Дисертації: "Signalling"

1

Schelfaut, Roselien. "Integrin Signalling." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6265.

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Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions.

The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes.

Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins.

To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained.

Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface.

β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.

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2

Demuth, Dirk Geoffrey. "Cannabinoid signalling." Thesis, University of Hertfordshire, 2004. http://hdl.handle.net/2299/14229.

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The general aim of the study was to investigate the signalling pathways utilised by cannabinoids. Cannabinoid CB1 receptor stimulation in DDT, MF-2 smooth muscle cells induces a rise in [Ca2], which is dependent on extracellular Cat' and modulated by thapsigargin-sensitive stores and MAP kinase suggesting capacitative Ca2+ entry (CCE). Non-capacitative calcium entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine Hl receptor-evoked increases in [Ca2+]; in DDTI MF-2 cells. In the current study both Ca 2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+];, were investigated. In the whole-cell patch clamp configuration, the cannabinoid receptor agonist CP 55,940 evoked a transient Cat+-dependent K+ current, which was not blocked by inhibitors of CCE, 2-APB and SKF 96365, although SKF 96365 did inhibit the outward current evoked by the refilling component of the response to histamine. AA but not its metabolites evoked a transient outward current and inhibited the response to CP 55,940 in a concentration-dependent manner. CP 55,940 induced a concentration-dependent release of AA, which was inhibited by the CB1 receptor antagonist SR 141716A. The non-specific Ca2+ channel blockers, La3+ and Gd3+, inhibited the CP 55,940-induced current at concentrations that had no effect on thapsigargin-evoked CCE. La3+ also inhibited AA-mediated currents. The effect of CP 55,940 on AA release was abolished by phospholipase A2 inhibition with quinacrine. This compound also inhibited outward currents mediated by CP 55,940. The data supports the possibility that in DDT, MF-2 cells AA is an integral component of the CBI receptor signalling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase. In a parallel line of work the present study aimed to identify the signalling events that might mediate a cannabinoid-induced inhibition of neurotransmission in the myenteric plexus, leading to a reduction in intestinal motility. Myenteric neurons were grown in primary culture enabling electrophysiological recordings to be made from individual cells to study the effects of cannabinoids on ion conductance. Immunohistochemistry validated these neurons as a model for those in situ, demonstrating that all CB1 receptor-positive cells express the cholinergic marker choline acetyltransferase. CP 55,940 was not shown to activate G-protein inwardly rectifying K+ channels but did inhibit evoked Ca2+ currents in myenteric cultures, a signalling mechanism that may underlie the CB1 receptor-mediated inhibition of neurotransmitter release from presynaptic sites. Nicotinic ACh (nACh) receptors are also expressed on cultured myenteric neurons. Stimulation of these receptors by nicotine evoked a transient inward current, which was inhibited by CP 55,940 and the endogenous cannabinoid anandamide, in an SR 14716A-insensitive manner. In fact, SR 141716A alone inhibited currents mediated by nACh receptors. PEA, a cannabinoid ligand whose effects are thought to occur independently of CB1/ CB2 receptor activation, also inhibited nicotine-induced currents. Pertussis toxin, a Gil,, inhibitor, did not reverse the cannabinoid-induced inhibition of nicotinic currents. In addition, CP 55,940 inhibited the sustained inward current evoked by 5-11T application in cultured myenteric neurons. The results suggest that cannabinoids inhibit nACh channels through a CB1 receptor-independent pathway in myenteric neurons, which would lead to a reduction in excitatory neurotransmission in the intact myenteric plexus. The inhibitory effect on the 5-HTinduced sustained inward current also suggests a cannabinoid-evoked inhibition of currents possibly mediated by the 5-HT1p receptor.

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3

Potter, Timothy James. "Transmembrane signalling." Thesis, University of Sheffield, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275041.

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4

O'Byrne, Declan. "CD28 and associated signalling elements of T lymphocyte signalling." Thesis, University of Bath, 1998. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266468.

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5

Shivdasani, Anish Anil. "Hedgehog signalling, TGF-Î² signalling and spermatogenesis in Drosophila melanogaster." Thesis, University of Sheffield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408836.

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6

Jordan, Lindsay. "Signalling Towards IRES." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19946.

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XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

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7

Anderson, Ian Paul. "Met receptor signalling." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526784.

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8

Clynes, David Alexander. "Signalling to chromatin." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496840.

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9

Samuels, Michael L. "Yeast stress signalling." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368116.

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10

Is'Harc, Hayaatun. "JAK/STAT signalling." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272414.

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11

Stephens, Sebastien. "Novel Osteoclast Signalling." Thesis, Griffith University, 2010. http://hdl.handle.net/10072/365823.

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When you say osteoporosis, people think of their grandma’s brittle bones, but scientists think of the osteoclast. When you say cancer, people think of death, but before this, many succumb to the osteoclast. The fact is all these things are true yet it is even truer to say that each disease in fact also has more of the other – osteoporosis and death, and cancer and brittle bones. However, the commonality is undeniably the osteoclast. Scratching the surface of the osteoclast reveals that it is the basis of a diversity of bone-related disorders yet the osteoclast itself is, even given the large amount of effort devoted to it to this day, an ill- defined cell. Not surprisingly, deaths related to osteoporotic fractures and skeletal pain due to metastases to bone remain far higher than ideal. Today’s dogma describes the osteoclast as a multinucleated cell derived from the haematopoietic cell lineage that is capable of bone resorption. It is suspected that it is through multiple signalling mechanisms, that this bone-resorbing activity is augmented in certain bone diseases eventually causing osteolysis or the break down of bone. Currently, it still stands that any way to advance our understanding of the osteoclast will provide stepping stones for eventual treatments and perhaps cures for these diseases. Thus the goal of this thesis was to better characterise the osteoclast. For this, four different projects were undertaken; analysis in the osteoclast of (1) the chemokine MCP-1, (2) the RhoGTPase family, (3) DMSO and (4) genes dependent on RANKL (by array). Project results are summarised in order.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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12

Halilagic, Aida. "Retinoid signalling in forebrain development and COUP-TF and retinoid signalling." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399574.

