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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

The oxygen sensor PHD2 affects energy metabolism and cell function in macrophages

Güntsch, Annemarie 23 September 2016 (has links)
No description available.
2

Spatial Regulation of the Polarity Protein aPKC During Asymmetric Cell Division of Drosophila Neuroblasts

Drummond, Mike 18 August 2015 (has links)
The Par complex protein, atypical protein kinase C (aPKC), plays an instrumental role in diverse cell polarities. aPKC is able to restrict substrate localization through a phosphorylation-induced cortical exclusion mechanism, allowing for the generation of molecularly distinct cortical domains. Thus, controlling the localization of aPKC is central to Par-mediated polarity but the mechanism by which aPKC is polarized remains poorly understood. In this dissertation I investigated the restriction of aPKC to the apical cortex of Drosophila neural stem cells, neuroblasts, as these cells dynamically polarize aPKC through repeated asymmetric cell divisions. The polarity created through aPKC phosphorylation must be tightly regulated in order to ensure proper balance between self-renewal and differentiation. To begin, I investigated whether or not aPKC’s so called ‘maturation’ by PDK1 phosphorylation is required for aPKC activity and localization. We found that aPKC’s phosphorylation by PDK1 is required for both polarity and full activity. An aPKC containing an unphosphorylatable activation loop mutation localizes symmetrically around the cortex in a manner independent of its binding partner, Par-6, suggesting that aPKC could interact with the cortex by an unknown mechanism. To investigate how aPKC is able to localize to the cortex independent of Par-6, I used an in vivo structure function analysis of domains within aPKC, accompanied by biochemical approaches. I identified a necessity for the aPKC C1 domain for binding to the neuroblast cortex. This interaction is mediated by negatively charged phospholipids. Neither aPKC interaction, with phospholipids or Par-6, is sufficient to restrict aPKC to the apical cortex. Thus, aPKC polarization utilizes a dual interaction mechanism that takes advantage of both protein-lipid and protein-protein interactions, and proper control of each of these signals is required to prevent neuroblast division defects. One interaction, mediated by the C1, is a general cortical targeting mechanism, whereas the other specifies polarization mediated by Par complex interactions. We conclude that a conformational change induced by these interactions activates aPKC’s catalytic activity, thereby coupling localization and activity. This dissertation includes unpublished co-authored material.
3

SHP-1 and PDK1 Form a Phosphotyrosine-Dependent Nucleo-Cytoplasmic Shuttling Complex: Implications for Differentiation

Sephton, Chantelle Fiona 28 June 2007
SHP-1 is a protein tyrosine phosphatase that often targets the phosphatidylinositol 3'-kinase (PI3K)/Akt signalling pathway. PI3K/Akt signalling regulates cell growth and survival, proliferation and differentiation. Growth factor-stimulated PI3K phospholipid production at the plasma membrane helps to recruit 3'-phosphoinositide-dependent protein kinase-1 (PDK1) and Akt, where PDK1 phosphorylates and activates the pro-survival kinase Akt.<p>Tyrosine phosphorylation of PDK1 may regulate its function and, perhaps more importantly, its nuclear localization. Yet, it is unclear how PDK1 is imported into the nucleus as it does not contain a nuclear localization signal (NLS), although it does contain a nuclear export signal (NES). Interestingly, several tyrosines in PDK1 are targets for Src kinase and are putative target motifs for SHP-1, which does have an NLS.<p>Hypothesis: SHP-1 and PDK1 form a tyrosine-dependent, nucleo-cytoplasmic shuttling complex. <p>Removal of serum from C6 glioma cell cultures induces a platelet-derived growth factor receptor (PDGFR)-sensitive redistribution of PI3K lipid kinase activity to the nucleus. PDK1 tyrosine phosphorylation and its association with SHP-1 are also increased, as is the accumulation of both SHP-1 and PDK1 in the nucleus. Site-directed mutagenesis of tyrosine residues in PDK1 reveals that tyrosine 9 (Tyr9) and Tyr376 are important for the interaction of PDK1 with SHP1, whereas Tyr333 and Tyr 373 are not. Using pharmacological and genetic manipulations, it was demonstrated that SHP-1 and PDK1 shuttle between the nucleus and cytoplasm, and that the C-terminal-expressed NLS of SHP-1 facilitates shuttling, while dephosphorylation of PDK1 Tyr9 and Tyr376 regulates the rate of PDK1 (and by virtue of association, SHP-1) export from the nucleus. The SHP-1/PDK1 complex, which is constitutive in most cell lines, is functionally relevant as indicated by its requirement for NGF-induced differentiation of preneuronal cells to a neuronal phenotype.
4

