Dissertations / Theses on the topic 'SIGNALLING MECHANISM'
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Promel, Simone. "Investigating the molecular mechanism of latrophilin signalling." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533843.
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Lewis, Miranda Faye. "Dissecting the molecular mechanism of toll signalling." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.707987.
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Nyamay'Antu, Alengo. "Elucidating the mechanism of angiopoeitin-mediated Tie2 signalling." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/elucidating-the-mechanism-of-angiopoeitinmediated-tie2-signalling(4269f3c3-fc71-455d-ae5b-4bc47dda78ca).html.
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Research on angiogenesis has been focused on developing anti-angiogenic therapies to target endothelial cell-specific signalling pathways, as a mean to limit tumour outgrowth and metastasis. One of the main targets is the endothelial cell-specific Tie2 receptor and its ligands, the angiopoietins, which controls the later stages of angiogenesis. Although the angiopoietin/Tie2 signalling pathways have been well characterized, the molecular mechanism by which the ligands regulate Tie2 activity remains unclear. To address this question, we determined whether the activation mechanism of Tie2 is induced by dimerisation alone, or whether subsequent relative rotation of the kinase domain is required. Here we employed a coiled-coiled based protein engineering approach to identify the relative orientations of the kinase domains that are optimal for Tie2 activation. By replacing the extracellular domain of Tie2 with the dimeric parallel coiled-coil motif Put3cc, we generated ligand-independent homodimers of the kinase domains Put3cc-Tie2 I-VII that have distinct orientations. We show that dimerisation is sufficient to induce Tie2 activation and downstream activation of Akt, and that varying the interface of the kinase domain in Tie2 dimers can increase its catalytic efficiency. In addition we examined for the presence of potential dimerisation within the transmembrane and intracellular domain of Tie2. We show that the KD and potentially the TM contain dimerisation motifs that stabilise Tie2 in the inactive and active conformations. In addition, we show that deletion of the potential coiled-coil motif in the JM does not disrupt dimerisation but decreases the catalytic efficiency of Tie2. Finally, we propose that the activation mechanism of Tie2 may be similar to the previously described asymmetric dimer formation of EGFR and FGFR receptors.
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Wong, Tin Lok. "Mechanism of action of silicon in cell signalling." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709339.
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Seymour, Lyndsey A. "Characterising the signalling mechanism of the mTOR-dependent phosphatase." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/55115/.
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The mechanistic Target of Rapamycin Complex 1 (mTORC1) complex is central in the regulation of many crucial cellular processes including translation, transcription, proliferation and autophagy. Deregulation of the complex is evident in a number of diseases including Tuberous Sclerosis, Alzheimer's Disease and cancer. Whilst the signalling events leading to activation of mTORC1 are well understood, the inhibitory phosphatase activity that prevents aberrant signalling has received comparatively little attention. In yeast, phosphatases are an integral part of TORC1 signalling. Poor nitrogen supply leads to activation of the phosphatases Pph21/22 and Sit4 and subsequent dephosphorylation of TORC1 substrates. Under these conditions, the phosphatase negative regulatory protein Tap42 is sequestered by Tip41. In good nitrogen supply, TORC1 phosphorylatesTip41 leading to release of Tap42 and subsequent inhibition of Pph21/22 and Sit4. This allows the accumulation of phosphorylated TORC1 substrates. This thesis investigated the role of Tip41 in mTORC1 signalling. Purification of Tip41 identified direct interaction with PP2Ac (human Pph21/22). As overexpression of Tip41 resulted in inhibition of mTORC1 signalling, Tip41 is proposed as a bona fide positive regulatory subunit of PP2Ac. Further investigation indicated that hypophosphorylated PP2A-rjp4i may directly oppose Rheb-mediated activation of mTORC1 thus promoting Raptor degradation. In addition, a specific nuclear isoform of Tip41 was identified, which may specifically regulate the transcription factor HIF1. Studies using the adenoviral protein E40RF4 also identified the PP2ABa complex in regulation of mTORC1 signalling. The data in this thesis show that PP2ABa acts downstream of the TSC 1/2 complex to inhibit mTORC1. Results also indicate that PP2ABa may be negatively regulated by ubiquitin-mediated proteasomal degradation of Ba in an mTORC1-specific manner. Therefore PP2ABa may be subject to an mTORC1 feedback mechanism that is required for activation of downstream substrates. These data indicate that phosphatase activity is critical in regulation of mTORC1, reflecting the mechanism in yeast.
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Bryan, Andrea. "A study of electrophysiological and pathological signalling mechanism in the Xenopus laevis oocyte." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267991.
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Jethwa, Susanna Anjali. "Exosomes : vesicular carriers of autotaxin, a novel mechanism of LPA signalling." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608189.
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Jopling, Helen Mary. "A Mechanism for VEGFR2 Trafficking, Signalling and Degradation in Human Endothelial Cells." Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515561.
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McDowell, Natasha. "The mechanism of long-range signalling between cells of the Xenopus embryo." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624744.
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McLaughlin, Declan. "Investigation into the mechanism of Nox2 NADPH oxidase-dependent signalling in doxorubicin cardiotoxicity." Thesis, Queen's University Belfast, 2014. https://pure.qub.ac.uk/portal/en/theses/investigation-into-the-mechanism-of-nox2-nadph-oxidasedependent-signalling-in-doxorubicin-cardiotoxicity(4250d47c-175c-48d9-bc79-7d1a5ab01443).html.
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Although the antibiotic, doxorubicin (DOX), is a widely-used and successful first-line treatment for cancers, it can induce cardiotoxicity which is associated with increased production of myocardial reactive oxygen species (ROS). Recent work has suggested that ROS generated specifically from Nox2 NADPH oxidase, contributes to key processes underlying cardiac dysfunction associated with chronic DOX treatment. The aim of this study was therefore (1) to characterise the role of Nox2 NADPH oxidase-derived ROS in DOX-induced cardiotoxicity in a 4 week murine experimental model as the basis for detailed mechanistic studies, (2) identify Nox2-regulated genes and signalling pathways which may play a key role in the development of DOX-induced cardiotoxicity in vivo, and (3) investigate detailed Nox2-dependent signalling mechanisms in DOX-treated cardiomyocytes in vitro. Wild type (WT) and Nox2-/y mice were treated with or without DOX (4mg/kg) and studied after 4 weeks. DOX-induced cardiac contractile dysfunction and cardiomyocyte remodelling was attenuated in Nox2-/y as compared to WT mice. Furthermore, DOX treatment was found to increase Nox2 mRNA expression in WT mice, whilst Nox2 mRNA was not detected in Nox2-/y animals and was not induced by DOX. Inhibitor studies conducted in WT animals confirmed that the observed effects were mediated by ROS. Taken together, these data give further support to our previous data suggesting that ROS specifically-derived from Nox2 NADPH oxidase make a significant contribution DOX-induced cardiac remodelling and provide a solid basis for use of the 4 week experimental model for detailed mechanistic studies. In this regard, a whole-genome gene expression array (Illumina MouseWG-6 v2.0) was performed on ventricular tissue from these animals in order to identify key Nox2-regulated pathways which may mediate protection against DOX cardiotoxicity. Pathway analysis identified 156 differentially expressed genes (P<0.1) and highlighted ‘Cell Death and Survival’ as the network of most significance to the dataset. Further examination using heat maps highlighted cell death and survival functions, with a focus on 5 genes associated with cardiomyocyte apoptosis. Of these genes, peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC1α), and the mitochondrial membrane protein, mitofusin 2 (Mfn2), which is known to be up- vi regulated by PGC1α, were selected as good candidates for further investigation. qPCR confirmed increased expression of Mfn2 mRNA in hearts from WT (1.3-fold) but not Nox2-/y DOX-treated animals, although no changes in PGC1α mRNA were apparent between groups. To further investigate the signalling mechanism by which Mfn2 may mediate the protective effects of Nox2, an in vitro model of DOX-induced apoptosis in HL-1 cardiomyocytes was employed. Initial characterization studies showed that DOX treatment for 24 hours resulted in increased expression of both Nox2 and Mfn2 and superoxide production. Targeted siRNA knockdown of Nox2 and Mfn2 produced significant downregulation of protein expression, and exhibited a protective effect against DOX-induced decreases in cell viability and caspase/viability activity. Furthermore, pharmacological inhibition of NADPH oxidase using the inhibitor, VAS2870, significantly reduced DOX-induced HL-1 cardiomyocyte apoptosis and cytotoxicity. Taken together, the findings of this study add further support to a key role for Nox2 NADPH oxidase-derived ROS in DOX cardiotoxicity, and suggest that apoptotic cell death may represent a central mechanism in this process, which may involve mitochondrial fusion, thereby contributing to the associated contractile dysfunction.