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13

Altosaar, Katrin. "Dimer-dependent allosteric modulation within GPCR signalling complexes can influence signalling diversity." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114353.

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G protein-coupled receptors (GPCRs) comprise the largest group of cell surface receptors, translating environmental signals into cellular responses via cognate G protein partners. Contrary to our initial understanding, most GPCRs do not function in living cells as monomers, but most likely dimers, or even larger arrays of receptors. Standard drug design approaches rely on the notion that drugs binding the two receptors in a given dimer likely function independently of one another. However, this view has been challenged by recent work showing that ligand binding at both receptors can modulate dimeric receptors via allosteric communication. While one receptor may actually be needed to drive signalling, the other acts to control or modulate these signals, without a direct signalling outcome itself. Based on the notion of allosteric modulation within homo- and heterodimers, I tested and compared changes in signalling downstream as well as at the level of the receptor-G protein-effector (RGE) complex in response to different combinations of ligands at each protomer. Using a combination of calcium, cyclic adenosine monophosphate, and mitogen-activated protein kinase signalling assays, I have demonstrated functional interactions for a putative D2 dopamine receptor, oxytocin receptor heterodimer (D2R/OTR), in HEK 293 cells. Immunoprecipitation, bioluminescence resonance energy transfer (BRET) and confocal microscopy experiments reveal D2R and OTR do in fact form a heterodimer in vitro, which may explain the nature of these potential allosteric functional interactions. Using BRET, I assessed the RGE complex conformational dynamics in HEK 293 cells for two other heterodimers, β2-adrenergic receptor with cannabinoid CB1 receptor (β2AR/CB1R) and β2AR/OTR, in order to determine how they manifest in parallel to signalling events themselves. These studies reveal functional interactions can occur in terms of signalling complex conformation. Thus GPCR signalling can be modulated by its partner receptor at the level of downstream effector signalling or at the level of the signalling complex itself. With that said, putative heterodimers need to be reanalyzed in vivo for their allosteric properties, which may explain some of the side effects of so many drugs, and may have implications in drug design.
Les récepteurs couplés aux protéines G (RCPG) constituent le plus grand groupe de récepteurs de la surface cellulaire, qui traduisent les signaux environnementaux en réponses cellulaires via leurs protéines G associées. Contrairement à notre compréhension initiale, la majorité des RCPG ne fonctionnent pas en tant que monomères, mais possiblement en tant que dimères ou même oligomères. Les approches actuelles de conception de médicament estiment que lors de la liaison d'un médicament aux deux récepteurs d'un dimère quelconque, ces derniers fonctionnent potentiellement indépendamment l'un de l'autre. Cependant, cette notion a été reconsidérée par une étude récente montrant que la liaison d'un ligand aux deux récepteurs peut les altérer par voie de communication allostérique. Alors qu'un premier récepteur peut être requis pour initialiser la signalisation, un second peut contrôler ou modifier ces signaux, n'ayant pas nécessairement une signalisation directe comme résultante. Dans l'étude suivante, basée sur la notion de modulation allostérique au sein d'homodimère et d'hétérodimère, les changements de signalisation en aval ainsi qu'au niveau du complexe récepteur/protéine G/effecteur (RGE) ont été étudiés et comparés en réponse à différentes combinaisons de ligands pour chaque protomère. En utilisant une combinaison d'essais de signalisation de calcium, d'adénosine monophosphate cyclique (cAMP) et de protéine kinase activée par des agents mitogènes (MAPK), une interaction fonctionnelle entre le récepteur dopaminergique D2 et le récepteur de l'ocytocine (D2R/OTR) a été démontrée dans les cellules HEK 293. Des expériences d'immunoprécipitation, de transfert d'énergie de résonance par bioluminescence (BRET) et de microscopie confocale ont révélé la présence d'hétérodimère entre le D2R et l'OTR in vitro, ce qui pourrait expliquer la nature des interactions fonctionnelles allostériques. En utilisant la technique de BRET, la dynamique fonctionnelle du complexe RGE dans les cellules HEK 293 a été examinée chez deux autres hétérodimères, soit celui composé du récepteur adrénergique β2 et du récepteur cannabinoïde CB1 (β2AR/CB1R) et l'hétérodimère β2AR/OTR, afin de déterminer comment ils traduisent les évènements de signalisation. Ces études démontrent donc qu'une interaction fonctionnelle peut survenir sur le plan de la conformation du complexe de signalisation. Par conséquent, la signalisation d'un RCPG peut être modulée par son récepteur partenaire au niveau des effecteurs ou au niveau du complexe de signalisation lui-même. Pour cette raison, il serait impératif de réanalyser in vivo les propriétés allostériques d'hétérodimères putatifs, ce qui pourrait expliquer certains effets secondaires d'une multitude de médicaments et ce qui pourrait impliquer des changements majeurs dans la façon de concevoir de nouveaux médicaments.

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14

Bryon, K. H. R. "Multiple dopamine signalling pathways antagonize RHO-1 signalling in the nervous system." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1419852/.