SHP-1 and PDK1 Form a Phosphotyrosine-Dependent Nucleo-Cytoplasmic Shuttling Complex: Implications for Differentiation

Sephton, Chantelle Fiona 28 June 2007 (has links)
SHP-1 is a protein tyrosine phosphatase that often targets the phosphatidylinositol 3'-kinase (PI3K)/Akt signalling pathway. PI3K/Akt signalling regulates cell growth and survival, proliferation and differentiation. Growth factor-stimulated PI3K phospholipid production at the plasma membrane helps to recruit 3'-phosphoinositide-dependent protein kinase-1 (PDK1) and Akt, where PDK1 phosphorylates and activates the pro-survival kinase Akt.<p>Tyrosine phosphorylation of PDK1 may regulate its function and, perhaps more importantly, its nuclear localization. Yet, it is unclear how PDK1 is imported into the nucleus as it does not contain a nuclear localization signal (NLS), although it does contain a nuclear export signal (NES). Interestingly, several tyrosines in PDK1 are targets for Src kinase and are putative target motifs for SHP-1, which does have an NLS.<p>Hypothesis: SHP-1 and PDK1 form a tyrosine-dependent, nucleo-cytoplasmic shuttling complex. <p>Removal of serum from C6 glioma cell cultures induces a platelet-derived growth factor receptor (PDGFR)-sensitive redistribution of PI3K lipid kinase activity to the nucleus. PDK1 tyrosine phosphorylation and its association with SHP-1 are also increased, as is the accumulation of both SHP-1 and PDK1 in the nucleus. Site-directed mutagenesis of tyrosine residues in PDK1 reveals that tyrosine 9 (Tyr9) and Tyr376 are important for the interaction of PDK1 with SHP1, whereas Tyr333 and Tyr 373 are not. Using pharmacological and genetic manipulations, it was demonstrated that SHP-1 and PDK1 shuttle between the nucleus and cytoplasm, and that the C-terminal-expressed NLS of SHP-1 facilitates shuttling, while dephosphorylation of PDK1 Tyr9 and Tyr376 regulates the rate of PDK1 (and by virtue of association, SHP-1) export from the nucleus. The SHP-1/PDK1 complex, which is constitutive in most cell lines, is functionally relevant as indicated by its requirement for NGF-induced differentiation of preneuronal cells to a neuronal phenotype.
5

Regulation of osteoclast activation and autophagy through altered protein kinase pathways in Paget's disease of bone