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Thomas, Paul William. "Long-distance systemic irradiance signalling : the extent, nature and mechanism in vascular angiosperms." Thesis, University of Sheffield, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421164.
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Wardyn, J. D. "Investigating the molecular mechanism of crosstalk between NF-κB and Nrf2 signalling pathways." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3000015/.
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Investigating the Molecular Mechanisms of Crosstalk Between NF-κB and Nrf2 Signalling Pathways In order to maintain tissue homeostasis, cells must respond swiftly to inflammatory and oxidative challenges, ensuring appropriate processes are sequentially activated and repressed when stress conditions are normalised. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Nuclear Factor-κB (NF-κB) are the key regulators of cellular antioxidant and inflammatory responses respectively. Imbalance between these processes is a contributing factor in many human diseases, including neurodegeneration, autoimmune disorders and cancers. Pharmacological and genetic studies suggest that there is functional crosstalk between these two pathways, however mechanistic details of conditional dominance between them remain unclear. To address this, we have generated multiple, low expression, fluorescent molecular tools that allow monitoring of the distribution and activity of Nrf2 and NF-κB proteins in primary living neurons and astrocytes. This allowed us to characterise the cell-specific antioxidant and inflammatory signalling, as well as to understand the patterns in basal activity. We also investigated patterns of crosstalk in a cancer-related context utilising a previously generated SK-N-AS neuroblastoma cell line, engineered to stably express Nrf2-Venus, from a bacterial artificial chromosome (BAC). This model cell-line was first extensively characterised in order to define conditional Nrf2 responses to antioxidant compounds in real-time. We then proceeded to investigate the effect of acute inflammation on the cellular antioxidant activity, in order to define the extent and nature of functional crosstalk between NF-κB and Nrf2. Data from these studies provide definitive proof of a self limiting reciprocal mechanism of interplay in neuroblastoma cells, in which NF-κB-mediated inflammatory signalling promotes an increase in Nrf2 transcription which then in turn supresses further NF-κB signalling. In addition, results from single cell imaging and population level studies show that Nrf2 responses are fine tuned, by either Keap1-mediated mechanism of repression. To define the intricate mechanisms of basal Nrf2 activity, we utilised a photoswitchable fluorescent protein fusion, which provided the first direct measurements of Nrf2 nuclear import and export dynamics in live-cells. Finally, we used the sophisticated live cell imaging approaches to define the mechanism of action of the Nrf2 inhibiting drug brusatol. Significantly, these results contradict published data and reveal a more general explanation for the potential therapeutic utility of brusatol.
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Meehan, B. "Bacteria-induced Wnt signalling as a mechanism for malignant development in the intestinal epithelium." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3012547/.
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Bates, Katharine Jane. "The neuroplastin cell adhesion molecules : activation of signalling pathways and characterization of the homophilic binding mechanism." Thesis, Royal Holloway, University of London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499983.
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The neuroplastins np65 and np55 are synapse-enriched members of the immunoglobulin superfamily of cell adhesion molecules. The neuroplastins contain two extracellular immunoglobulin-like domains, a single transmembrane domain followed by a short intracellular domain. Np65 differs from np55 by the presence of a third N-termmal Ig domain. They arise by alternative splicing of the same gene transcript. Np65, but not np55 exhibits homophilic binding.
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Gruber, Claudia. "Investigation into the regulatory mechanism of BRCA2 stability." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:e69ab649-f955-48d2-a7c5-48b65f15df45.
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Inherited mutations in the BRCA2 gene predispose individuals to the development of breast and ovarian cancers. The BRCA2 protein plays a fundamental role in the repair of DNA double strand breaks by homologous recombination (HR). BRCA2 mediates the recruitment of the RAD51 recombinase to DNA damage sites, which in turn promotes homologous pairing and strand exchange during HR. It has been reported that increased BRCA2 mRNA levels correlate with poor cancer prognosis, and recently it has been shown that increased levels of BRCA2 suppress HR. As HR is regulated through the cell cycle and can only be employed during S and G2 phases of the cell cycle, in this study, the cell cycle-dependent regulation of BRCA2, as a key player of HR, was investigated. In this study I report that BRCA2 stability is regulated by the ubiquitin-proteasome system (UPS), which has become increasingly evident as an important regulator of DNA repair. In line with this, I found that BRCA2 can be ubiquitylated in vivo and that it interacts with proteins of the UPS. Interestingly, I observed that BRCA2 levels and its ubiquitylation status change during the cell cycle. Using a siRNA-based approach, I identified a candidate E3 ubiquitin ligase, the SCFFBXW7 complex, which is also a known major cell cycle regulator. siRNA-mediated knockdown of FBXW7 led to stabilization of BRCA2 and overexpression of FBXW7 resulted in BRCA2 ubiquitylation in vivo. Furthermore, I have refined the regions that the SCFFBXW7 interacts with on BRCA2, which likely occurs in a phosphorylation-dependent manner. Taken together, these observations suggest that BRCA2 stability is regulated by the UPS in a cell cycle-dependent manner, which may be an important regulatory mechanism for BRCA2 function.
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Britton, G. "Proteomic analysis of Vascular Endothelial Growth Factor (VEGF) signalling : studies of the mechanism of VEGF-induced Heat Shock Protein 27 phosphorylation and its role in endothelial cell signalling and function." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19979/.
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Vascular Endothelial Growth Factor (VEGF) is essential for angiogenesis and endothelial function. Proteomic analysis of Human Umbilical Vein Endothelial Cells (HUVEC) identified Heat Shock Protein 27 (Hsp27) as a major VEGF-regulated protein. Hsp27 is implicated in actin organization, cell survival and migration, and is a potential mediator of these VEGF functions in the endothelium. Studies of pharmacological inhibitors indicated that VEGF-stimulated Hsp27 serine 82 (S82) phosphorylation was resistant to p38 mitogen-activated protein kinase inhibition and mediated by Protein Kinase C (PKC). VEGF activated Protein Kinase D (PKD), and this effect was inhibited by small interfering (si)RNAs targeting selected PKC isoforms. PKD2 siRNA inhibited VEGF-induced Hsp27 S82 phosphorylation, and PKD2 immunoprecipitated from VEGF-treated cells selectively phosphorylated Hsp27 at S82. Hsp27 siRNAs markedly inhibited VEGF-induced cell migration, increased apoptosis and reduced tubulogenesis. Furthermore, inhibition of PKC but not p38 kinase inhibited VEGF-stimulated cell migration. Overexpression of S82A and S82D Hsp27 mutants using adenoviral vectors (Ad) had no significant effect on migration. However, VEGF reduced Hsp27 oligomeric size, and Ad-overexpressed S82D Hsp27 also formed smaller oligomers than wild-type Hsp27. These findings identify a VEGF/PKC/PKD/Hsp27 S82 pathway, indicate a role for PKD and HSP27 in VEGF-induced endothelial migration, and also suggest a specific role for Hsp27 S82 phosphorylation in regulation of Hsp27 oligomerisation. Further proteomic analysis of HUVECs identified Stomatin-Like Protein 2 (SLP2) as a major component of anti-phosphotyrosine immunoprecipitates. The function of SLP2 is little understood. VEGF did not alter the amount of anti-phosphotyrosine-associated SLP2, and further investigations suggested that SLP2 may not be directly tyrosine phosphorylated. SLP2 was localized to mitochondria and co-immunoprecipitated with Prohibitin, a protein implicated in mitochondrial function. However, siRNA-mediated SLP2 knockdown did not affect mitochondrial membrane potential, apoptosis or migration of endothelial cells, and the function of this protein remains unknown.
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Séquaris, Gilles Frédéric Brice [Verfasser], Siegfried [Akademischer Betreuer] Roth, and Angelika A. [Akademischer Betreuer] Noegel. "Molecular mechanism of Indian Hedgehog signalling in human sebocytes / Gilles Frédéric Brice Séquaris. Gutachter: Siegfried Roth ; Angelika A. Noegel." Köln : Universitäts- und Stadtbibliothek Köln, 2012. http://d-nb.info/1038227216/34.