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Alterations in RhoA (RHO-1) signalling cause neuronal signalling defects in both mammals and C. elegans. In the case of C. elegans, exaggerated RHO-1 signalling in cholinergic neurons (nRHO-1*) has numerous effects that include increased release of acetylcholine (ACh) and exaggerated body curvature during locomotion. A suppressor screen identified the mutation, nz99, that suppresses nRHO-1* exaggerated curvature, but not increased ACh release. Non-neuronal defects caused by heat-shock, expression of RHO-1* (sterility, death, tail-swelling and protruding vulva) were not suppressed, suggesting that DAT-1 is required only for RHO-1 neuronal signaling. Whole genome sequencing and rescue experiments identified the nz99 suppressor as a mutation in the dopamine transporter DAT-1. DAT-1 is a negative regulator of dopamine (DA) in both mammals and C. elegans by transporting extracellular DA into cells. dat-1 mutations were unable to suppress nRHO-1* locomotion defects when DA synthesis was inhibited either pharmacologically, using the drug reserpine, or genetically by a cat-2, (tyrosine hydroxylase) mutation. This is consistent with a model in which elevated DA signaling acts to inhibit the locomotion defects of nRHO-1*. Exogenous DA causes a dose dependent slowing of locomotion in C. elegans, and this was suppressed by nRHO-1*. Thus nRHO-1* and DA co-suppress each other's locomotion behaviours, suggesting that DA and RHO-1 signalling act in parallel to control locomotion. C. elegans possess at least four DA receptors of the G protein coupled class (DOP-1-4) that fall into either the D1 (DOP-1, 4) or the D2 class (DOP-2, 3). All four receptors are required for elevated DA signaling to suppress nRHO-1* locomotion defects. In other studies, the D1 and D2 classes of DA receptor have been shown to act antagonistically in controlling locomotive behaviours including, swimming, basal slowing and paralysis. My data suggests that a co-ordinated DA response that is mediated by multiple DA receptors, possibly acting in numerous cell types, also acts to modulate locomotion in C. elegans possibly by altering both small synaptic vesicle and dense core vesicle release.

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15

Li, Xiaolei. "Quadgate forward-signalling pipelines." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442597.

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16

Melford, Steven K. "Calcium signalling in megakaryocytes." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364104.

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17

Rebocho, Ana Paula da Mota Torres. "Cellular signalling by Araf." Thesis, University of London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531334.

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18

McVeigh, P. "Neuropeptide signalling in nematodes." Thesis, Queen's University Belfast, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411747.

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19

Lindqvist, Susanne. "Colonic crypt calcium signalling." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327537.

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20

Sun, Yan. "Cannabinoids and PPARa signalling." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431260.

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21

Greig, Aina Vibeke Hiller. "Purinergic signalling in skin." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407987.

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22

Brickell, Laura. "Wound signalling Arabidopsis thaliana." Thesis, University of York, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286054.

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23

Jai-Yoon, Sul. "Calcium signalling in astrocytes." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391921.

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24

Sarafimidis, Ioannis. "Polyketide signalling in Dictyostelium." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615971.

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25

Lau, C. I. "Hedgehog signalling in haematopoiesis." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1428443/.

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The Hedgehog (Hh) family proteins and their signalling pathway are key mediators of, and important in, many mammalian developmental processes. Malfunction of the Hh signalling pathway contributes to developmental disorders and birth defects. This project aims to investigate the role of the Hh signalling pathway in murine haematopoiesis. In our study, we found that Dhh plays a negative regulatory role in normal erythropoiesis and under stress conditions. However, it is not required for regulating erythropoiesis in the fetal liver during embryo development. In contrast, analysis of conditional deletion of Smo in haematopoietic cells revealed that Smo controls early haematopoietic differentiation in the fetal liver but is dispensable for regulating haematopoiesis in adult bone marrow and spleen. Furthermore, pervious studies have demonstrated that Hh signalling is involved in T-cell development throughout maturation. We tested the hypothesis that Foxa2, a downstream target gene of Hh during pre-TCR signalling, is also required for late T-cell development and activation. Analysis of mice conditionally Foxa2-deficient in mature T-cells revealed that Foxa2 is important in the process of maturation in late thymocyte development. In addition, Foxa2 is also involved in regulation of T-cell activation, and the differentiation of T helper cells. Gene expression experiments confirmed that Foxa2 is also a Hh target gene in the thymus. Taken together, our findings revealed that the Hh signalling pathway and its target genes play critical roles in haematopoiesis during embryogenesis and in adult mice.

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26

Benford, Heather Elizabeth. "Signalling in hypothalamic tanycytes." Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/62112/.

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Hypothalamic tanycytes are a specialised type of glial cell found lining the 3rd ventricle. They possess a cell body that contacts the cerebrospinal fluid (CSF) and a single long process that projects into the hypothalamic parenchyma, some of which are known to extend into the Arcuate nucleus (ARC) and Ventromedial nucleus (VMN), two key hypothalamic regions involved in control of energy homeostasis. Owing to their unique position, it has been hypothesised that hypothalamic tanycytes may be able to detect nutrient related signals in the CSF and influence the neurons of the ARC and VMN leading to changes in food intake and body weight. Tanycytes have recently been shown to be glucosensitive, responding to brief application of glucose by generating Ca2+ waves. However the signalling pathways inducing this response remain elusive. Here we investigated the nature of tanycyte glucosensing. Using Ca2+ imaging techniques we show that tanycytes can respond to glucose and non-nutritive sweeteners implicating the sweet taste receptor as the mediator of tanycyte glucosensitivity. We further show that activation of the sweet taste receptor induces release of ATP from tanycytes via pannexin hemichannels allowing propagation of the Ca2+wave. In addition we also investigated whether hypothalamic tanycytes can communicate to secondary cells within the hypothalamus. Using multiphoton stimulation of a single tanycyte, we show that Ca2+ waves can be generated, which spread between neighbouring cells as well as along the tanycyte process towards the hypothalamic parenchyma. We also demonstrate signalling in secondary cells following tanycyte stimulation is likely the result of tanycyte communication. Thus hypothalamic tanycytes are able to detect sweet tasting compounds in the CSF via the sweet taste receptor, this induces Ca2+ signalling which may be communicated to secondary cells, including the neurons of the ARC and VMN, where it may be integrated to induce changes in energy homeostasis.

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27

Gagliardi, M. "Endocytosis and wingless signalling." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1353109/.

