. Pour l'instant, il y a 3 hypothèses concernant le mécanisme du DMD. L'absence de la dystrophine peut supprimer le lien physique entre les protéines structurales de la membrane basale (laminines) et les protéines structurales du cytosquelette (filaments intermédiaires et actine), ou la distribution et la fonction des canaux ioniques, ou des voies de signalisation nécessaires à la survie du muscle. Caenorhabditis elegans ne possède qu'un homologue du gène de la dystrophine humaine, le gène dys-1. La protéine DYS-1 présente 37% d'homologie avec la dystrophine humaine. Le double mutant dys-1(cx18) ; hlh-1(cc561) présente une forte dégénérescence musculaire. Comme le sarcomère de C. elegans ressemble au sarcomère de mammifère, C. elegans est modèle pertinent d'étude la maladie. En vue de comprendre la raison du DMD chez les mammifères et chez les vers, le groupe L. SEGALAT a effectué des cribles pour identifier les molécules et les gènes qui peuvent supprimer la dégénérescence musculaire. On a trouvé un gène pkc-2 qui est capable de supprimer la dégénérescence musculaire chez C. elegans. La protéine PKC-2 est l'orthologue de la Protein Kinase C Alpha (PKC) humaine et appartient à la famille du serine/threonine protéine kinase. Afin d'étudier la fonction du gène pkc-2, on a analysé l'expression du gène avec les construits différents in vivo et a utilisé la technique de double-hybride dans la levure. De plus, le crible par EMS (éthane méthyle sulfonâtes) a identifié une molécule sérotonine (5-HT) qui est un neuromédiateur, et supprime partiellement la dégénérescence musculaire des doubles mutants dys-1; hlh-1. La sérotonine a aussi un effet fort sur le mutant pkc-2(ok328), puisqu'elle provoque un phénotype blister. Ça nous permet de rechercher le lien entre la signalisation sérotoninergique et pkc-2. Le crible génétique peut contribuer à la connaissance du rôle pkc-2. [...]. Elle sert aussi de plate-forme de voie de signalisation intracellulaire. L'identification de Y59A8A.3 propose la possibilité que pkc-2 modifie la filamin A par l'intermédiaire de la filamin A interacting protéine 1. Le crible génétique par EMS pour rechercher des suppresseurs de l'effet blister de la sérotonine sur les mutants pkc-2(ok328) a donné 8 candidats sur 5000 F1s : cx253, cx254, cx259, cx263, cx267, cx268, cx270, cx276. Les mutations ont été localisées sur les chromosomes par SNP mapping avec une souche de C. elegans très polymorphe, mais le temps a manqué pour leur identification exacte. L'expérience valide notre approche à étudier le lien entre la signalisation sérotoninergique et pkc-2. En résumé, le but de la thèse était de rechercher la fonction du gène pkc-2 dans les mécanismes moléculaires conduisant à la nécrose musculaire en absence de dystrophine. Les résultats présentés dans la thèse apportent des réponses aux questions fondamentales sur pkc-2 et aussi demandent des expériences supplémentaires afin de élucider plus avant les mécanismes de la dégénérescence musculaire dystrophine-dépendante.
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Aaltonen, V. (Vesa). "PKC and neurofibromin in the molecular pathology of urinary bladder carcinoma:the effect of PKC inhibitors on carcinoma cell junctions, movement and death." Doctoral thesis, University of Oulu, 2007. http://urn.fi/urn:isbn:9789514285899.
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Abstract
This study examined the role of tumor suppressor neurofibromin and Protein kinase C (PKC) in urinary bladder cancer, and the effect of PKC inhibitors on cancer cell behaviour.