McManus, Stephen January 2016 (has links)
Résumé : La maladie osseuse de Paget (MP) est un désordre squelettique caractérisé par une augmentation focale et désorganisée du remodelage osseux. Les ostéoclastes (OCs) de MP sont plus larges, actifs et nombreux, en plus d’être résistants à l’apoptose. Même si la cause précise de la MP demeure inconnue, des mutations du gène SQSTM1, codant pour la protéine p62, ont été décrites dans une proportion importante de patients avec MP. Parmi ces mutations, la substitution P392L est la plus fréquente, et la surexpression de p62P392L dans les OCs génère un phénotype pagétique partiel. La protéine p62 est impliquée dans de multiples processus, allant du contrôle de la signalisation NF-κB à l’autophagie. Dans les OCs humains, un complexe multiprotéique composé de p62 et des kinases PKCζ et PDK1 est formé en réponse à une stimulation par Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), principale cytokine impliquée dans la formation et l'activation des OCs. Nous avons démontré que PKCζ est impliquée dans l’activation de NF-κB induite par RANKL dans les OCs, et dans son activation constitutive en présence de p62P392L. Nous avons également observé une augmentation de phosphorylation de Ser536 de p65 par PKCζ, qui est indépendante d’IκB et qui pourrait représenter une voie alternative d'activation de NF-κB en présence de la mutation de p62. Nous avons démontré que les niveaux de phosphorylation des régulateurs de survie ERK et Akt sont augmentés dans les OCs MP, et réduits suite à l'inhibition de PDK1. La phosphorylation des substrats de mTOR, 4EBP1 et la protéine régulatrice Raptor, a été évaluée, et une augmentation des deux a été observée dans les OCs pagétiques, et est régulée par l'inhibition de PDK1. Également, l'augmentation des niveaux de base de LC3II (associée aux structures autophagiques) observée dans les OCs pagétiques a été associée à un défaut de dégradation des autophagosomes, indépendante de la mutation p62P392L. Il existe aussi une réduction de sensibilité à l’induction de l'autophagie dépendante de PDK1. De plus, l’inhibition de PDK1 induit l’apoptose autant dans les OCs contrôles que pagétiques, et mène à une réduction significative de la résorption osseuse. La signalisation PDK1/Akt pourrait donc représenter un point de contrôle important dans l’activation des OCs pagétiques. Ces résultats démontrent l’importance de plusieurs kinases associées à p62 dans la sur-activation des OCs pagétiques, dont la signalisation converge vers une augmentation de leur survie et de leur fonction de résorption, et affecte également le processus autophagique. / Abstract : Paget’s disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover. In PDB, osteoclasts are larger, more active, more numerous, and resistant to apoptotic stimuli. While no single root cause has been identified, mutations to the gene encoding the p62 protein, SQSTM1, have been described in a significant population of patients with PDB. Among these mutations, the P392L substitution is the most prevalent, and overexpression of p62P392L in osteoclasts generates at least a partial pagetic phenotype in vitro. Normally this protein mediates a number of cell functions, from control of NF-κB signaling to autophagy. In human osteoclasts, a multiprotein complex containing p62 and protein kinases PKCζ and PDK1 (the principal kinase of Akt), form in response to stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL), the principal osteoclastogenic-signaling cytokine. We found that PKCζ is involved in RANKL-induced activation of NF-κB, and that it contributed to a basal activation of NF-κB observed in p62P392L mutants. This may be regulated in part by a PKCζ dependent increase in p65 phosphorylation at Ser536 which we characterized, independent of IκB. This could represent one alternative pathway by which mutant p62 leads to increased NF-κB activation. We observed increased basal phosphorylation of survival regulators ERK and Akt in PDB that was reduced upon PDK1 inhibition. The activity of 4EBP1 and Raptor, associated with mTOR activity, were also altered in pagetic osteoclasts and regulated by PDK1 inhibition. We then identified autophagic defects common to pagetic osteoclasts; with higher basal levels of LC3II (associated with autophagic structures), regardless of p62 mutation, and reduced sensitivity to autophagy induction in PDB. These results suggest an accumulation of non-degradative autophagosomes. Inhibition of PDK1 not only induced apoptosis in PDB and controls, but significantly reduced resorption in PDB, and with regards to autophagy, PDK1 inhibition was more potent in PDB than in controls. Therefore PDK1/Akt signaling represents an important checkpoint to PDB osteoclast activation. In sum, these results demonstrate the importance of several p62-associated kinases in the over-activation of pagetic osteoclasts, through increased survival and altered signaling. As p62 mutations alone do not account for most cases of PDB, the characterization of these pathways may identify a common factor linking pagetic osteoclasts. Therefore these studies represent a novel approach to osteoclast apoptosis, activation, and autophagy associated with PDB.
6

Regulation of PDK1 Protein Kinase Activation by Its C-Terminal Pleckstrin Homology Domain