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Wingless (Drosophila Wnt-1) is secreted glycoprotein that triggers an evolutionary conserved signal transduction pathway. The role of endocytosis in Wnt/Wingless signaling is not clearly understood and highly debated. In my thesis I explore the role that endocytosis/endocytic trafficking has on Wingless signalling activation and termination. In the canonical pathway Wingless binds to a member of the Frizzled family of seven-pass transmembrane receptor (Frizzled1 or Frizzled2) and to Arrow. Formation of this trimeric complex leads to the inactivation of the Armadillo degradation complex and translocation of Armadillo into the nucleus where it contributes to the activation of target genes. I show that internalization of the ligand-receptor complex is not required for signalling activation. I also show that Wingless has different effects on the trafficking route of its receptors: it induces the degradation of Frizzled2 and the recycling of Arrow. To identify post translational modifications that regulate Arrow trafficking I conducted an RNAi screen in Drosophila S2R+ cells for de-ubiquitylating enzymes (DUBs) and ubiquitin conjugating enzymes (E2) that modulate signalling. To carry out this screen, improvements on the current TOPFlash Wnt/Wingless signalling reporter were made. I also directly assessed the role of endocytic trafficking on signalling using a chemical inhibitor of endocytosis, Dynasore. I find that Dynasore inhibits signalling by causing a strong decrease in Armadillo levels. Future experiments will determine whether it is the stability or the rate of production of Armadillo that is affected.

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28

Bond, Alistair. "Purinergic signalling in osteoblasts." Thesis, University of Liverpool, 2012. http://livrepository.liverpool.ac.uk/11293/.

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ATP is now well established as an extracellular signalling molecule and has been shown to play a role in many different tissues including bone. ATP acting on P2 receptors has been reported to cause an increase in osteoblast proliferation, apoptosis, IL-6 production, AA release and a decrease in bone mineralization. It has also been shown in vivo, by using KO mice, that ATP can cause both an anabolic and catabolic response depending on the P2 receptor involved, with P2Y2 KO mice showing an increased bone formation whilst P2X7 mice have a decrease in the periosteal bone formation. ATP is released from osteoblasts in vitro basally, following trauma to the cell and in response to mechanical strain. This mechanical strain comes in the form of medium displacements, where the medium is taken up in a pipette and then immediately washed over the cells. Work carried out in this thesis looked to refine the experimental procedure of these tests in an attempt to reduce the variation of ATP supernatant measurements following medium displacement experiments. A new experimental procedure has now been implicated that ensures that the luciferase enzyme, used to measure ATP, is kept under constant conditions, the measurements from the wells are taken from the same area and basal levels are measured in each well as it was determined that ATP measurements are not consistent throughout a single well. Further to this a sensitive measure of cell death was developed in order to ensure that the observed ATP release was not due to cell trauma. This used qPCR to measure mtDNA present in the supernatant. Tests were also carried out to investigate the synergy between ATP and PTH as ATP synergises with PTH to increase the expression of c-fos in osteoblasts. c-fos is a master regulator gene that causes an increase in osteoblast proliferation and differentiation. It also causes the secretion of RANKL and decreases secretion of OPG, the RANKL decoy receptor, thus osteoclast fusion is also upregulated with an increase in c-fos production. Given that ATP is a local hormone that is released by mechanical stress and acts in an autocrine/paracrine manner and that PTH is an indiscriminate system wide hormone this synergy could be one mechanism whereby Wolff’s Law is achieved. That is, BMD is increased in areas of higher loading. The mechanism of this synergy is still to be elucidated and is likely to be different between cell types. Experiments on UMR-106 cells has shown that ATP and PTH can cause a potentiation of intracellular calcium release that ultimately leads to increased c-fos transcription. In SaOS-2 cells two separate signalling pathways converge on different areas of the c-fos promoter to cause an increase in c-fos. Work carried out for this project now shows for the first time that ATP sensitises osteoblasts to the action of PTH. Given the short half life of both molecules this increases the likelihood that they do play a significant role in bone remodelling in vivo. The mechanism behind this sensitisation appears to be independent of the known mechanisms described above, as it was shown that the sensitisation was conferred in the medium and was not due to ATP break down products ADP, AMP and adenosine. Thus it is likely another, longer lasting, paracrine factor is involved. BL-1249 is a putative K+ channel opener and inhibits ATP+PTH induced c-fos expression. The inhibition was shown not to be due to BL-1249s effect on K+ channels. It appears more likely that it acts on the cAMP/PKA pathway to inhibit c-fos transcription, as BL-1249 did not decrease FCS induced c-fos expression but did inhibit sp-cAMP induced c-fos expression. Further to this BL-1249 at concentrations of 100 μM caused a massive increase in cell death to the osteosarcoma cell lines SaOS-2, MG-63 and TE-85, however it had no effect on primary human osteoblasts. The apoptotic effect of BL-1249 is very likely due to the activation of K+ channels as it has been reported that opening K+ channels will cause apoptosis through K+ efflux and subsequent cell shrinkage. This was further confirmed by use of TEA, a K+ channel blocker, which acted to reverse the effect of BL-1249. Thus BL-1249 can act to decrease osteosarcoma cell number via inhibition of c-fos and, via K+ xiii channels, cause apoptosis. It is still unclear how apoptosis is targeted to osteosarcoma lines however it is likely through BL-1249s effect on c-fos expression. Occasionally the true effect of a gene or protein can only be elucidated when studying it in the context of a pathology. It has been reported that P2X7 can cause astrocytes to be toxic to motor neurones by secreting factors that contribute towards their death. In situations of increased oxidative stress, such as those found in SOD1 -/- mice, this effect can be increased and cause basal levels of ATP to activate the neurotoxic phenotype of the astrocytes. SOD1 -/- mice have also been examined for the bone and muscle phenotypes. They were shown to have a massive reduction in muscle mass with the peak difference being less than 50% their wild-type counterparts, their bones also had a decrease in BMD, a decreased length of their femur and had a decreased stiffness. Given this link between SOD1, P2 signalling and an altered bone phenotype SOD1-/- mice were examined in more detail. μCT scans were used to analyse the bone structure of these mice, however in this study no differences were observed. P2X4 was increased in SOD1-/- calvarial cells whilst P2Y2 was decreased in these cells suggesting that oxidative stress does cause an altered P2 receptor expression and this may change the BMD of bone.