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Lo, William Wing-Yan. "A study of receptor-mediated phosphoinositide signalling mechanism using the human pituitary cell line flow 9000 as a model system." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328913.
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Woolson, Hayley D. "Investigation of the cAMP-mediated inhibitory mechanism on the signalling pathways of 2 cytokines : IL-6 and leptin in endothelial cells." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/648/.
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There is a wealth of evidence to support the anti-inflammatory properties of the prototypical second messenger cyclic-AMP (cAMP), notably with regard to endothelial function. Many studies have shown that cAMP can limit vascular permeability by enhancing barrier function and reducing pro-inflammatory effects of cytokines. Although the protective effects of cAMP elevation on limiting endothelial dysfunction have been well documented, the exact molecular mechanisms remain unclear. Using two endothelial cell types, namely human umbilical vein endothelial cells (HUVECs) and a novel human endothelial angiosarcoma-derived cell line (AS-M), this study has further characterised the cAMP-mediated inhibitory mechanism on the signalling pathways of two cytokines; interleukin-6 (IL-6) and leptin. Both cytokines have been implicated in the regulation of the immune response and both have been shown to play important pathological roles in various inflammatory diseases. In preliminary studies, cAMP elevation was shown to induce suppressor of cytokine signalling 3 (SOCS3) in HUVECs. Further investigation of this SOCS protein in the context of IL-6 and leptin signalling in endothelial cells would be of interest in terms of possibly elucidating the molecular mechanisms underlying the protective effects of cAMP. Results from this study demonstrated a cAMP-mediated inhibition of soluble IL-6Rα (sIL-6R)/IL-6-stimulated extracellular regulated mitogen-activated protein kinase 1, 2 (ERK1,2) and signal transducer and activator of transcription 3 (STAT3) activation in HUVECs, which was independent of cAMP-dependent protein kinase A (PKA). Instead, results demonstrated the involvement of the other major cAMP sensor; exchange protein activated by cAMP 1 (Epac1). Moreover, this inhibition was shown to be SOCS3-dependent. There also appeared to be a requirement for ERK1,2 activation in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated STAT3 activation in HUVECs. In contrast to these findings, cAMP-mediated inhibition of leptin-stimulated STAT3 activation in HUVECs was shown to occur via a SOCS3-independent mechanism. The responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated ERK1,2 activation in AS-Ms were variable, since basal levels of ERK1,2 activation were high. Furthermore, the responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated STAT3 activation in AS-Ms were either very modest or showed no effect, respectively. SOCS3 was not shown to be involved in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated ERK1,2 and STAT3 activation in AS-Ms. In conclusion, this study further characterised the cAMP-mediated inhibitory mechanism in HUVECs and AS-Ms, with a particular focus on the ERK1,2 signalling pathway of IL-6 and leptin. Despite varying results between both cell types, this study also identified AS-Ms as a useful and tractable cell model to study in the context of endothelial biology. Thus, a potentially new pathway has been identified which inhibits cytokine receptor activation of ERK1,2 and STAT3 in endothelial cells. A better understanding of this mechanism could contribute towards new therapeutics in the area of chronic inflammatory diseases, such as atheroscleriosis.
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Salvi, Michele [Verfasser], Christian [Akademischer Betreuer] Griesinger, Blanche [Gutachter] Schwappach, and Groot Bert [Gutachter] De. "Structural Studies on Transmembrane Signalling Mechanism of Histidine Kinase CitA / Michele Salvi ; Gutachter: Blanche Schwappach, Bert De Groot ; Betreuer: Christian Griesinger." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1201884659/34.
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Phan, Michael. "Viral Sensitizers Potentiate the Infection of Cancer Cells Via NF-kB." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40524.
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Genetically engineered oncolytic viruses (OVs) have been proven to be effective anti-cancer agents. However, the heterogeneity of tumours and obligate attenuation of OVs to achieve safety can limit their efficacy. Our lab has previously shown that diverse small molecules, which we have termed “Viral Sensitizers”, used in combination with OVs can potentiate the infection of cancer cells by OVs over 1000-fold in some cases, resulting in cancer-specific killing in both in vitro and in vivo tumour models. We observed that a subset of viral sensitizer compounds ultimately acts by reducing the expression of IFNb, thereby inhibiting antiviral signaling. Here, we aimed to further refine the mechanism of action of this class of compounds. Our results suggest that VSe1 and more stable analogs such as VSe1-28 inhibit nuclear accumulation of NF-kB p65 and expression of various antiviral cytokines including, TNFa, IL-6, IFITM1, and MX2 in multiple oncolytic VSV-resistant cancer cell lines but not in normal cells. This was also observed in vivo in CT26wt immune-competent mouse tumour models, where our group has already demonstrated the therapeutic benefit of combining VSe1-28 with oncolytic VSV. Using various biochemical methods, we have determined that VSe1 and its analog VSe1-28 lead to these effects at least in part through covalent modification of NF-kB p65. In sum, this study provides a new understanding of how these novel viral sensitizers work at the molecular level. This new understanding will not only aid in the discovery and development of improved molecules but also their clinical translation in combination with oncolytic viruses.
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Symington, Hannah Lucy. "Mechanism of IL-2 mediated BACH2 regulation in the control of Human naive B cell differentiation into plasma cells." Thesis, Rennes 1, 2016. http://www.theses.fr/2016REN1B009.
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La différenciation terminale des lymphocytes B qui se déroule dans les centres germinatifs des organes lymphoïdes secondaires est l’étape ultime de la réponse T dépendante et aboutit à la production de plasmocytes (PC) à longue durée de vie qui sécrètent des anticorps hautement affins spécifiques de l’antigène et caractéristiques de la réponse immune adaptative. La transition d’une cellule B naïve vers un PC est gouvernée par un réseau de régulation génique bien décrit et est largement influencée par l’intégration de stimuli externes qui contrôlent le devenir des cellules B tels que l’interaction BCR-antigène et les cytokines produites par les cellules T. La stimulation précoce des lymphocytes B humains activés par IL-2, induit la différenciation en PC via une signalisation ERK prolongée entraînant la baisse d’expression de BACH2, un facteur de transcription clef des cellules B. La répression transitoire de BACH2 est suffisante pour déclencher la différenciation en plasmablastes en l’absence d’IL-2, suggérant ainsi qu’il joue un rôle de « verrou moléculaire » de la différenciation en PC. Il est à noter que cette répression forcée de BACH2 aboutit à la production de plasmablastes caractérisés par un phénotype lymphoplasmocytaire. Ce travail de recherche s’est focalisé sur la caractérisation des mécanismes moléculaires régulant l’expression de BACH2 via la voie de signalisation ERK induite par IL-2. Nous avons identifié ELK-1 comme un médiateur de la répression de BACH2 par la voie IL-2/ERK, comme l’atteste sa capacité à se lier avec un élément de régulation d’un enhancer localisé dans l’intron 1 de BACH2, induisant ainsi la répression de l’enhancer et déverrouillant la différenciation en PC. La caractérisation de cet enhancer de BACH2 a confirmé qu’il est régulé de manière dynamique au cours de la différenciation terminale B et qu’il est localisé dans une région sujette aux mutations suggérant qu’il pourrait être impliqué dans la lymphomagenèseThe terminal differentiation of B cells, which takes places within germinal centres of secondary lymphoid organs, is the ultimate step of a T cell dependent response and results in the generation of long-lived plasma cells (PCs) that secrete protective, antigen-specific, high-affinity antibodies as part of adaptive immunity. The transition of a naive B cell into a PC is governed by a well-characterised gene regulatory network and is heavily influenced by the integration of externally received signals, including BCR-antigen binding and T cell help, such as cytokines which guide B cell fate. The early IL-2 priming of human primary activated B cells triggers PC differentiation through sustained ERK signalling resulting in the down regulation of B cell transcription factor BACH2. Transient BACH2 repression is sufficient to trigger plasmablast differentiation in the absence of IL-2 suggesting that it acts as a key lock of PC differentiation. Importantly, this enforced BACH2 repression results in the generation of plasmablasts with a lymphoplasmacytic phenotype. The focus of this thesis was to characterise the molecular mechanisms regulating BACH2 expression via the IL-2 ERK transduction pathway. We identify ELK-1 as the mediator of IL-2 ERK induced BACH2 downregulation as it binds to a regulatory enhancer element located within intron 1 of BACH2 instigating its repression and unlocking the PC programme triggering differentiation. The characterisation of this BACH2 enhancer confirms that it is dynamically regulated during PC differentiation and is located within a region targeted for mutation suggesting that it may have a potential role in lymphomagenesis
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Gyles, Shan Lindsey. "Intracellular signalling mechanisms in steroidogenesis." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269684.
