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Dromey, Jasmin Rachel. "Elucidating novel aspects of hypothalamic releasing hormone receptor regulation". University of Western Australia. School of Medicine and Pharmacology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0133.
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[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.
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Jama, Abdirahman Mohamud. "Functional regulation of kisspeptin receptor by calmodulin and Ca2+/calmodulin-dependent protein kinase II". Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/15914.
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The kisspeptin receptor (KISS1R), functioning as a metastasis suppressor and gatekeeper of GnRH neurons, is a potent activator of intracellular Ca2+. The surge in cytoplasmic Ca2+ mediates the exocytosis of GnRH from GnRH neurons. However, the regulatory processes which enable KISS1R to sense increasing intracellular Ca2+ and avoid Ca2+ excitotoxicity via a signalling off-switch mechanism remain unclear. This thesis provides evidence for the interaction between KISS1R and the Ca2+ regulated proteins of calmodulin (CaM), and αCa2+/CaM-dependent-protein kinase II (α-CaMKII). Binding of CaM to KISS1R was shown with three independent approaches. Firstly, cell-free spectrofluorimeter assays showed that CaM selectively binds to intracellular loop (IL) 2 and IL3 of the KISS1R. Secondly, KISS1R co-immunoprecipitation experiments identified ligand/Ca2+-dependent binding of KISS1R to HEK-293 endogenous CaM. Thirdly, confocal experiments showed CFPCaM co-localises with YFP-KISS1R. The functional relevance of CaM binding was examined with alanine substitution of critical residues of the CaM binding motifs in IL2 and IL3 of KISS1R. This approach revealed that the receptor activity (relative maximum responsiveness) was increased in the mutated residues of the juxtamembrane regions of IL3 and the N-terminus of IL2 relative to wild-type KISS1R. The Ca2+/CaM regulated αCaMKII was also found to interact with KISS1R by selectively phosphorylating T77 of IL1. Phosphomimetic mutations of T77 into E or D created a receptor that was unable to elicit inositol phosphate production upon ligand stimulation. Finally, in vivo studies using ovariectomised rats that were intracerebroventricularly administered with a cell-permeable αCaMKII inhibitor augmented the effects of kisspeptin ligand stimulation of plasma luteinizing hormone levels. Taken together, this thesis demonstrates that the KISS1R-G protein coupling is regulated by Ca2+-dependent CaM binding and αCaMKII-mediated KISS1R phosphorylation.
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Åkerberg, Helena. "Functional Studies of the Neuropeptide Y System : Receptor-Ligand Interaction and Regulation of Food Intake". Doctoral thesis, Uppsala universitet, Institutionen för neurovetenskap, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9533.
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The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.
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Richardson, Kathryn. "Mechanisms of GPCR signal regulation in fission yeast". Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/63554/.
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Cells communicate with each other and respond to environmental cues by sending and receiving signals. Many external signals (ligands) are detected through G protein-coupled receptors (GPCRs), a major class of transmembrane proteins. GPCRs transduce these external signals into appropriate intracellular responses, enabling the cell to adapt to its environment. Malfunctions in these signalling pathways can lead to a range of human diseases and hence GPCRs have become attractive candidates for pharmacological design. The activation of a single receptor has the ability to induce numerous intracellular responses. Coupling this with the great number of different GPCR-types expressed in human cells means that understanding the basic principles of signal transduction and termination in humans is complicated. This study utilises the more simplistic eukaryotic yeast Schizosaccharomyces pombe (S. pombe) to overcome this complexity, as it contains only two GPCR types and hence the cross-talk between pathways is greatly reduced, whilst the structure and signalling functions of GPCRs are often evolutionarily conserved between yeast and humans. Mathematical modelling was used to aid the understanding of GPCR signalling in S. pombe and to inform experimental design. Speci�cally, an ordinary differential equation model �rst developed by Croft et al. (2013) was extended to include all known downstream signal transduction, regulation and termination events. This model is the �rst of its kind to describe a whole GPCR signalling pathway within S. pombe. Although it accurately predicts the cellular response to GPCR signalling it could only reproduce the biological plateau in temporal response with the addition of a 'yet unknown mechanism' GPCR degradation term. This motivated the investigation of how GPCRs in S. pombe are internalised from the plasma membrane in response to ligand stimulation. The primary mechanism for signal termination is via internalisation of the GPCR. This study identi�ed three potential casein kinases (Cki1, Cki2 and Cki3) that promote internalisation of the S. pombe GPCR Mam2. Microscopy analyses in combination with quantitative transcriptional, cell growth and cell cycle position assays uncovered a novel role for these kinases: that Cki2 regulates cell size during vegetative growth, Cki1 and Cki3 regulate the GPCR-response pathway and that Cki3 is essential for completing cytokinesis in S. pombe that have already undergone formation of a conjugation tube in response to ligand. Confocal microscopy of uorescent labelled Mam2 indicated a role for Cki2 in the internalisation and hence termination of the GPCR-response pathway. These findings add to the growing body of evidence that casein kinases are implicated in GPCR desensitisation.