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29

Káradóttir, Raghildur Póra. "Neurotransmitter signalling to oligodendrocytes." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1446463/.

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Neurotransmitter signalling to neurons and glial cells plays a key role in brain development, information processing and pathological processes. This thesis focuses on neurotransmitter signalling to oligodendrocytes, the glial cells which provide myelin to speed the propagation of action potentials along neuronal axons. In cerebellar and corpus callosal slices, I used patch-clamping and immunocytochemistry to examine the properties of precursor, immature and mature oligodendrocytes, characterizing their morphology and basic electrical properties, their response to glutamate, GABA and other neurotransmitters, and the neurotransmitter receptor subunits that they express. In contrast to the currently held view, I found that oligodendrocytes express NMDA receptors. These receptors show extremely weak magnesium-block, allowing them to be activated at the resting potential, and they may be composed of NR1, NR2C and NR3 subunits. To investigate the role of these NMDA receptors in pathology, experiments on hippocampal neurons were first used to establish how best to block glycolytic and mitochondrial production of ATP to mimic the energy deprivation which occurs in ischaemia. Ischaemia-evoked glutamate release was found to activate oligodendrocyte NMDA and non-NMDA receptors. Although the normal role of the oligodendrocyte NMDA receptors may be to regulate myelination, they probably contribute to the glutamate-mediated damage which occurs to oligodendrocytes in periventricular leukomalacia (leading to cerebral palsy), stroke, spinal cord injury and multiple sclerosis. Block of these receptors may therefore offer a potential therapeutic approach to treating these disorders.

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30

Hiskens, Richard Andrew. "The interaction of activator of G protein signalling 1 (AGS1) with signalling components." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422078.

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31

Brodin, Greger. "Smad7 in TGF-β Signalling". Doctoral thesis, Uppsala University, Ludwig Institute for Cancer Research, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1949.

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Members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors regulate a vast array of biological functions in the adult, and are of great importance in governing cell fate determination and patterning in the developing embryo. The TGF-β signal is propagated intracellularly by Smad proteins resulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known to downregulate the TGF-β signal and thereby possibly modulating the biological response. This thesis describes a functional analysis of the inhibitory Smad7 from an in vitro and in vivo perspective.

The prostate gland is dependent on androgens for its growth and differentiation. Androgen withdrawal can cause regression and apoptosis in normal and malignant prostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. We investigated the expression levels of Smad proteins in the rat ventral prostate as well as in an androgen sensitive prostate tumor model (Dunning R3327 PAP) by immunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4 and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, as well as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 were also increased in both normal and malignant prostate in response to castration.

Several studies have shown that Smad7 is upregulated in response to TGF-β stimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response. We investigated the molecular mechanism behind that response by studying the transcriptional regulation of the Smad7 gene. We identified a palindromic Smad binding element (SBE) in the promoter. Point mutations introduced into the SBE abolished transcriptional activation via TGF-β. We also observed that mutating or deleting binding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediated transcriptional induction as well as the basal promoter activity.

Gene ablation of Smad proteins has revealed specific physiological and developmental roles. We analysed mice targeted on the Smad7 locus. The mice appeared viable and fertile with a slight reduction in litter size, suggesting a perinatal loss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no major difference between wild type and mutant MEFs.

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32

Cargill, James. "Multitone signalling on telephone lines." Thesis, University of Ottawa (Canada), 1988. http://hdl.handle.net/10393/5543.

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33

Law, Robert. "PDE3A signalling in blood platelets." Thesis, University of Hull, 2016. http://hydra.hull.ac.uk/resources/hull:13761.

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Cyclic 3’, 5’ adenosine monophosphate (cAMP) signalling downstream of prostacyclin (PGI₂) is a key inhibitory pathway in blood platelets. This pathway is dynamically regulated by phosphodiesterase 3A (PDE3A), which hydrolyses cAMP into metabolically inactive AMP. Although PDE3A is an established drug target in anti-platelet therapies, the molecular mechanisms that underlie its function in platelets remain unclear. Therefore, the major aim of this study was to further explore PDE3A signalling in human platelets. Using a combination of cell fractionation and immunoblotting we identified two PDE3A splice variants in platelets, PDE3A1 and PDE3A2, that were differentially localised within the cell. PDE3A1 was located in the membrane fraction, whereas PDE3A2 was primarily located in the cytosolic fraction. Treatment of platelets with PGI2 induced a transient phosphorylation of PDE3A2 at Ser³¹² in a PKA dependent manner. In contrast, no phosphorylation of PDE3A1 was detected. The phosphorylation of PDE3A2 was associated with increased PDE3A enzymatic activity, which suggested that cAMP signalling activated only the cytosolic form of the enzyme. In many cells, A-kinase anchoring proteins (AKAPs) orchestrate a coordinated response between PKA and its effector proteins. The phosphorylation and activation of PDE3A2 in response to PGI2 was blunted by a cell permeable peptide inhibitor of PKA-AKAP interactions suggesting that PKA-mediated activation of PDE3A2 was dependent on an AKAP. Using a cAMP-pull down approach to enrich cAMP binding proteins combined with immunoblotting, we confirmed the presence of two AKAP7 isoforms (δ and γ) in platelets. Additionally, we found that AKAP7δ co-precipitated with PDE3A2 and possessed associated PDE3A activity. Furthermore, AKAP7 also possessed PKA activity, which was a result of its constitutive association with PKA-II. Critically, immunoprecipitated PDE3A was found to be co-associated with both PKA-II and AKAP7δ. The findings in this thesis suggest that blood platelets express multiple differentially-regulated PDE3A splice variants, of which PDE3A2 is regulated by PKA-II within a novel cytosolic AKAP7δ facilitated signalling complex. The selective inhibition of PDE3A splice variants and/or pharmacological disruption of PDE3A signalosomes may provide safer and more specific ways of controlling pathological platelet activation.