Tumor suppressor protein neurofibromin is a product of the NF1 gene, a mutation of which causes the most common hereditary tumor syndrome, type 1 neurofibromatosis. NF1 gene mutations and changes in expression have been demonstrated in malignancies, unrelated to type 1 neurofibromatosis. The best known function of neurofibromin is its Ras GTPase accelerating function. Thus, it functions as a Ras inactivator. This study demonstrated for the first time that the NF1 gene is expressed in normal and malignant urinary bladder epithelium and in cultured bladder carcinoma cells in mRNA and at the protein level. Furthermore, neurofibromin expression is decreased during bladder carcinogenesis. It can be speculated that this may lead to increased Ras activity in urinary bladder cancer.
The PKC family is composed of several different isoenzymes which are responsible for a number of important intracellular events and cellular functions. Many of these are also important in cancer development and progression. The results demonstrate changes in expression of PKC α and βI isoenzymes in urinary bladder carcinoma. Furthermore, the results relate the increased expression of isoenzymes to increased PKC enzyme activity and the high proliferation rate of the cancer cells. In addition, this study utilizes small molecular inhibitors of PKC isoenzymes in order to study the effect of the inhibition of these isoenzymes on cancer cell behaviour in vitro and in vivo. The study mainly focuses on the function of PKC α and βI isoenzymes and on the effects of inhibition of these by using Go6976. The results show that Go6976 inhibits cancer cell growth, migration and invasion in vitro, and tumor growth in a mouse model. The use of Go6976 induces changes in desmosomes and adherens junctions, and in focal adhesions and hemidesmosomes. The results also show that Go6976 functions as a cell cycle checkpoint abrogator and increases the cytotoxicity of two classical chemotherapeutic agents, doxorubicin and paclitaxel. In the future, it may be possible that Go6976 or related drugs could be used in clinical cancer treatments.
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Hessabi, Tarik. "Über die Rolle von PKC epsilon während der Wundheilung." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=977243265.
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Garratt-Lalonde, Michelle. "The role of atypical PKC iota in glioblastoma multiforme." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26484.
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Glioblastoma multiforme, a high grade malignant astrocytoma, is the most common and mortal intracranial tumor in adults. The median survival time for glioblastoma patients remains from 9--12 months despite aggressive treatment programs. These high-grade central nervous system tumors bear genetic and molecular aberrations that result in innate chemoresistance to commonly used chemotherapeutic agents.
The main focus of this thesis is on exploring signaling events downstream of phosphoinositide 3-kinase (PI3-K) in glioblastoma multiforme in attempts to discover molecular targets that may be highly specific and thus less toxic therapeutic targets. The PI3-K pathway is constitutively activated in glioblastoma. The Protein Kinase C family of serine/threonine kinases is activated downstream of PI3-K. Our focus is placed on a member of the atypical protein kinase C subfamily called, atypical PKC iota.
We finally began examining the role of atypical PKC iota and invasion in U87MG glioblastoma cells using scratch/wound assays. (Abstract shortened by UMI.)
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Ettinger, Susan Lorraine. "Cytokine-induced activation of PKC isoenzymes in hemopoietic cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ27139.pdf.
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Nakagawa, Rinako. "Role of PKC during B cell development and transformation." Thesis, University of Glasgow, 2006. http://theses.gla.ac.uk/9056/.