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Ranganathan, Anirudh. "The impact of GPCR structures on understanding receptor function and ligand binding". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-129879.
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G protein-coupled receptors (GPCRs) form the largest superfamily of eukaryotic membrane proteins and are responsible for the action of nearly 30% of all marketed drugs. For a long period, efforts to study these receptors were limited by the paucity of atomic-resolution structural information. Numerous receptors spread across the GPCR superfamily have recently been crystallized, revealing crucial clues about receptor function and ligand recognition. The work in this thesis has primarily focused on using computational techniques to capitalize on this increasing amount of structural information. In papers I, II, and III protocols were developed to identify novel ligands for pharmaceutically important targets from in silico screens of large chemical libraries. In these papers, the fragment-based lead discovery (FBLD) approach was evaluated for GPCR targets using molecular docking screens. The high hit-rates obtained in these studies indicate promise for the use of computational approaches for fragment screening. In paper IV, molecular dynamics was used to identify a possible role for a conserved ionizable residue (Asp792.50) as a protonation switch during the activation process of the β2 adrenergic receptor. Analyses from this paper indicated that this residue could also perform a similar function in other class A GPCRs. Papers V and VI detail the modeling strategy followed during the GPCR Dock 2013 assessment to blindly predict the structure of two serotonin receptor subtypes (5-HT1B and 5-HT2B) bound to ergotamine. The developed ligand-steered homology modeling protocol was largely successful resulting in the best-ranked predictions for the 5-HT1B subtype. It is hoped that the work described in this thesis has highlighted the potential for structure-based computational approaches to identify novel ligands for important pharmaceutical targets and improve understanding of GPCR function.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
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Hillier, Stephen Gilbert. "Regulation of ovarian function". Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/26607.
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(i) Basic Experimental studies Publications 1-35 deal mainly with the use of cultured rat and marmoset monkey granulosa cells to study endocrine and paracrine mechanisms underlying gonadotrophin action on the ovaries. Primary cell cultures were used to define the roles of FSH and LH in controlling granulosa cell function and to assess the intrafollicular functions of sex steroids and putative nonsteroidal regulatory factors, such as inhibin. A particular contribution was the demonstration that androgens produced by thecal cells exert specific (receptor-mediated) modulation of granulosa cell differentiation - notably expression of aromatase, the enzyme uniquely responsible for oestrogen synthesis. Synthesis of inhibin and expression of messenger RNA species encoding inhibin and activin subunits in granulosa cells were also shown to be under gonadotrophic control and modulated by sex steroids, leading to the suggestion that the androgen/oestrogen and inhibin/activin axes of the ovarian paracrine system are functionally interlinked. (ii) Basic Clinical Studies Publications 36-56 are concerned with in vitro research on 'normal' ovarian tissues obtained from women undergoing elective surgical procedures. Techniques and experience acquired from experimental work on animal ovarian tissues were used to study the regulation of steroid hormone synthesis in human follicular and luteal cells. This work demonstrated that granulosa cells are primary cellular sites of oestradiol biosynthesis in the human ovary. It also confirmed the potential that theca-derived androgens have to modulate FSH-induced granulosa cell function, including aromatase activity and inhibin production. Conversely, androgen production by thecal cells was shown to be promoted by inhibin. Based on these findings it is postulated that an intrafollicular positive feedback loop exists mediated by theca-derived androgen and granulosa-derived inhibin, which may underpin preovulatory follicular 'selection' and oestrogen synthesis in the human menstrual cycle.