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34

Tadros, Amir. "Novel pathways in microvascular signalling." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29504.

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The regulation of microvascular survival impacts both developmental remodelling of the vasculature, and various pathologies. The aim of this thesis was to examine the role of a recently identified A20 binding inhibitor of NF-kB, a zinc finger protein termed ABIN-2 in endothelial protection. More specifically, looking at its effects on inflammation and apoptosis in endothelia both at the cellular level and in vivo. The involvement of ABIN-2 in the Tie2 receptor pathway was also examined. Tie2 is an endothelial receptor essential for blood vessel formation and promotes endothelial survival. A transfection protocol was established allowing expression ABIN-2 in up to 90% of endothelial cells. ABIN-2 was shown to reduce apoptosis in endothelial cells as well as improve cell survival following growth factor deprivation. This effect was inhibited by Wortmannin and LY294002 which are known inhibitors of phatidylinositol-3 kinase. Expression of the truncated form of ABIN-2 lacking the carboxy terminal of ABIN-2 did not protect cells from apoptosis. In addition, expression of the truncated form prevented cell rescue by angiopoeitin-1 from apoptosis. The chick chorioallantoic membrane was used as an in vivo model for testing the role of ABIN-2 in vessel inflammation. It was possible to use this model to transfect plasmids into live microvessels using electroporation resulting in high yield expression of target protein. This model was adapted to look at microvessel inflammation and apoptosis. Expression of ABIN-2 in microvessels reduced leukocyte rolling following tumour necrosis factor-a and lipopolysaccharide induced inflammation. The truncated form of ABIN-2 lacking the carboxy terminal did not reduce microvessel inflammation. The NF-kB inhibitor PTDC was found to suppress leukocyte rolling. ABIN-2 expression also appeared to give limited protection against apoptosis in vivo.

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35

Kaplan, Andrew Daniel. "Purinoceptor signalling in skeletal cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq28500.pdf.

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36

Day, Jonathan Robert Stewart. "Therapeutic inhibition of thrombin signalling." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434321.

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37

Haines, Nicola. "Mutational analysis of hedgehog signalling." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365823.

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38

Saltmarsh, Esther Joy. "Calcium signalling in neutrophil behaviour." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55796/.

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Neutrophils are at the front line of defence in humans against attack by invading pathogens. They undergo complex behaviours, many of which are controlled by changes in the concentration of cytosolic free calcium ions (Ca2+). However, the control and mechanisms of these behaviours are still the subject of much research and debate. Ca2+ is known to be important in many different cell systems. There are a plethora of Ca2+ signalling events in the "Ca2+ toolkit", but not all cells have each element. It is not fully understood elements of the toolkit that neutrophils employ to make up their well- characterised global Ca2+ changes. This thesis is an investigation into the involvement of Ca2+ in neutrophil behaviours that underlie much of their functionality, particularly focussing on subcellular Ca2+ release events (puffs) and Ca2+ signalling during neutrophil spreading on surfaces and during phagocytosis. Reports were published by Petty et al of a travelling zone of elevated Ca2+ (z-wave) in neutrophils. However, research laid out in this thesis demonstrates that these z-waves are questionable in neutrophils. Instead, conventional Ca2+ puffs and waves were seen and characterised in response to fMLP stimulation and uncaging IP3. Neutrophils undergo dramatic, rapid morphological changes during Ca2+ signals triggered by both fMLP and uncaging IP3, representative of behaviour during adhesion and extravasation. It was shown that the high concentration of Ca2+ under the plasma membrane that results from Ca2+ influx activated the protease calpain, which released the membrane from the cytoskeleton for rapid shape change. Similar signals and behaviours are seen when neutrophils are stimulated by binding of beta3-integrins. Indeed, adhesion to ICAM-1 expressing cells and phagocytosis resulted in global Ca2+ signals and Ca2+ influx accompanied by rapid shape change. However, no Ca2+ puffs were seen in these latter experiments, implying that the signalling in these conditions occurs through a different pathway, possibly PIP3. In conclusion, neutrophils utilise conventional Ca2+ signalling events (release puffs and influx waves) not z-waves to control many of their functions. They are capable of producing Ca2+ puffs, but the lack of ER means this is unlikely to be a primary signalling pathway. Binding through jS-integrins does trigger Ca2+ signals, possibly through the PIP3 pathway, but not store release. The characterisation of Ca puffs and the functionality of the Ca2+-activated protease calpain in these conditions are novel and merit future investigation. The findings in mis thesis enhance the general understanding of how neutrophil behaviours are regulated by Ca2+ and provide a new methodology for further research.

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39

Ludlow, Melanie Joanna. "Purinergic signalling in dictyostelium discoideum." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/7450.

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The identification of five predicted proteins (dP2XA-E) with homology to vertebrate P2X receptors in Dictyostelium discoideum proved a unique opportunity to explore purinergic receptor function in a simple unicellular eukaryote from outside the animal kingdom. dP2XE was shown to be expressed as a trimer and trafficked to the cell surface in Xenopus oocytes. However, no currents were detected to extracellularly applied ligands. This lack of function was potentially due to inaccurate post-translational modifications since dP2XE expressed in oocytes and D. discoideum displayed different molecular weights. Unexpectedly for a potential ligand-gated ion channel, dP2XE-eGFP displayed a solely intracellular distribution in D. discoideum, localising to the endolysosomal and contractile vacuole systems. Ablation of the p2xE gene by homologous recombination revealed a role for dP2XE in axenic growth in suspension, associated with a modest defect in cytokinesis, and a potential involvement in the calcium signalling and homeostasis functions of the contractile vacuole. Both this distribution and disruption phenotype were mirrored for p2xA. The existence of purinergic signalling in D. discoideum was demonstrated by utilising an apoaequorin expressing strain to show that extracellular ATP and ADP evoked increases in intracellular Caˆ{2,+}. Indicative of P2X receptor activation, responses were rapid and transient, required extracellular Caˆ{2,+}, inhibited by Gdˆ{3,+}, modified by extracellular pH and remained unaffected by deletion of either the single heterotrimeric G\beta or iplA genes. ATP/ADP responses were unaffected by ablation of either the p2xA or p2xE genes leaving dP2XB-D as potential candidates. Inhibition of the large P2X-like response with Znˆ{2,+} revealed the presence of a much smaller response with a slower time course indicating that P2Y-like receptors may also be present. The work presented in this thesis demonstrates that D. discoideum possesses cell surface purinergic receptors for extracellular ATP/ADP and extends our knowledge of the intracellular role of purinergic signalling in this organism.