Al-Ali, Hassan 28 April 2010 (has links)
Phosphoinositide-dependent protein kinase-1 (PDK1) plays an integral role in signaling cellular growth and proliferation, one that's dependent on its ability to autophosphorylate Ser-241 in its T-loop. This process appears to have a strict requirement for its C-terminal pleckstrin homology (PH) domain. Thus, the overall objective of this work was to determine the mechanism by which the PH domain induces an active kinase conformation in unphosphorylated PDK1, capable of Ser-241 autophosphorylation. First, computational modeling and protein cross linking studies were combined with site-directed mutagenesis and kinetic assays in order to provide initial assessment of how the PH domain scaffolds Ser-241 autophosphorylation. A significant number of contacts were identified between the enigmatic "N-bud" region of the PH domain and the kinase domain. Specifically, these studies implicated Glu-432 and Glu-453 of the N-bud region of the PH domain that bind and serve as mimics of the phosphorylated Ser-241 in the T-loop and the phosphorylated C-terminal tail of PDK1 substrates, respectively. Next, a novel method for protein trans-splicing of the regulatory and catalytic kinase domains of PDK1 was developed. The method utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. The cross-reacting KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH fusion constructs generated full length spliced-PDK1 with kobs = (2.8 +- 0.3) x 10-5 s-1. Finally, NMR was used to further characterize the structural and dynamical properties of the PH domain in both its isolated form and in full length PDK1. Whereas, it was not possible to obtain chemical shift assignments of any backbone or side chain nuclear resonances, methods were optimized for 2H,13C,15N-isotopic labeling of the recombinant PH domain. Furthermore, the protein trans-splicing method was significantly improved and utilized for segmental isotopic labeling of the PH domain in full length PDK1. These new findings and developments may provide specific insight and technological improvements towards future studies aimed to better understand and target autoinhibited conformations of PDK1 for translational purposes.
7

Molecular studies of intra-oocyte phosphatidylinositol 3 kinase (PI3K) signaling pathway in controlling female fertility

Dubbaka Venu, Pradeep Reddy, January 2009 (has links)
Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 2 uppsatser. Även tryckt utgåva.
8

Celecoxib: Its non-cox-2 targets and its anti-cancer effects

Lin, Ho-Pi 24 August 2005 (has links)
No description available.
9

COMPUTATIONAL DESIGN OF 3-PHOSPHOINOSITIDE DEPENDENT KINASE-1 INHIBITORS AS POTENTIAL ANTI-CANCER AGENTS

AbdulHameed, Mohamed Diwan Mohideen 01 January 2009 (has links)
Computational drug design methods have great potential in drug discovery particularly in lead identification and lead optimization. 3-Phosphoinositide dependent kinase-1 (PDK1) is a protein kinase and a well validated anti-cancer target. Inhibitors of PDK1 have the potential to be developed as anti-cancer drugs. In this work, we have applied various novel computational drug design strategies to design and identify new PDK1 inhibitors with potential anti-cancer activity. We have pursued novel structure-based drug design strategies and identified a new binding mode for celecoxib and its derivatives binding with PDK1. This new binding mode provides a valuable basis for rational design of potent PDK1 inhibitors. In order to understand the structure-activity relationship of indolinone-based PDK1 inhibitors, we have carried out a combined molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling study. The predictive ability of the developed 3D-QSAR models were validated using an external test set of compounds. An efficient strategy of the hierarchical virtual screening with increasing complexity was pursued to identify new hits against PDK1. Our approach uses a combination of ligand-based and structure-based virtual screening including shape-based filtering, rigid docking, and flexible docking. In addition, a more sophisticated molecular dynamics/molecular mechanics- Poisson-Boltzmann surface area (MD/MM-PBSA) analysis was used as the final filter in the virtual screening. Our screening strategy has led to the identification of a new PDK1 inhibitor. The anticancer activities of this compound have been confirmed by the anticancer activity assays of national cancer institute-developmental therapeutics program (NCI-DTP) using 60 cancer cell lines. The PDK1-inhibitor binding mode determined in this study may be valuable in future de novo drug design. The virtual screening approach tested and used in this study could also be applied to lead identification in other drug discovery efforts.
10

Investigarion of Activated Phosphaidylinositol 3’ Kinase Signaling in Stem Cell Self-renewal and Tumorigenesis

Ling, Ling 31 August 2012 (has links)
The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

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