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The objective of this thesis is to determine the role of specific PKC isotypes during B cell development and transformation. B cell generation systems were validated both in vitro and in vivo, by coculturing haematopoietic progenitor cells (HPCs) on the calvanial cell line, 0P9, or by adoptively transferring HPCs into recombinase-activating gene 1-deficient (RAG-1-/-) mice, respectively. In both cases, mature B cells were generated as determined by analysing surface B cell marker expression. Coupling of these in vitro and in vivo B cell generation systems with a retroviral gene transfer technique, plasmids-encoding PKC mutants in the retroviral vector MIEV were stably expressed in foetal liver (FL)-derived HPCs from wild type mice and cultured to assess the ability of individual PKC isoforms to modulate the development or transformation of B cells. Of note, expression of a plasmid-encoding dominant negative PKCalpha (PKCalpha-KR) in HPCs and placement in B cell generation system in vitro or in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity. Analysis of PKCalpha-KR-expressing cells in vitro revealed that these cells incorporated BrdU significantly more than the MIEV control, and unexpectedly upregulated cell cycle regulators, p21waf-1 and p27kip-1. Of surprise, PKCalpha-KR-expressing cells phenotypically resemble human B cell chronic lymphocytic leukaemia (CLL) cells. Expression of constitutively active PKCalpha, PKCalpha-CAT, or dominant negative PKCalpha, PKCalpha-KR in HPCs caused significant decrease in cell number. CLL is characterised by the accumulation of long-lived phenotypically mature B cells with the distinctive phenotype: CD19hi, CDS+, CD23+, IgMdim, which are deficient in apoptosis and have undergone cell cycle arrest in the G0/G1 phase. Closer analysis of PKCalpha-KR-expressing cells uncovered that these cells undergo cell cycle arrest in the absence of growth factors and stroma and consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 and Mcl-1 expression. Upon stimulation with IL-7, PKCalpha-KR-expressing cells showed explosive proliferation, suggesting that IL-7 is a proliferation factor for these cells. In accordance with this, IL-7R expression was upregulated in these cells, which may contribute to the increased sensitivity to IL-7. Mice injected with wildtype PKCalpha-KR-HPCs bore solid intraperitonial tumours at the injection site and the cells from both tumour and spleen showed CLL-like phenotype. Interestingly, splenocytes from these mice were cycling whereas the tumour cells were arrested at the G0/G1 stage, probably reflecting the two phases of this disease, a quiescent stage and an extensive proliferative stage, respectively. The expansion of the leukaemic cells was halted when they were cultured on 0P9-DL1, 0P9 cells with ectopic expression of Notch ligand, DL1, suggesting that Notch signalling mediates tumour suppression in CLL cells.
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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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Mermet-Joret, Noëmie. "Etude du développement postnatal des interneurones de la couche II interne dans le sous-noyau caudal du trijumeau chez le rat." Thesis, Clermont-Ferrand 1, 2016. http://www.theses.fr/2016CLF1DD02.
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Les premières semaines postnatales sont essentielles pour le développement de la sensibilité à la douleur et sont associées à une réorganisation structurelle et fonctionnelle des systèmes sensoriels. Les interneurones localisés dans la couche II interne (IIi) du sous noyau caudal du trijumeau (Sp5C), premier relais de l’information tactile et nociceptive orofaciale, sont des éléments clés des circuits responsables de l’allodynie mécanique orofaciale. L’objectif de ce travail de thèse est d’étudier le développement postnatal, à la fois morphologique (en utilisant l’immunohistochimie et l’analyse morphologique tridimensionnelle) et fonctionnel (enregistrements en patch-clamp sur tranches de Sp5C), de ces interneurones. Nous nous sommes d’abord intéressés à une population très particulière d’interneurones de la couche IIi, qui expriment l’isoforme gamma de la protéine kinase C (PKCγ). Au stade le plus précoce de notre étude (3 jours postnataux, P3), les interneurones PKCγ sont présents dans toutes les couches superficielles sauf, précisément, la couche IIi. Ce n’est qu’à P6 que les premiers interneurones PKCγ peuvent être observés dans cette couche. Leur nombre y croît ensuite progressivement jusqu’à P11-15. A cet âge, leur nombre dans la couche IIi est quasiment identique à celui observé aux âges plus tardifs. De plus, nous montrons que cette augmentation du nombre d’interneurones PKCγ dans la couche IIi n’est liée ni à une prolifération cellulaire ni à l’arrivée progressive des fibres afférentes nociceptives dans le Sp5C. Nous avons également étudié le développement des interneurones de la couche IIi dans leur ensemble. Ces neurones sont l’objet d’un grand nombre de changements morphologiques, aussi bien au niveau de leur soma (augmentation du volume) que de leurs neurites (augmentation de leur longueur combinée à une diminution de leur nombre et de leurs ramifications). Sur le plan fonctionnel, les neurones de la couche IIi, à la naissance, sont plus dépolarisés, ont une rhéobase plus basse – ils seraient donc plus excitables – et montrent plus fréquemment un profil de décharge avec un seul potentiel d’action, comparés aux mêmes interneurones chez l’adulte.Toutes ces modifications structurelles et fonctionnelles des interneurones de la couche IIi du Sp5C pourraient contribuer au développement de la sensibilité orofacialeThe first postnatal weeks are pivotal for the development of pain sensitivity and are associated with structural and functional reorganization of sensory systems. Interneurons located in the inner