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Van, den Bosch Marion T. J. "Signalling mechanisms regulating platelet secretion." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.685919.
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Platelets are vital in haemostasis to prevent bleeding after injury. Uncontrolled platelet activation, however, leads to thrombosis. Platelet granule secretion is critical in thrombus formation and, due to the wide variety of granule contents released, also plays a role in many other physiological and pathological processes. The signalling molecule PKC is key in secretion of both dense and a-granules, but the downstream pathways that link it to secretion still need to be defined. A regulator of a-granule release, specifically, is ADP, which acts on the P2Y12 receptor after being secreted by dense granules. How it may control the release of functionally different cargoes is poorly understood. I first identified an extensive range of candidate platelet PKC substrates through proteomics screening using an anti-phosphopeptide antibody-based approach. Among these, I validated cytohesin-2, an Arf-GEF not previously characterised in platelets. This study demonstrated that, upon platelet activation, PKC phosphorylation of cytohesin-2 relieves the basal suppression of dense granule release, shedding new light on how PKC regulates secretion. I next examined the role of ADP in a-granule secretion with a specific focus on whether P2Y12 can induce differential cargo release, considering the implications for the clinical use of the P2Y12 blocker, clopidogrel. I investigated this using a P2Y12 antagonist and the Munc13-4-1 - mouse model where dense granule secretion is ablated and therefore autocrine ADP is not released. Results revealed that the secretion of certain a-granule contents is more dependent on ADP than others and that, in the absence of ADP signalling, granules may release contents without complete fusion with the plasma membrane. Taken together, this thesis provides new findings on the regulation of dense and a-granule secretion, which can be used in the design of improved therapies for thrombosis and other disorders where platelet granule contents play a role.
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Brumell, John H. "Phosphorylation-dependent signalling mechanisms in human neutrophils." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0018/NQ27612.pdf.
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Kwan, Yuen-wah, and 關琬樺. "Signalling mechanisms of Epac1-mediated vascular responses." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49618106.
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Cyclic adenosine monophosphate (cAMP) is an important intracellular secondary messenger. The major target of cAMP was traditionally considered as protein kinase (PK) A. This belief has been challenged by the discovery of exchange protein activated by cAMP 1 (Epac1), a cAMP-dependent guanine-nucleotide-exchange factor (GEF).
Epac1 is ubiquitously expressed in all tissues and plays important roles particularly in the cardiovascular system. As cAMP activates both PKA and Epac1, the development of 8-pCPT-2'-O-Me-cAMP (8-pCPT), which has 107-fold higher affinity to bind and activate Epac1 than PKA, aids the researches on Epac1-mediated responses.
In the present study, the protein expressions of Epac1 in the porcine coronary arteries and rat aortas were confirmed by Western blot analysis. In organ chambers, 8-pCPT induced acute relaxations in isolated porcine coronary arteries contracted to thromboxane receptor (TP-receptor) antagonists, and the relaxation was endothelium-independent. The 8-pCPT-induced Epac1 activation selectively altered the vasoactive responses to the TP-receptor agonists. The Epac1-mediated relaxation was found not related to PKA, PKG and the opening of ATP-sensitive potassium channels.
Although Epac1 was first cloned as a Rap-linked GEF, in the porcine coronary artery, small GTPase Rac1 is the downstream target of Epac1 instead of Rap1 for relaxation. Activation of TP-receptors stimulates Rho-kinase to cause contraction, and the 8-pCPT-induced relaxation was Rho-kinase dependent, probably through pathway that is distinct from Rac1. Activation of Epac1 also inhibited the contraction to PKC, which is also downstream of TP-receptor but independent to Rho-kinase activity.
On the contrary, in the aorta from male Sprague-Dawley rats aged 10-12 weeks, 8-pCPT induced relaxation in rings contracted to phenylephrine (PE) and the relaxation was endothelium-dependent. The relaxation depended mainly on endothelial nitric oxide synthase (eNOS) and partly on cyclooxygenase (COX). Western blot analysis found that 8-pCPT did not enhance eNOS phosphorylation, which is one of the mechanisms for eNOS activation. Activation of Epac1 also did not alter the phosphorylation of Akt and ERK1/2 which play important roles in cAMP-dependent eNOS. More experiments are needed to examine whether or not Epac1 alters nitric oxide (NO) and prostanoids synthesis, which are the major endothelium-derived mediators responsible for vascular tone regulation.
In summary, the selective Epac activator 8-pCPT induced significant relaxations by distinct mechanisms in porcine coronary arteries and rat aortas. It is most likely that the relaxing effects of Epac1 activator are tissue and/or species specific. Owing to the effects of 8-pCPT on vascular relaxation, Epac1 might be an alternative therapeutic target for the treatment of vasospasm and hypertension. Further studies are necessary to explore the detailed mechanisms of Epac1 and its in vivo effects and in diseased models.published_or_final_version
Pharmacology and Pharmacy
Master
Master of Philosophy
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Healey, Eleanor G. "Molecular mechanisms of Repulsive Guidance Molecule signalling." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:3ba6699b-7919-47db-a58e-95970e5e8fcf.
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Repulsive Guidance Molecules (RGMs) control fundamental and diverse cellular processes including axon guidance, immune cell regulation and systemic iron metabolism. RGM dysfunction has been linked to diseases such as multiple sclerosis, cancer and the iron overload disorder juvenile hemochromatosis (JHH). RGMs signal by binding directly to the transmembrane receptor Neogenin (NEO1) to trigger cytoskeleton rearrangements and subsequent axon repulsion. Additionally, RGMs are important activators of the essential developmental Bone Morphogenetic Protein (BMP) signalling pathway. RGM-activated BMP signalling is crucial for the regulation of iron metabolism and mutations in RGMC cause JHH. This thesis outlines structural and functional studies of the molecular mechanisms of RGM signalling. In Chapter 3, an analysis of an RGMB-NEO1 crystal structure is presented. Combined with mutagenesis studies, analytical ultracentrifugation experiments and neurite outgrowth assays, this allowed a mechanism for RGM signalling through NEO1 to be proposed. RGM acts as a molecular staple, bringing together the juxtamembrane regions of two NEO1 receptors to activate downstream signalling. In Chapter 4, crystal structures of the N-terminal domains of all RGM family members in complex with the BMP2 ligand are presented. Together with biophysical and cellular assays these structures allowed an endocytosis-linked mechanism for RGM-activated BMP signalling to be proposed, which is dependent on the subcellular localisation of the BMP-receptors. The work outlined in both of these chapters also revealed a molecular rationale for the disease-causing mechanism of RGMC JHH-linked mutations. In Chapter 5, the crystal structure of the ternary BMP2-RGMBNEO1 complex is described along with super-resolution uorescence microscopy data demonstrating BMP-induced clustering of RGM-NEO1 complexes in the membrane. In summary, this work sheds light on the molecular mechanisms of RGM signalling through NEO1 and BMPs, and demonstrates for the first time that RGM forms a structural bridge between these two fundamental signalling pathways.
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Flack, Joshua Edwin. "Mechanisms of molecular switching in the Wnt signal transduction pathway." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/284206.
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Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the 'degradasome', which targets the central effector β-catenin for degradation in the absence of Wnt; the 'signalosome', which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the 'enhanceosome', which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt 'transcriptional switch'. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.
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Tamiya, Shigeo. "Autocrine and paracrine signalling mechanisms in lens cells." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365025.
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Bhangu, P. S. "Vesicular 'pre-synaptic' glutamatergic signalling mechanisms in bone." Thesis, University of York, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288814.
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Spencer, Gary James. "Glutamate recycling and signalling mechanisms in bone cells." Thesis, University of York, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399248.
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Fiúza, Ulla-Maj. "Mechanisms of regulation of notch signalling in Drosophila." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611170.
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Jenkinson, Stephen. "Molecular mechanisms of lithium action on phosphoinositide signalling." Thesis, University of Leicester, 1993. http://hdl.handle.net/2381/35263.