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Bittencourt, Fabiola M. "Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo". University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234044.
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Munjal, Akankshi. "Regulation of a bio-mechanical network driving shape changes during tissue morphogenesis". Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4038/document.
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Forces requises pour les changements de forme au cours de la morphogenèse des tissus sont générés par d’actine et de myosine. Durant ma thèse, je étudié le rôle de la réglementation MyoII par la voie Rho1-Rok durant l’élongation de l’ectoderme ventro-latéral par intercalation cellulaire. Les pulsations de MyoII médio-apicale se déplacent de manière anisotrope vers les jonctions parallèles avec l’axe dorso-ventral (ou jonctions verticales). Ceci provoque le rétrécissement graduel des jonctions qui sont stabilisées par une population de MyoII polarisée dans le plan du tissu et enrichie au niveau de ces jonctions. Les mécanismes cellulaires qui régulent la pulsatilité, la stabilité et la polarité de la myosine II restent à élucider. J’ai identifié deux propriétés cruciales de la dynamique de la myosine II régie par phospho- à savoir la cinétique d’échange gouvernée par les cycles de phosphorylation-déphosphorylation des chaines légères régulatrices de la MyoII (RLC) et l’advection due à la contraction des moteurs sur le réseau de F-actine. Contrôle spatial sur le chiffre d'affaires MyoII établit 2 régimes stables des taux élevés et faibles dissociation résultant dans MyoII polarité. Pulsatilité est un comportement auto-organisé qui émerge à taux de dissociation intermédiaires permettant d'advection MyoII et les régulateurs en amont. Dans la deuxième partie de ma thèse, je l'ai montré que la protéine GPCR- GRsmog et la brume, et la voie G-protéines en aval permettent l'activation progressive des MyoII, établissant pulsatilité et de la stabilité pour produire des déformations de forme polarisées cours de la morphogenèseForces required to power shape changes during tissue morphogenesis are generated by non-muscle MyosinII (MyoII) pulling filamentous actin. During my PhD, I investigated the role of MyoII regulation through the conserved Rho1-Rok pathway during Drosophila germband extension. The morphogenetic process is powered by cell intercalation involving shrinkage of junctions in the dorsal-ventral axis (‘vertical junctions’) followed by junction extension in the anterior-posterior axis. Advances in light microscopy revealed that the actomyosin networks exhibit pulsed contractions to power junction shrinkage, and alternate with steps of stabilization by MyoII enriched on vertical junctions (planar-polarity) to result in irreversible shape changes. Although described in many different contexts, the underlying mechanisms of this ratchet-like behavior remained unclear. Using genetic and biophysical tools, quantitative imaging and subtle perturbations, I identified 2 critical properties underlying MyoII dynamics- turnover governed by phospho-cycling of the MyoII Regulatory Light Chain, and advection due to contraction of the motors on actin networks. Spatial control over MyoII turnover establishes 2 stable regimes of high and low dissociation rates resulting in MyoII planar polarity. Pulsatility is a self-organized behavior that emerges at intermediate dissociation rates enabling advection of MyoII and upstream regulators. In the second part of my thesis, I showed that G protein coupled receptors- GRsmog and Mist, and the downstream G-protein pathway allow step-wise activation of MyoII, establishing pulsatility and stability, to drive polarized shape deformations during morphogenesis
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Clay, L. "CDC20 function, regulation and proteolysis". Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597750.