part of lamina II(IIi) of the caudal trigeminal subnucleus (Sp5C), the first central node in orofacial tactile and nociceptive pathways, are key elements in circuits underlying the orofacial mechanical allodynia. The aim of this thesis is to study the morphological (by using immunohistochemistry and tridimensional morphological analysis) and functional (by using whole-cell patch-clamp recordings) postnatal development of these interneurons. First, we looked at a very specific population of lamina IIi interneurons expressing the gamma isoform of the protein kinase C (PKCγ). At the earliest stage of our study (3 postnatal days, P3), PKCγ interneurons are present in all superficial layers but PKCγ interneurons can be observed in lamina IIi only at P6. The number of PKCγ interneurons within this lamina then increases gradually up to P11-15. At this age, the number of PKCγ interneurons in lamina IIi is almost the same as that at later ages. Interestingly, we show that neither cell proliferation nor the gradual projection of nociceptive fibers within the Sp5C accounts for such increase. We also studied the development of the whole population of lamina IIi interneurons. These interneurons undergo a large number of morphological changes, in their soma (increased volume) as well as neurites (concomitant increase in length and decrease in number and branching). Furthermore, according to electrophysiological properties, lamina IIi interneurons, at birth, are more depolarized, have a lower rheobase – suggesting that they are more excitable – and exhibit more frequently a single action potential discharge profile compared with mature ones. All these structural and functional changes of lamina IIi interneurons might contribute to the development of orofacial sensitivity
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Palmer, Romy [Verfasser]. "Untersuchung von PKCα als Schlüsselprotein der β-Arrestin2 vermittelten Nephrin-Endozytose / Romy Palmer." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1053761619/34.
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Duarte, Mariana Lemos. "Identificação e validação funcional de novos alvos das PKCs em célula tronco embrionária." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-16012014-105728/.
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Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCβI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCβI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCβI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a β-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCβI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e β-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na β-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCβI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCβI em CTE indiferenciadas.Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCβI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCβI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCβI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and β-tubulin, as a phosphorylation site of α-tubulin by PKCβI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and β-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in β-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCβI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCβI in undifferentiated ESC.
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Kelly, Joanna. "Non-canonical PKCε activation is required for the cellular response to TopoIIα inhibition." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10045030/.
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The cell cycle is tightly regulated to safeguard the complete and equal division of the nuclear contents between the daughter cells. To ensure accurate segregation of the chromatin, DNA catenation generated during replication must be resolved by topoisomerase IIα (TopoIIα). In some tumour-derived cells, Protein kinase C epsilon (PKCε) has been shown to play a crucial role in mediating a delay signal at the metaphase-to-anaphase transition when catenation is still present, allowing time for catenation resolution prior to anaphase entry and sister chromatid separation. PKCε has also been shown to affect the decatenation process itself, with an increase in metaphase catenation being observed upon PKCε knock down or inhibition. Active PKCε is predominantly membrane associated as lipid binding is required for canonical activation. However, a lipid-independent activation pathway has been observed which involves complex formation with the scaffold protein 14-3-3. In this thesis, we have identified a second lipid-independent pathway. In mitosis, we have identified a caspase-mediated, proteolytic cleavage of PKCε within a chromatin-associated sub-compartment. The cleavage occurs at two distinct sites, one of which leads to the generation of a free kinase domain. Blocking PKCε cleavage at this site, via mutation or caspase-7 inhibition, results in a reduction in the PKCε dependent delay to the metaphase-to-anaphase transition. This can be rescued by artificially recapitulating the cleavage event. Cells expressing a non-cleavable PKCε also retain larger amounts of metaphase catenation, indicating that cleavage is required to support resolution as well as the delay. As both of these phenotypic responses require active PKCε this proteolytic cleavage represents a novel, non-canonical activation pathway. The mechanism by which the free kinase domain generated influences the response to TopoIIα inhibition is still to be fully resolved but phosphorylation of the key mitotic regulator Aurora B and subsequent phosphorylation of TopoIIα have been shown to play a crucial role. Due to an increased dependence on this response in a number of tumour-derived cells, identifying the pathways involved in its regulation may offer a potentially novel target for cancer therapeutics.