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40

Allen, John Cronin. "Neutrophil signalling via Fcγ receptors". Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494099.

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It has previously been shown that removal or blocking of the neutrophil receptor, FcyRIIIb (CD16b) completely prevented the ability of neutrophils to secrete reactive oxidants and granule enzymes in response to soluble immune complexes (SIC), but had no effect on the phagocytosis and killing of serum-opsonised Staphylococcus aureus. As soluble immune complexes are the major neutrophil activating factors in the synovial fluids of patients with rheumatoid arthritis, the aim of this work is to determine the intracellular signalling pathways regulated by FcYRIIIb to understand, in more detail, neutrophil function in disease.

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41

Brennan, Damian Frederick. "Structural studies of MAPK signalling." Thesis, Institute of Cancer Research (University Of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511162.

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42

Lee, Sue Chin. "Spatial signalling of phosphatidic acid." Thesis, University of Strathclyde, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510804.

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43

Dawson, M. A. F. "JAK-STAT signalling at chromatin." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598423.

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The aim of my work was to explore the possibility that the mammalian JAK2 signalling pathway influences the structure and function of chromatin. I have demonstrated that JAK2 is present in the nucleus of both human haematopoietic cell lines and primary cells. My results suggest that JAK2 functions as a histone tyrosine kinase and phosphorylates histone H3 at tyrosine-41 (H3Y41). This novel histone modification, the first described tyrosine phosphorylation on any of the non-variant histones, regulates the binding of heterochromatin protein 1-alpha (HP1α) at a new binding site on chromatin. HP1α uses its chromo-shadow domain to bind the H3Y41 region. Phosphorylation of H3Y41 by JAK2 reduces its affinity for chromatin. This reciprocal relationship was given a functional context by demonstrating its relationship to the expression of a key haematopoietic oncogene Imo2. Genome-wide studies demonstrate that H3Y41ph is present at the 5’ end of genes and is highly correlated with active transcription. This is the first comprehensive genome wide mapping of a histone phosphorylation mark and potentially highlights a role for this novel modification in the regulation of transcription. H3Y41ph was also present at specific cis-regulatory elements on JAK2-STAT5 target genes and genome-wide mapping of STAT5 binding confirmed that STAT5 binding and H3Y41ph was coincident at a significant number of sites within the human genome. This interesting observation suggests that canonical JAK2-STAT5 signalling is not confined to the cytoplasma but also occurs at chromatin. These findings extend the existing paradigm of JAK-STAT signalling and provide a platform for a better understanding of this critical signalling pathway, which is important in both normal development and oncogenesis.

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44

McGouran, Joanna. "Probing sugar-plant-soil signalling." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526091.

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45

Moon, Alice E. "Immune signalling in insect cells." Thesis, Kingston University, 2009. http://eprints.kingston.ac.uk/20407/.

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Immune responses in insects include components and mechanisms that are highly evolutionarily conserved. In addition to providing insight into the insects themselves, knowledge of the conserved mechanisms involved in insect immunity can offer valuable insight that is broadly relevant to a wide variety of other species. Three aspects of insect immune cell signalling have been studied here. Cell signalling responses have been investigated in two insect cell lines following treatment with double stranded (ds) RNA, a common intermediate of viral replication. It has been established that both cell lines investigated, from the fruitfly Drosophila melanogaster and vector mosquito Aedes albopictus, do not exhibit activation of mitogen activated protein kinases (MAPKs) or NF-KB proteins as a direct response to dsRNA. Secondly, a detailed analysis of the mechanisms of transcriptional regulation has been carried out on the Drosophila drosomycin gene, a key factor both in terms of its function during the immune response and in terms of its role during previous characterisation of Drosophila immune signalling. The drosomycin promoter was found to be regulated independently by the Toll and IMD signalling pathways via distinct sequence elements. Finally, investigation of the responses of an A. albopictus cell line to treatment with bacterial cell wall components has revealed key differences in the mechanisms involved in immune-induced regulation of transcription compared with the model established in Drosophila. A role for p38 MAPK has been identified in the negative regulation of transcription of A. albopictus cecropin Ai, an inducible antimicrobial peptide gene.

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46

Zhou, Zhigang. "TNF signalling in endothelial cells." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435077.

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Pleiotropic cytokine tumour necrosis factor a (TNF) is the archetypal member of a large super-family of ligands and associated receptors. Through its two surface presenting receptors, TNFRI and TNFR2, TNF can elicit responses such as differentiation, proliferation, and apoptosis, depending on several factors such as microenvironment, cell cycle, and cell type. Recent evidence indicated that TNF can be up-regulated in endothelial cells (EC) upon their stimulation and plays an important role in regulating EC responses which have been implicated in several cardiovascular diseases, such as heart failure and atherosclerosis. Anti-TNF therapy has been successfully applied on several inflammatory related diseases, such as rheumatoid arthritis and Crohn's disease. Conversely, for heart failure patients, anti-TNF therapy has been considered but has been disappointing in trials thus far. Therefore, a greater understanding of TNF's ability to signal disparate responses is essential for the development of more effective TNF- or signalling-based therapeutics. Here we demonstrate that the regulation of TNF activity in endothelial cells is controlled by two distinct receptor signalling pathways via its associated adaptor and downstream effector proteins, cross-linked by potential crosstalk mechanisms at different cascades. Our findings clearly demonstrate that TNF activates both the canonical and non-canonical NF-KB pathways, mainly through TNFRI signalling while TNFR2-driven NF-KB nuclear translocation is mainly inhibitory. TNF regulated NF-KB dependent genes including adhesion molecules which were up-regulated via the TNFRI receptor pathway; MAPKs activation may interact with TNF-driven signal at several cascades for fine tuning of its overall effects. We also discovered that TNFR2 is able to switch on survival signals via induction of the protective genes heme oxygenase-1 (HO-1) and ferritin or through the auto-regulation of TNFR2 itself. Through our findings, we hypothesised that TNF elicits in EC its pro-inflammatory effects mainly through TNFRI while balancing the overall signalling response by the survival signals mediated via TNFR2. Our results have implications for improving anti-TNF therapeutic strategies by selective inhibition of TNF receptor signalling rather than inhibiting TNF as a whole.