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The work described in this thesis examines the phosphoinositide (PI) signalling system and its disruption by the anti-manic agent lithium. The effects of lithium upon the accumulation of labelled and unlabelled inositol (poly) phosphates in muscarinic cholinoceptor-stimulated rat brain slices and Chinese hamster ovary (CHO) cells expressing the human M1-muscarinic receptor subtype (CHO-Ml) were examined. Similarly, the effects of this ion on other intermediates of this second messenger signalling system were examined in order to give an overall picture of the action of lithium. These included the accumulation of CMP-phosphatidic acid (CMP-PA), a precursor of the (poly) phosphoinositide lipids, and the agonist-stimulated levels of the (poly) phosphoinositide lipids. Initial experiments examined phosphoinositide metabolism in cortex, hippocampus and striatum to determine whether there were regional variations in both this signalling system and the effects which lithium had upon it. Both cortical and hippocampal PI metabolism were similar, however, striatum was significantly different such that in the continued presence of agonist this region was unable to maintain the initial elevated levels of Ins(l,3,4,S)P4, unlike the other regions examined. Lithium appeared to have a similar disruptive effect on PI metabolism in all regions, with statistically similar EC50 values for the accumulation of InsP1 in all regions. The effects of lithium upon PI metabolism stimulated by a variety of different agonists was examined to determine whether the action of lithium was agonist dependent Lithium appeared to have a similar disruptive effect upon PI metabolism stimulated by these various agonists. Studies examining the effects of lithium upon the carbachol-stimulated accumulation of the inositol (poly) phosphate isomers revealed the presence of a lithium-sensitive accumulation of the inositol bisphosphate Ins(4,5)P2. This study was unable to determine the source of this isomer, however, the formation of this isomer in both cerebral cortex slices and CHO cells suggests the possibility that Ins(4,5)P2 may be a dephosphorylation product of Ins(l,4,5)P3. The lithium-sensitivity of the accumulation of this isomer also suggests that a novel Uthium- sensitive 4- or 5-phosphatase activity may be present in these preparations. The effects of lithium on phosphoinositide metabolism in CHO cells expressing the human M1-muscarinic receptor subtype was also examined to determine whether this cell line would represent a suitable model of cerebral PI metabolism. It was hoped that the use of this cell-line would result in clearer more interpretable data. Indeed, a definitive analysis of PI metabolism in this cell line clearly demonstrated that the addition of lithium to agonist-stimulated cells resulted in a decrease in the intracellular myo-inositol reserves within the cell which resulted in a decrease in the (poly)phosphoinositide precursor of Ins(l,4,5)P3, PtdIns(4,5)P2. In turn this resulted in a time-dependent decrease in the agonist-stimulated levels of Ins(l,4,5)P3 after a lag period of 5-10 min, similar to that observed in cerebral cortex slices. The data demonstrate that the CHO-Ml cell-line is a valuable tool in elucidating the actions of lithium upon PI signalling. In conclusion, the results described in this thesis clearly demonstrate the profound effects that this monovalent ion has on phosphoinositide signalling in the preparations examined. The main action of lithium appears to be the inhibition of the inositol monophosphatase, however, this agent may also have other more subtle effects upon this complex system. These possibilities and their implications are also discussed.
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Ambily, Anju. "Studies of calcium signalling mechanisms in human platelets." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/studies-of-calcium-signalling-mechanisms-in-human-platelets(0754e26d-374a-485e-9387-41e074926048).html.
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Platelet activation is essential in haemostasis and thrombosis and central to this role is cytosolic calcium (Ca2+) elevation. The two ways in which this can occur are release from intracellular Ca2+ stores and the entry of Ca2+ across the plasma membrane (PM). Changes in cytosolic Ca2+ are required to enable a variety of platelet responses. In platelets and other non-excitable cells, the major route of Ca2+ entry occurs as a result of Ca2+ depletion from intracellular stores, and is known as store operated Ca2+ entry (SOCE). STIM1 is the Ca2+ sensing protein of the endoplasmic reticulum (ER) which activates Orai channels in the plasma membrane allowing Ca + entry. However, not all of the details of Ca2+ entry are understood. In platelets, two other pathways also enable Ca2+ entry. The P2X1 receptor is a ligand gated ion channel activated by the binding of ATP. Second messengers (such as 1,2-diacyl-sn- glycerol [DAG]) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) may directly gate plasma membrane cation channels such as the transient receptor potential canonical 6 (TRPC6) channel. -- The main aim of this study was to examine the role of STIM1 and TRPC proteins in Ca2+ entry in human platelets. The TRPC1 protein was originally proposed to be a major component of the SOC channel based on the observation that a commercial preparation of an anti-TRPCl antibody against the channel impaired SOCE in platelets. However, in the current study I demonstrate the failure of this reagent to bind to TRPC1. The previously reported inhibitory effect on SOCE may be due in part to be a result of the azide component of the antibody preparation.
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Castello, A. "Regulatory mechanisms of antigen encounter and BCR signalling." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1407025/.
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Activation of B cells is dependent on two events: the encounter of cognate antigen and the triggering of an adequate B cell receptor (BCR) signal. In vivo B cells encounter antigen within secondary lymphoid organs, such as lymph nodes (LNs). Here, specialised CD169+ macrophages, strategically located across the fenestration of the inner wall of the subcapsular sinus (SCS), trap lymph-borne antigen and present it to B cells. I have shown that within CpG-inflamed LNs, CD169+ macrophages undergo a dramatic reorganisation by retracting from the inner wall fenestration and scattering along the edges of the tissue. These events are partly caused by the arrival of dendritic cells pushing away the macrophages form the SCS fenestration to gain access to the LN. In vitro CD169+ macrophages were found to respond directly to CpG by downregulating CD169 and some scavenger receptors, while upregulating the expression of interferon-γ. Because of these structural and phenotypic changes, antigen retention at the SCS is impaired resulting in fewer B cells encountering their cognate antigen and weaker antibody responses. Antigen encounter is important because it initiates BCR signalling. Within a signalling cascade, adaptor proteins act as signal integrators and amplifiers. The adaptor protein Nck is best known for linking receptor signalling to cytoskeleton regulation. However upon B cell activation I found that Nck controls the PI3K/Akt pathway by recruiting the B-cell adaptor for PI3K (BCAP). Nck can carry out these functions by directly binding to the BCR via the non-ITAM phospho-tyrosine 204 in the Igα tail. Importantly, genetic ablation of Nck resulted in defective BCR signalling leading to hampered survival and proliferation in vivo. Antibody responses in Nck-deficient animals were thus severely impaired. Collectively these findings are important as they shed light on some of the regulatory mechanisms for antigen encounter and BCR signalling.
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Orlando, Giulia. "Molecular mechanisms of ARF regulation in response to DNA damage." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:fde8ab19-bc7f-4ad8-8396-6c5f5f385d34.
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DNA is a highly unstable molecule. Endogenous souces of DNA damage, such as reactive oxygen species (ROS), can cause DNA damage and it has been estimated that 20000 lesions occur in a cell per day. BER is the major pathway for the repair of these lesions and therefore maintains genome stability, thus preventing the development of human diseases such as neurodegenerative diseases and cancer. Therefore, if BER cannot accomplish the repair, accumulation of DNA damage occurs, triggering different cellular responses, such as cell cycle delay and senescence. The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating upregulation of p53 protein. Moreover, ARF expression is upregulated in senescent cells, suggesting that ARF induction might be triggerred in response to persistent DNA damage. Although ARF has been reported to be important in the regulation of proteins involved in the DNA damage response, its role is still controversial. Here, it has been shown that ARF gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage generated by disabling BER. These data suggest that PARP1-dependent poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD+, thus inhibiting SIRT1 activity and consequently activating E2F1-dependent ARF transcription. These findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.
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Kirkeby, Håkon. "In silico Investigation of Possible Mitotic Checkpoint Signalling Mechanisms." Thesis, Norwegian University of Science and Technology, Department of Mathematical Sciences, 2007. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-9663.