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El, Houfi Younas. "PKCα interagit avec la sous-unité catalytique de la m1A58 ARNt méthyltransférase Trm6-Trm61." Thesis, Montpellier 1, 2011. http://www.theses.fr/2011MON1T008/document.
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La protéine kinase C alpha (PKCα) est une sérine/thréonine kinase ubiquitaire. Elle intervient dans la régulation de différentes fonctions cellulaires en interagissant avec de nombreuses protéines. Parmi ces dernières, nous avons réussi à identifier Trm61, la sous-unité catalytique de la m1A58 ARNt méthyltransférase qui joue un rôle essentiel dans la stabilité de l'ARNtiMet. Les études de localisation de PKCα et des deux sous-unités Trm6 et Trm61 ont permis de démonter que ces deux sous-unités ne partagent pas toujours les mêmes compartiments cellulaires : si la sous-unité Trm6 est toujours nucléaire, la Trm61 est pancellulaire et se co-localise avec PKCα dans le cytoplasme. Nous avons apporté la preuve que l'augmentation de l'expression de PKCα entraîne une diminution de Trm61, alors que la diminution de l'expression de PKCα s'accompagne d'une augmentation aussi bien de Trm61 que d'ARNtiMet et se traduit par une importante augmentation de la prolifération à forte densité cellulaire. Ce travail a permis également de démontrer que la sous-unité Trm61 est essentielle pour la survie des cellules C6. La surexpression de Trm6 et/ou de Trm61 a permis de pointer la Trm6 comme le déterminant essentiel du niveau de la m1A58 ARNt méthyltransférase fonctionnelle et de suggérer un rôle secondaire de Trm61 cytoplasmique dans la régulation de la prolifération de façon indépendante de l'action du complexe Trm6-Trm61. De façon intéressante, les gliomes de bas grade présentent des taux plus élevés d'ARNm PKCα que les glioblastomes et inversement pour les taux des ARNm TRM6 et TRM61, apportant un argument en faveur de la relevance de nos observations dans la tumorigenèse gliale humaineProtein kinase C alpha (PKCα) is a ubiquitous serine/threonine kinase. It is involved in the regulation of various cellular functions by interacting with many intracellular proteins. Among these, we were able to identify Trm61, the catalytic subunit of the tRNA m1A58 methyltransferase which plays an essential role in the stability of the tRNAiMet. Localization studies of PKCα, Trm6 and Trm61 demonstrated that these two subunits do not always share the same subcellular compartment: while Trm6 is strictly nuclear, Trm61 is both in the nucleus and in the cytoplasm where it co-localizes with PKCα. We also provided the evidence that the increased expression of PKCα induces a decrease in that of Trm61, while reduced PKCα expression is accompanied by an increase in both Trm61 and tRNAiMet levels. These changes in expression are accompanied by a significant increase in cell proliferation at high-density. This work has also shown that Trm61 subunit is essential for the survival of the C6 glioma cell line. Our results suggest that Trm6 is the essential determinant of functional tRNA m1A58 methyltransferase level and we discuss the possibility of a secondary role for cytoplasmic Trm61 in the regulation of the proliferation independently of Trm6-Trm61 action. Interestingly, human grade II and III gliomas expressed higher levels of PKCα mRNA than glioblastomas and inversely for TRM6 and TRM61 mRNA levels, arguing for a relevance of our observations for human gliomagenesis
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Bom, Vinícius Leite Pedro. "A importância da proteína fosfatase sitA na adesão, integridade da parede celular, biofilme e virulência de Aspergillus fumigatus." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-20072016-092319/.