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47

Iessi, Elisabetta. "TRAIL signalling regulation by ezrin." Phd thesis, Université de Bourgogne, 2011. http://tel.archives-ouvertes.fr/tel-00695336.

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Background and Aim: TRAIL has sparked a growing interest in oncology due to its ability to selectively trigger cancer cell death while sparing normal cells. The Fas/actin association through ezrin, a member of the ERM protein family, has been reported to regulate early steps of Fas-mediated apoptosis. In this project, we addressed the role of ezrin regarding TRAIL-induced cell death in B lymphoma cell lines, or adherent cancer cell lines (HeLa WT, HCT116, SW480). Methods: Molecular and biochemical approaches were employed to study the relevance of ezrin and its phosphorylation status in TRAIL signaling. Results: We found that ezrin displays a negative function towards TRAIL- and Fas-mediated apoptosis and that the ezrin-mediated TRAIL-induced cell death inhibition led to ezrin activation through phosphorylation/dephosphorylation events at serine 66 and tyrosine 353, but is mainly independent of TRAIL DISC (Death Inducing Signalling Complex) formation or activation. Mutations of these residues to alanine (S66A) or aspartic acid (Y353D) selectively enhanced TRAIL-induced cell death, whereas point mutations mimicking ezrin phosphorylation on S66 (S66D) or a nonphosphorylable variant on Y353 (Y353F) strongly protected cancer cells from apoptosis induced by TRAIL. Moreover, inhibition of the ezrin serine 66 PKA target site, using H89, increased cancer cell sensitivity to TRAIL, while treatment with 8bromocyclic AMP, a PKA activator, decreased TRAIL-induced cell death. In addition, combined TRAIL/cisplatin treatments abrogated ezrin-mediated inhibition of TRAIL-induced apoptosis.

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48

Chen, Daliang. "Cell signalling in Paracoccidioides brasiliensis." Thesis, Durham University, 2006. http://etheses.dur.ac.uk/2655/.

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Paracocidioides brasiliensis (P. brasiliensis or Pb) is the etiological agent of paracocidioidomycosis which is the most prevalent systemic mycosis in South America. About 60% of the clinical cases are found concentrated in Brazil and the disease is a major threat for the public health there, p. brasiliensis is a dimorphic fungus because it undergoes a morphological switching from a mycelium to yeast form after shifting the temperature from 26 c to 37 c. Similar morphological changes have been implicated to be important in the pathogenicity of other dimorphic fungi and they appear to be under the control of the с AMP signalling transduction pathway. In order to establish the relationship of с AMP signalling pathway and the morphological change in p. brasiliensis, a project was initiated to clone the key components of the с AMP signalling pathway and analyze their functions. To this end, a genomic DNA library and two cDNA libraries oiPb were constructed. Degenerate primers were synthesized based on the sequence of genes of several other important fungi in GenBank. After amplification, specific PCR products were obtained and sequenced. The specific PCR products were used for labeling to screen libraries and their sequences were used for designing specific primers to do genomic walking and RACE-PCR. Both cDNA and genomic DNA sequences have been determined for the key components of the с AMP signalling pathway including adenylate cyclase (PbCYRl), three G protein α subunits (PbGPAl,PbGPA2 and PbGPA3), a G protein β subunit (PbGPBl), a G protein γ subunit (PbGPGl), a с AMP dependent protein kinase catalytic subunit (PbTPKJ), a с AMP depentdent protein kinase-like gene (PbTPKLl) and a TUP gene (PbTUPA).The interactions of the proteins encoded by the genes in the с AMP signaling pathway were studied with Clontech's Matchmaker yeast-two-hybrid System III. This approach demonstrated that the N-terminal third of adenylate cyclase PbCyrl 1 •678 can interact with PbGpal and PbGpbl. To further test if PbGpa2, PbGpa3, PbRasl, and PbActin can interact with adenylate cyclase, random mutagenesis libraries were made for these genes and screened with PbCyr1 1 •678, PbCyr1 600- 1316, PbCyri 1302 1876 and PbCyr1 1648-2100. It was demonstrated that PbCyrl 1-678, where the Ras association domain resides, can interact with truncated versions ofPbGpa2, PbGpa3, PbRasl, and PbActin, i.e., PbGpa21 -102, PbGpa21-183, PbGpa3 1-160, PbGpa3 1-213, PbRas1 1-83,PbRas 126-238, and PbActin 1-314. For the first time, the major components of Pb cAMP signaling pathway have been cloned and characterized; and we provide direct evidence that the G protein a subunits, G protein p subunit, RAS protein and actin interact directly with adenylate cyclase in fungal biology. This thesis funds the basis for the further study of the cAMP signalling pathway in P. brasiliensis. It may facilitate delineation of the mechanism for the dimorphic switching and the development of potentially novel antifungal drugs.

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49

Schweinzer, Paul Gernot. "Three essays on signalling games." Thesis, Birkbeck (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442064.

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50

Woodroffe, P. J. "Mathematical modelling of cell signalling." Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416886.

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