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The mitotic checkpoint is the major bio-chemical pathway acting to ensure stable genome content in cell division. A delay in chromosome segregation is enforced as long as at least one kinetochore is in lack of proper attachment to the mitotic spindle, something that prevents premature initiation of anaphase and uneven chromosome distribution. The backbone of the mitotic checkpoint control system is established as the production of a wait-anaphase signal at the unattached kinetochores. However, how this signal is able to support a functional checkpoint is unclear. To explore the performance of the wait-anaphase signal in terms of providing the mitotic checkpoint with high fidelity, a mathematical modelling framework is constructed that simulates the spatially distinct production of anaphase inhibitors, their diffusion in the cytoplasm and interference with the anaphase-promoting machinery. The model is used to analyse the performance of several different signalling mechanisms, with emphasis on testing the ability to maintain tight inhibition and allow rapid release of the anaphase promoter.
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Atwal, Jasvinder K. "Signalling mechanisms underlying Trk function in neonatal sympathetic neurons." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37645.
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NGF acting through the TrkA tyrosine kinase receptor promotes survival and axon growth of developing sympathetic neurons in vivo and in vitro. This thesis examines the signalling pathways and proteins used by Trk receptors to mediate neuronal survival, local axon growth, and growth cone regulation in neonatal rat sympathetic neurons of the superior cervical ganglion (SCG).First, I examined the involvement of key signalling motifs on the Trk receptor in mediating neurotrophin-dependent survival and local axon growth. Using recombinant adenoviruses, I expressed the BDNF receptor TrkB, which is not endogenously expressed in SCG neurons, either in wild-type form or mutated at defined effector binding sites. Ectopically expressed wild-type TrkB activated signalling pathways similarly to endogenous TrkA, and supported both neuronal survival and local axon growth in compartmented Campenot chambers. However, TrkB mutated at the Shc-binding site was impaired in its ability to activate PI3-kinase/Akt and MEK/ERK, and was a poor mediator of both neuronal survival and local axon growth. Furthermore, by using pharmacological inhibitors, I found that TrkB-mediated survival and local axon growth required both PI3-kinase and MEK/ERK signalling.
Next, I investigated the contribution of Trk signalling to growth cone maintenance in sympathetic neurons. Acute inhibition of NGF/TrkA signalling rapidly collapsed growth cones, as did inhibition of either PI3-kinase or MEK/ERK. I then asked whether the chemorepellent Sema3F collapsed sympathetic growth cones by inhibiting TrkA-mediated growth signals. Sema3F did not disrupt NGF-induced activation of TrkA, Shc, or PLC-gamma but significantly reduced PI3-kinase and MEK/ERK activation. Furthermore, sustained hyper-activation of PI3-kinase and MEK/ERK partially blocked Sema3F-induced collapse, suggesting that Sema3F acts, at least in part, by inhibiting Trk-dependent pathways.
Together, these data describe the role of Trk signalling in survival, local axon growth, and growth cone maintenance in cultured sympathetic neurons. The Shc-binding site was found to be critical for TrkB-mediated survival and local axon growth. The PI3-kinase and MEK/ERK pathways were found to be important for survival, local axon growth and growth cone maintenance. Furthermore, the chemorepellent Sema3F collapses sympathetic growth cones, at least in part, by interfering with key neurotrophin-induced signalling pathways.
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Neve, Joshua Thomas. "Identification of novel regulators and mechanisms of auxin signalling." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612611.
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The plant hormone auxin is essential for plant development and controls growth and patterning of cells and tissues by strict regulation of gene expression. Past studies have elucidated a succinct auxin signal transduction pathway but some functions of the core components have only been partially explored. Several published studies have provided evidence for the existence of different mechanisms for the repressive functions of Auxin/Indole Acetic Acid (Aux/IAA) repressors and part of this study has sought to quantify the extent to which TOPLESS (TPL) and TOPLESS-RELATED proteins contribute to their [unction in plants. Yeast Two Hybrid (Y2H) suggests that differential interactions occur between Aux/IAAs and TPL/TPRs but genetic analyses carried out with Arabidopsis thaliana mutants did not substantiate findings in yeast. Published studies show that Auxin Response Factors (ARPs) interact with proteins other than Aux/IAAs in planta to carry out wild type function. Current understanding supports a formal possibility that novel' regulators of auxin signalling have yet to be discovered, and so two distinct screening procedures have been carried out to find novel components. 7000 Full-length Arabidopsis cDNA Over-eXpression (FOX) lines, each over-expressing on average 1.2 Arabidopsis cDNAs have been systematically screened for resistance to exogenous auxin and have revealed a novel allele of A UX I but no novel regulators of auxin signalling. Alternatively the transcriptional activator, ARF7, and repressor, ARFI, have each been used in Y2H screens resulting in the identification of novel interacting protein partners that have been corroborated by biochemical analysis. Results suggest possible links between auxin and jasmonic acid signalling, and suggest a physical interaction between both AR FL and ARF7 and several members of the Type-One Protein Phosphatase (TOPP) family of Protein Phosphatase I (PP I) catalytic subunits. Up-regulation of TOPP1 expression confers changes in more than one auxin-regulated developmental process and therefore suggests a novel role of phosphorylation in auxin-regulated development.
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Cameron, A. L. "Intracellular signalling mechanisms of the unicellular parasite, T.b. brucei." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597238.
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The primary aim of this work was to investigate phospholipid metabolism in trypanosomes, including a phosphoinositide signalling, and the metabolism of the VSG and its GPI anchor. The phosphoinositides of T.b brucei were labelled using 32P (from [32P)Pi or [γ32P]ATP) and [3H)inositol through a variety of methods. Most work was carried out with phospholipids labelled using [γ32P]ATP as the donor, with cells permeabilised by being placed in a buffer of low osmotic strength (swell dialysis). Species labelled with 32P were identified as PI 4-P and PI 4,5-P2 through HPLC analysis. There was no evidence of 3-phosphorylated phosphoinositides under the conditions used. Results showed that T.b brucei has GTP- and Ca2+-regulated phosphoinositide metabolism, similar to that seen in vertebrate cells. Additionally, an unidentified factor in rabbit serum caused a 2-fold increase in the phosphorylation of the polyphosphoinositides. Although Ca2+ is known to cause the release of VSG through the action of GPI-PLC, it is not clear whether this enzyme plays an important role in membrane-form (mf) VSG metabolism in intact cells. The relationship between VSG release and subsequent GPI anchor metabolism was examined using a GPI-PLC inhibitor (pCMPSA) and conditions known to cause the release of soluble form (s) VSG (Ca2+). Metabolism of the mfVSG GPI anchor was taken to correspond with [32P]PA generation in cells incubated in the presence of [γ32P)ATP. Anti-VSG antibodies were used to detect VSG in supernatant samples of T.b. brucei. A great deal of phospholipid metabolism sensitive to pCMPSA, so possibly related to GPI-PLC activity, was shown to occur in the absence of sVSG release. The primary role of GPI-PLC in viable trypanosomes may therefore be in the regulation of processes other than mfVSG metabolism. T.b. brucei was incubated with anti-VSG IgGs to observe whether interaction with anti-VSG antibodies caused the destruction of mfVSG and concomitant increase in PA labelling. Anti-VSG IgGs had no effect on PA metabolism. mfVSG endocytosed in bloodstream from T. brucei may therefore be returned intact to the cell surface.
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Lahne, Manuela. "Damage-induced signalling mechanisms in the neonatal rat cochlea." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444911/.
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Sound overstimulation and exposure to ototoxic drugs damage cochlear hair cells (HCs) and cause their death. The surrounding support cells maintain an epithelial barrier and the appropriate physiological environment for surviving HCs during pathological conditions. Coordination of this homeostatic process requires cellular signalling. However, the signalling events that are activated during damage in the mammalian cochlea, are poorly understood. Neonatal rat cochlear explants were subjected to mechanical damage or exposed to neomycin - an ototoxin. Mechanical damage triggered the immediate propagation of an intercellular wave of increased intracellular Ca2+ from the lesion site into distinct cochlear regions. The properties of the Ca2+ wave and the source of Ca2+ required were specific to the cochlear region. IP3-mediated release from intracellular stores and influx of extracellular Ca2+ contribute differentially to the rise in intracellular Ca2+. The release of extracellular ATP is crucial for the propagation of the damage-induced Ca2+ wave. Gap junctions or connexin hemichannels also contribute to its formation. A subsequent damage-induced signalling event is the transient phosphorylation of ERK1/2 that arises within minutes of the insult occurring in support cells specifically. Similarly to the formation of the Ca2+ wave, release of extracellular ATP and gap junctions are critical for ERK1/2 activation. UTP-induced activation of ERK1/2 reveals the involvement of P2Y receptors. In addition, a requirement for the influx of extracellular Ca2+ also suggests a role for ion channels - potentially P2X receptors. P2X2,3,4 and P2Y2.4, n receptors were expressed in cochlear explants with P2X2 and P2Y2 being exclusive to support cells. Damage-induced currents were recorded from Deiters' cells in a syncytium during mechanical damage of the cochlea. Finally, when HCs were specifically targeted using neomycin, ERK1/2 activation occurred in support cells surrounding pyknotic HC nuclei. Inhibition of ERK1/2 delayed HC death.