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Aspergillus fumigatus é um fungo patogênico oportunista capaz de infectar pacientes imunocomprometidos causando eventualmente infecções disseminadas difíceis de serem controladas e com alta taxa de mortalidade dos indivíduos infectados.. Para um melhor entendimento de como esse fungo age no hospedeiro é importante saber como as vias de sinalização que regulam esses fatores de virulência são orquestradas. Proteínas fosfatases são centrais em uma grande variedade de vias de transdução de sinal. Neste trabalho, nós caracterizamos a proteína fosfatase 2A SitA, a proteína homóloga de Sit4 em Saccharomyces serevisiae. O gene sitA não é essencial e por isso fomos capazes de construir um mutante nulo em A. fumigatus. A cepa ?sitA apresenta aumento na fosforilação da MpkA, é mais sensível à agentes que causam dano na parede celular, tem um aumento na quantidade de ?-1,3 glicano e quitina, e também tem problemas na adesão e formação de biofilme. O mutante ?sitA é mais sensível a vários metais e íons, como MnCl2, CaCl2, LiCl, entretanto é mais resistente à ZnSO4. O mutante ?sitA é avirulento em modelo de aspergilose pulmonar invasiva em camundongos. Esses resultados revelam que a fosfatase SitA está envolvida na via de integridade da parede celular de A. fumigatus possivelmente modulando a atividade de PkcA/MpkAAspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients causing eventually disseminated infections that are difficult to be controlled, and lead to high mortality rates. It is important to understand how are orchestrated the signalling pathways that regulate these factors involved in virulence. Protein phosphatases are central to numerous signal transduction pathways. Here we characterize A. fumigatus protein phosphatase 2A SitA, the S. cerevisiae Sit4p homologue. The sitA gene is not an essential gene and we were able to construct an A. fumigatus null mutant. The ?sitA strain had increased MpkA phosphorylation, was more sensitive to cell wall damaging agents, had increased ??1,3?glican and chitin, and was impaired in biofilm formation. The ?sitA strain is more sensitive to several metals and ions such as MnCl2, CaCl2, and LiCl, however, it is more resistant to ZnSO4. The ?sitA strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway
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Kumar, Varun. "Protein Kinase C Signaling in Neurodegeneration." Kent State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1455721051.
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Dykes, Ava Caudill. "Diverse roles of PKC[alpha] in vascular smooth muscle contraction." Huntington, WV : [Marshall University Libraries], 2006. http://www.marshall.edu/etd/descript.asp?ref=680.
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Theses (Ph. D.)--Marshall University, 2006.Title from document title page. Includes abstract. Document formatted into pages: contains xiii, 122 p. including illustrations. Bibliography: Chap.I. p.28-33; Chap. II. p. 82-86; Chap. III p.115-116.
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Brandt, Dominique. "Über die Rolle von PKC bei der Reorganisation des Zytoskeletts." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968536875.
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Hall, Kellie Joann. "The Regulation and Role of Novel PKC Isoforms in Platelets." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486072.
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Platelets are central to physiological haemostasis and respond to stimulation by a variety of soluble and insoluble agonists. Most platelet activatory agonists couple to activation of protein kinase C (PKC) isoforms, including PKC8 and PKCe, members of the novel PKC family. So far the role of PKC8 in platelets has been fairly well characterised although its regulation in platelets, particularly by phosphorylation, is less well understood. Conversely the role of PKCe is less well understood due to a lack of selective inhibitors. This thesis aimed to investigate the regulation ofPKC8 by tyrosine phosphorylation and the role ofPKCe in platelets to improve our understanding of the signalling pathways leading to platelet activation. PKCo was found to be phosphorylated on T~11 and T~65 following thrombin stimulation of human platelets, downstream ofSrc family kinases and phospholipase C signalling. PKCo required membrane recruitment and allosteric modulation (by DAG or PMA) for tyrosine phosphorylation to occur. Tyrosine phosphorylation of PKCo was not required for membrane recruitment of the kinase but was able to potentiate the kinase activity. The presence of the two identified phosphorylation sites in the hinge region (fy(3l1) and the kinase domain (f~6~ suggests that phosphorylation of these sites may stabilise the active conformation ofthe kinase. The role ofPKCe in platelet functional responses is less well characterised than that of PKCo. Using platelets from PKCe-l- mice it was f~und that PKCe negatively regulated integrin activation and a-granule release downstream of GPVI signalling. Interestingly PKce positively regulated thrombin-induced integrin activation however this effect was not seen when using the PAR4 agonist AYPGKF. PKCe was found to positively regulate dense granule secretion downstream of PAR4 but negatively regulate it downstream of TxA2 although the mechanism by which this occurred was not investigated. The findings from this study suggest that PKCe plays a distinct role in platelet activation in an agonist-specific manner.
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Clelland, Lyndsay Jacquelyn. "Role of ROK and PKC in Permeabilized Rabbit Femoral Artery." VCU Scholars Compass, 2007. http://hdl.handle.net/10156/1581.
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Rao, Sudha. "Identification and characterisation of a novel form of PKC-zeta." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312797.
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Scott, Hannah Elizabeth. "PKC-δ, its C2 domain and breast cancer cell lines." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12668/.
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Protein kinase C δ (PKC-δ) is a novel member of the PKC family of serine-threonine kinases. PKC-δ structure is widely conserved within the PKC family and has a catalytic region and regulatory region. The regulatory region has two main sub domains C1 and C2. Although several studies have investigated the role of the C1 domain, little is known about the function of the C2 domain, however there is some evidence that it acts as a protein interaction domain. PKCs are involved in a wide variety of cellular functions within cancer. PKC-δ has been demonstrated to have a particular involvement with the apoptotic processes of a cancer cell. A pro-apoptotic role for PKC-δ has been identified, whereby tyrosine phosphorylation of particular residues induces translocation to the nucleus, alongside a similar translocation event for caspase-3. In the nucleus, caspase-3 cleaves the regulatory and catalytic regions to form a free catalytic domain that is uninhibited by the regulatory portion. This free catalytic region causes the induction of the apoptotic pathway. Conversely an anti-apoptotic role has also been identified for PKC-δ. This was found in MDA-MB-231 cells, which have a ras mutation. Due to this mutation in these cells, ERK1/2 phosphorylation is high. Without PKC-δ activity the high phosphorylation levels induce apoptosis. PKC-δ acts on a pathway to reduce ERK1/2 phosphorylation, thus facilitating cell survival. This study aimed to investigate the role of the PKC-δ C2 domain within breast cancer cells. Through use of these techniques, the role of the C2 domain was examined in order to consider its utility as a drug target to treat breast cancer. •Constructs were developed of pIRESneo2 vector with myc-tagged C2 domain and myc-tagged PKC-δ sequences •Breast cancer cell lines, MDA-MB-468, MDA-MB-231 and MCF-7, were stably transfected with a vector control