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Kemp, Daniel M. "Reporter gene analysis of regulatory mechanisms in cAMP signalling." Thesis, University of Kent, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310202.
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Davies, Clare Charlotte. "Mechanisms of CD40 signalling and apoptosis in carcinoma cells." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398887.
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JimeÌnez, Luis Albert. "Signalling mechanisms induced by particulate matter in lung cells." Thesis, Edinburgh Napier University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250084.
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Li, Xin. "TRPM2 channel-mediated signalling mechanisms for neuronal cell death." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/18576/.
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Transient receptor potential melastatin-related 2 (TRPM2) channel is gated by ADP-ribose (ADPR) and potently activated by reactive oxygen species (ROS) through stimulating ADPR-generating mechanisms. Recent studies provide evidence to show a crucial role for TRPM2 in neuronal death and cognitive impairment associated with ischemic stroke and Alzheimer’s disease. However, the underlying mechanisms are poorly understood. Studies described in this thesis adopted genetic and pharmacological interventions, in conjunction with immunofluorescent and live cell imaging, to investigate TRPM2-dependent cell death induced by H2O2 and the 42-residue of amyloid β (Aβ42) in cultured hippocampal neurons. H2O2 and Aβ42 induced significant neuronal death, which was reduced or prevented by TRPM2 knock-out (TRPM2-KO), TRPM2 channel inhibitors, or Zn2+ chelator TPEN. H2O2 and Aβ42 induced intracellular Zn2+ increase, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, dysfunction and ROS generation. Bafilomycin A1-induced lysosomal dysfunction also resulted in mitochondrial Zn2+ accumulation and ROS generation. These events were abolished by TRPM2-KO or suppressed by inhibiting poly(ADP-ribose) polymerase-1 (PARP-1) or TRPM2 channel. Immunofluorescent imaging suggests mitochondrial localization of TRPM2. ADPR enhanced Zn2+ accumulation in isolated mitochondria from wild-type (WT) but not TRPM2-KO neurons. Finally, the inhibition of protein kinase C (PKC) and NADPH oxidases (NOX), particularly NOX1/4, suppressed H2O2/Aβ42-induced neuronal death and Aβ42-induced intracellular Zn2+ increase, lysosomal and mitochondrial dysfunction, and mitochondrial ROS generation. The inhibition of the proline-rich tyrosine kinase 2 (Pyk2) and the downstream MEK/ERK kinases protected against Aβ42-induced neuronal death. Taken together, these results provide evidence to support a vicious positive feedback signalling loop that drives hippocampal neuronal death in response to ROS and Aβ42, in which the TRPM2 channel in mitochondria integrates multiple mechanisms comprising PKC/NOX-mediated ROS generation, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, mitochondrial dysfunction and ROS generation. In addition, the Pyk2-MEK-ERK signalling pathway is critically involved in Aβ42-induced TRPM2-dependent neuronal death. These findings provide novel insights into the mechanisms underlying neuronal death and cognitive impairment related to ischemic stroke and AD.
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Gooch, Sophie Louise. "Studies into NOD1 and NOD2 activation and signalling mechanisms." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608182.
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Hsu, Li-Heng. "Signalling mechanisms underlying priming and tolerance of T cells." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4263/.
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The primary mission of the immune system is to defend against invading pathogens. The normal healthy body can distinguish self from non-self antigens. When a new antigen is encountered, such discriminatory capacity would generate a productive immune response against invasive pathogens or exert antigen-specific tolerance, the latter to prevent harmful immune responses against self-components or non-dangerous food or environmental antigens. Peripheral tolerance plays an important role in preventing T cells response to self or harmless antigens. A breakdown in tolerance within an individual can result in the development of a variety of autoimmune disorders. Full T cell activation requires at least two signals. The first one is provided by the TcR recognizing cognate peptides derived from antigen in the context of appropriate MHC molecules expressed by antigen presenting cells (APC). The second is mediated by “co-stimulation” via interaction of CD28 on the T cell with CD80/86 on the APC. The clonal anergy is induced when the TcR is ligated in the absence of co-stimulation, one of the proposed mechanisms of peripheral tolerance, describes a state of long lasting unresponsiveness to antigen, in the T cell. Despite widely studies in this area, however, the mechanisms of induction of anergy and the efficient markers for diagnosis of anergy are still not clear. One of the mechanisms which contributes to forming tolerance is anergy, which can be defined as defect in cellular proliferation and IL-2 production. Furthermore, GTPase Rap1 has been reported to inhibit the generation of pERK signals and to accumulate in tolerant cells. However, most of previous studies have done by biochemical assessment of signaling in T cell lines or clones upon polyclonal stimulations in vitro, and thus has generated some conflicting data. For solving this problem, our lab has developed the technique, laser scanning cytometry (LSC), for observation of responses in individual antigen-specific T cells within their environmental niche within primary or secondary lymphoid tissue. By LSC, it has reported that there are significant differences in the amplitude and cellular localization of phosphorylated ERK signals when naïve and in vitro-primed and tolerized T cells respond to Ag. To further investigate the role of Rap1 by LSC, it revealed that counter regulation in Rap1 and phosphor-ERK expression during the maintenance phase of tolerance and priming of antigen-specific CD4+ T cells in vitro and in vivo. In T cells, the maintenance phase of anergy has been reported to reflect defective activation of transcription factor, such as c-Jun/c-Fos, that are involved in formation of the AP-1 complex, which is required for inducing transcription of the IL-2 gene and optimal activation and effector function of T cells. In turn, this appears to be determined by the lack of recruitment of the ERK, JNK and p38 MAPK signaling cascades. The small GTPase, Rap1, has long been implicated in such desensitisation of ERK, and the consequent reduced IL-2 production, observed in tolerised T cells. However, the most of these studies were processed with T cell lines or clones in vitro and as such are not necessarily representative of physiological responses of primary antigen-specific T cells. Consistent with the previous finding, we have extended these studies to investigate whether Rap1 plays a role in determining commitment to anergy and priming during induction and maintenance phases. As expected, analysis in the DNA synthesis during maintenance phase reported that the primed T cells exhibited a higher response than either naïve or anergic T cells, whilst the anergic T cells displayed an even lower DNA synthesis than naïve T cells undergoing a primary response. To further investigation in cytokine production of IL-2 and IFNγ at 24, 48 and 96 hour during the maintenance phase, consistent with previous studies, the primed T cells produced the highest levels of IL-2, relative to anergic cells with the lowest levels, at the first 24 hours after challenge with antigen. However, the IL-2 production from primed and anergic T cells both drop down from 48 hours and to very low level at 96 hours but accompanying with gradual increase of IFNγ production. This implicates both anergic and primed cells consumed IL-2 secreted in the early stage of maintenance phase for supporting following cellular differentiation. The assessment of cellular proliferation also indicates that both primed and anergic cells had undergone several rounds of division. Whereas the primed cells proliferated more and faster than anergic cells over the first two days, after that anergic cells were able to catch up with primed cells. Consistent with above proliferative responses, the primed T cells showed higher levels of ERK activation than anergic cells at day 1 but lower levels of ERK activation than anergic cells at day 3. Surprisingly, there is no difference in Rap1 activation between primed and anergic T cells during maintenance phase. The additional finding from cellular proliferation during maintenance phase revealed that both primed and anergic cells undergo clonal expansion during induction of priming and tolerance, which leads the further investigation in functional outcomes, MAPK signaling and mTOR pathways studies during induction phase. The primed cells exhibited higher levels of DNA synthesis than anergic cells at 48 hours whereas they had similar levels of DNA synthesis at 96 hours. The IL-2 and IFNγ production were only detectable within the first 48 hours but not 96 hour. Collaborating with the data from cellular proliferation indicates the IL-2 were consumed for promoting the cells survival and proliferation since both populations showed clear peaks representing differential numbers of cell division from day 2 (48 hour) onwards, whereas the primed cells proliferated more and faster than anergic cells during whole induction phase. Moreover, cyclic activation of ERK was seen in the primed T cells and at higher levels of activation than in the anergic population, which did not exhibit these kinetics in western blotting. Interestingly, the primed T cells exhibited slightly higher levels of Rap1 than anergic cells from 48 hour until 96 hour during induction phase. Consistent with data from in vitro, the proliferation response in mimicking physiological model also can be replicated. Additionally, the counter regulation in ERK and Rap1 activation also occurred during the induction of priming and tolerance, which is investigated by adenoviral gene transfer of Ad Rap1 S17N, an inactive mutant of Rap1. Furthermore, modulation of Rap1 expression with Ad Rap1 S17N in cells during induction of anergy, revealed that Rap1 activity acts to limit cellular proliferation and thus switching off Rap1 activity upregulates cellular proliferation to generate a phenotype more resembling priming of normal (or GFP-) T cells by antiCD3+anti-CD28, which showed higher proliferation that GFP- cells stimulated with anti-CD3 only. However, when these adenoviral transfer experiments were repeated in the more physiological model, the higher proliferation exhibited in anergic Ad Rap1 S17N transduced cells were not replicated, suggesting that the enhancing effect of Ad Rap1 S17N might be substituted by signals generated under these more physiological conditions. There did not appear any difference between anergic and primed cells in terms of ERK/Rap1 signalling during the induction phase and introduction of Ad Rap1 S17N did not modulate ERK activity in transduced cells treated with anti-CD3 or anti-CD3+anti-CD28, suggesting that Rap may target some other effector during the induction phase. To sum up these data, Rap signaling in anergy and priming as well as the use of the dominant negative construct suggested that Rap was not acting to suppress ERK activation during induction of anergy.