and myc-δC2 construct. MDA-MB-231 and MCF-7 were also transfected with myc-PKC-δ. •Cell lines developed were examined for alterations due to the presence of the C2 domain or PKC-δ over-expression As the C2 domain is proposed to be a protein interaction domain, we hypothesized that over-expression of this domain would interfere with endogenous PKC-δ interactions, through competitive inhibition, and thus we could identify C2 domain roles. The effect on the cells of the endogenous PKC-δ would be opposed by the C2 domain. Thus the role of endogenous PKC-δ could also be clarified for a particular situation - it would be the opposite of the effects induced by the myc-δC2 on the cells. In the MDA-MB-468 stable cell lines, immuno-fluorescence examination of the myc-δC2 cells showed the myc-δC2 was localised at the ends of actin protrusions from the bulk of the cell. The myc-δC2 expressing cells had a more extensive cytoskeleton than the Vector control cells, possibly suggesting improved attachment to a surface. An experiment examining this illustrated that the myc-δC2 cells appeared to attach in a shorter time period. This implies that the role of the endogenous PKC-δ is to discourage cell attachment and promote an invasive phenotype, this is in agreement with the literature. During sub-culture of the MDA-MB-468 cell lines it became apparent that the myc-δC2 cells were growing at an increased rate over the Vector cell lines. This was quantified and indeed the myc-δC2 cells did increase in cell number more than the Vector cells. This was also the case with the MDA-MB-231 cells but not with the MCF-7 cells. Growth is a balance of proliferation and apoptosis. This effect indicates that PKC-δ is pro-apoptotic, or anti-proliferative. MCF-7 cells lack caspase-3 and thus pro-apoptotic effects of PKC-δ would be affected in this cell line. As the myc-δC2 did not have an effect in these cells we examined apoptosis to see if these effects could be attributed to differences in apoptosis. The MDA-MB-468 cells expressing myc-δC2 had higher viability than the Vector cells. This fits with the cell number data, indicating that a lower level of apoptosis has led to a greater cell number, and advocates a pro-apoptotic role for PKC-δ. This was not the case in MDA-MB-231 cell lines where Vector cells had higher viability. This agrees with the literature describing PKC-δ displaying an anti-apoptotic role in this cell line, but does not fit with the cell number data. Thus, differences in cell number are likely due to effects on proliferation, although this was not investigated. MCF-7 cells showed no differences indicating the apoptotic program of these cells is indeed affected by the lack of caspase-3. Serum starvation is a commonly used method to induce apoptosis. MDA-MB-468 cell lines were serum starved in order to examine the effects. The apoptosis profile was altered and myc-δC2 cells showed lower viability than the Vector cells, indicative of an anti-apoptotic effect of PKC-δ. An anti-apoptotic effect is observed in MDA-MB-231 cells, where the effect was proteasome dependent. This was also the case in this situation. The anti-apoptotic effect is related to levels of phosphorylated ERK1/2, where high levels are pro-apoptotic. The phosphorylation status was examined and illustrated a much higher level of phosphorylation in myc-δC2 cells over Vector cells when starved. This indicates myc-δC2 is inhibiting the de-phosphorylating role of PKC-δ in these apoptotic cells. Thus it appears that the C2 domain acts as a ‘sensor’ to serum status, appropriating PKC-δ effects in apoptotic pathways according to the serum status, the method of which is unknown. This study has highlighted the importance of the PKC-δ C2 domain in breast cancer apoptosis. The effects appear to require a fully active PKC-δ pro-apoptotic pathway, and are dependent on the serum status of the cells. Further investigation would be required to identify a level of serum for use in vitro that is relevant to an in vivo tumour situation. If low levels are more relevant to a clinical state then it may be possible to target the C2 domain with drugs to allow induction of apoptosis. The differences between the cell lines clearly show that the phenotypic analysis of tumours would be vital to identifying whether such treatment would be applicable, as effects of any drug would vary greatly across tumour types. MDA-MB-468 and MDA-MB-231 are both triple negative cell lines (i.e. they do not express progesterone, oestrogen or HER2 receptors); however the strong differences seen in this case indicate further phenotypic analysis would be essential.