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Kneeshaw, Sophie. "Molecular mechanisms of redoxin-mediated signalling in plant immunity." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/18754.
Full textAbstract:
Posttranslational modification (PTM) of proteins is essential to creating a diverse proteome with the complex functions necessary to regulate key cellular processes. Redox-based PTMs exhibit many desirable characteristics to finely modulate transcriptional regulators; they occur rapidly and can alter protein conformation, localisation and activity. The plant immune system offers an excellent model in which to study redox-based modifications due to the rapid accumulation of oxidising agents that occurs during immune invasion. This so-called “oxidative burst” causes spontaneous oxidation of cysteine residues that are present in many regulatory proteins. These modifications fine-tune the activities of proteins that harbour them, enabling them to act in a concerted effort to reprogram the transcriptome, prioritising the expression of immune-related genes over housekeeping genes. Disulphide bonds (S-S) and S-nitrosothiols (SNO, i.e. the addition of an NO group to a cysteine moiety) have been shown to play particularly important roles in plant immunity. However, what still remains unclear is how these redox-based PTMs are rendered reversible, enabling them to act as molecular signalling switches. The work presented in this thesis explores a class of enzymes that are responsible for controlling the cellular levels of protein oxidation: the Thioredoxins. In addition to their well-established role in reducing disulphide bonds, I demonstrate in Chapter 3 that Thioredoxins are able to reverse protein S-nitrosylation during plant immune signalling. Immune-inducible Thioredoxin-h5 (TRXh5) was shown to be unable to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, but rescued impaired immunity and defence gene expression in nox1-mutants that exhibit elevated levels of free NO. This data indicates that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signalling in plant immunity. Furthermore, data is presented to show that TRXh5 reversed the effects of S.nitrosylation on many immune-related transcriptional regulators in vitro, forming the initial stages of an investigation into which proteins and pathways might be controlled by reversible S-nitrosylation in plant immunity (Chapters 3 & 4). Although the majority of transcriptional regulators are likely modified at their site of action, the nucleus, very little is currently known about nuclear redox signalling in plants. Therefore, in Chapter 5 a subclass of theThioredoxin superfamily was studied, the Nucleoredoxins, which have previously been shown to display disulphide reduction activity and localise in part to the nucleus. Here it is revealed that the activity and nuclear accumulation of Nucleoredoxin 1 (NRX1) is induced by the plant leaf pathogen Pseudomonas syringae, suggesting a key role for this protein in immune signalling. Target-capture experiments and subsequent mass spectrometry analysis identified the first in vitro targets of NRX1 and revealed many proteins with roles in oxidative stress, including the hydrogen peroxide scavenger Catalase 2 (CAT2). Moreover, overexpression of NRX1 was shown to be able to rescue the enhanced cell death phenotype of cat2 knockout mutants in response to the oxidative stressor, methyl viologen. Accordingly, nrx1 knockout mutants also exhibited an enhanced cell death phenotype in response to methyl viologen treatment. Together, these data indicate that NRX1 plays a key role in the control of oxidative stress-mediated cell death, potentially through direct regulation of Catalase proteins. Taken together, the work in this thesis implicates members of the Thioredoxin family as key regulators of transcriptional reprogramming during plant immunity and uncovers a novel role for Thioredoxin superfamily member, NRX1, in the control of oxidative stress.
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Alayash, Abdu I. "Hemoglobin-based blood substitutes : redox, signalling and clearance mechanisms." Thesis, University of Essex, 2010. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.528868.
Full text
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Sianati, Setareh. "μ-Opioid receptor signalling mechanisms: quantifying bias and kinetics." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/12794.
Full textAbstract:
Opioids such as morphine remain among the mainstay treatments for management of moderate to severe pain. However, the long-term use of opioids is limited by the development of severe adverse effects such as tolerance and addiction. Numerous studies have been devoted to understand the molecular and cellular mechanisms that are responsible for MOPr signalling, short-term regulatory events and cellular adaptations that lead to opioid tolerance in order to develop analgesics with safer therapeutic profiles. A large body of evidence indicates that activity of different opioids at the same receptor does not always stimulate a similar set of signalling pathways. Indeed agonists have various efficacies that can be different when the receptor interacts with distinct signalling effectors, a phenomenon termed agonist bias or functional selectivity. This discovery offers new approaches for development of novel pathway-selective drugs that stabilize particular conformations of the receptor while preferably activating signalling pathways associated with therapeutic outcomes but not those responsible for adverse effects. Although this concept is very interesting, the identification and quantification of biased agonism still remains a challenge. Several analytical methods have been developed to address this issue, but these methods are not applicable for all the agonists and systems. In this study, different analytical approaches for bias quantification were compared for a range of MOPr agonists. MOPr agonists showed distinct bias for different pathways. For example, Bilaid-C2, a novel MOPr agonist, had similar effectiveness to DAMGO for G-protein activation but showed significantly lower effectiveness for Ser375 phosphorylation and βarrestin-2 recruitment and more importantly was not able to promote receptor internalization. In contrast, morphine did not display bias towards any pathways and had significantly lower effectiveness for all pathways compared to DAMGO. The bias values determined in this study can be used for future studies to translate the bias into physiological responses. Furthermore, this study showed that operational model is the optimal approach to determine bias when the agonist affinity value was fixed to the functional dissociation constant. The next part of the study investigated the off-rate kinetics of MOPr agonists for potassium current, Ser375 phosphorylation and βarrestin-2 recruitment. Present study illustrated that the kinetics of MOPr for these signalling pathways were ligand-dependent. Furthermore, there was a robust positive correlation between off-rate kinetics; agonists with greater time constant for G-protein deactivation exhibited relatively slower Ser375 dephosphorylation and βarrestin-2 unbinding, suggesting that higher agonist affinity for GIRK activation reflects sustained occupancy of MOPr in the phosphorylated state with the greater affinity to interact with βarrestin-2. In addition, the results provide strong evidence that duration of receptor occupancy contributes to development of bias. Slowly dissociating agonists, e.g. endomorphins, display greater bias towards endocytosis versus G-protein activation, whereas rapidly dissociating agonists are more biased towards G-protein activation and away from βarrestin-2 recruitment and internalization. In summary, these findings underscore the prominent role of binding kinetics in characterizing of bias profile. The present study demonstrated that off-rate kinetics of all signalling readouts are agonist dependent, suggesting that phosphorylation and arrestin binding require agonist-bound receptors. Moreover, duration of receptor occupancy is highly associated with agonist ability to stimulate arrestin-linked pathways and could be used as a major determinant of biased agonism.