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The destruction of mitotic cyclins and other key regulators uses ubiquitin mediated proteolysis controlled via the activation of the ubiquitin ligase the Anaphase Promoting Complex/Cyclosome (APC/C), and its adaptor proteins Cdc20 and Cdh1. The spindle assembly checkpoint coordinates the APC/C with microtubule attachment and sets the timing from NEBD to anaphase. Cdc20 is inactivated by the spindle assembly checkpoint to prevent premature anaphase onset. Once the spindle assembly checkpoint is satisfied, Cdc20 can be released and activate the APC/C. However, cyclin A is degraded independently of the spindle assembly checkpoint before cyclin B1 and the anaphase inhibitor Securin. How Cdc20 can target different substrates for degradation at different times during mitosis is not yet clear. Using live-cell imaging and RNA interference and immunofluorescence techniques, I have studied the degradation of endogenous and ectopicly expressed APC/C substrates in cells with reduced Cdc20 levels. In this dissertation, I show that cyclin A degradation strongly depends on Cdc20, whereas cyclin B1 and Securin degradation do not. I identified the region of Cdc20 required for cyclin A binding and by mutating this site, I found that Cdc20 was no longer properly localised. I also show that Cdc20 proteolysis in human somatic cells does not require the KEN motif and gone on to find the motif required for Cdc20 degradation. I also show that the function of Cdc20 in the spindle assembly checkpoint can be influenced by the serine/threonine kinase Aurora A. Co-expression of a fluorescently tagged Cdc20 and Aurora A in human somatic cells causes an accelerated progression through mitosis and premature substrate degradation.
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Brandao, Haga Raquel. "Function and regulation of RhoBTB1". Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/function-and-regulation-of-rhobtb1(0904ff24-d566-4987-8c61-440c09854eeb).html.
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Rho GTPases are a family of proteins known to be involved in cytoskeletal regulation and are important for several processes including cell migration, cell polarity, vesicle trafficking and cytokinesis. RhoBTB1 is an atypical member of the Rho GTPase family. It consists of a non-functional GTP-binding domain followed by a proline-rich region and two tandem BTB domains. The only known interacting partner for RhoBTB1 is cullin3, a scaffold protein in ubiquitin ligase complexes. So far RhoBTB1 has not been shown to affect the cytoskeleton and it has no known cellular function. Most Rho GTPases are regulated by GEFs, GAPs, RhoGDIs and post-translational lipid modifications at the C-terminus. However, RhoBTB1 is not regulated by any of these mechanisms. RhoBTB1 has additional domains that could be involved in protein-protein interaction, leading to an alternative mechanism for RhoBTB1 regulation. This project has shown that RhoBTB1 can interact with RhoA and ROCK1 as well as cullin3. The interaction between RhoA and RhoBTB1 was explored since RhoBTB1 has the potential to recruit substrates for ubiquitination by cullin3 complexes. The region of interaction between RhoA and RhoBTB1 was mapped and RhoBTB1 influenced the protein level of RhoA, suggesting that it inhibits RhoA degradation by the proteasome. RhoBTB1 was found to localise diffusely in the cytoplasm or to punctate structures. Knockdown of RhoBTB1 led to a change in cell morphology in a 3D Matrigel matrix, indicating that it influences cell shape in 3D, although it did not alter cell shape on 2D substrata. RhoBTB1 can be phosphorylated by ROCK1 in vitro and the region of interaction between RhoBTB1 and ROCK1 was mapped using ROCK1 deletion mutants. I hypothesize that RhoBTB1 interacts with RhoA to affect its ubiquitination and degradation and hence affects cell morphology in a 3D matrix, and that RhoBTB1 activity is regulated by ROCK1-mediated phosphorylation.
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Berry, David (David A. ). "Glycosaminoglycan regulation of cell function". Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/34153.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.Includes bibliographical references (p. 252-285).
Glycosaminoglycans (GAGs) are complex polysaccharides that exist both on the cell surface and free within the extracellular matrix. The intrinsic sequence variety stemming from the large number of building blocks that compose this biopolymer leads to substantial information density as well as to the ability to regulate a wide variety of important biological processes. With the recent and progressive emergence of biochemical and analytical tools to probe GAG structure and function, efforts can be taken to understand the role of GAGs in cell biology and in disease in the various physiological locations where GAGs can exist. As a first step to probe the functions of GAGs, the heparin/heparan sulfate-GAG (HSGAG)-fibroblast growth factor (FGF) system was examined. Understanding the role of HSGAGs in inducing FGF2 dimerization led to the development of a novel engineered protein that was found to be effective at promoting functional recovery in stroke. Subsequently, methods to isolate HSGAGs from the cell surface were optimized and the ability of HSGAGs to support FGF signaling was investigated. Cell surface HSGAGs can define the responsiveness of a given cell to FGF1 and FGF2 through multiple receptor isoforms. Stromal cell derived HSGAGs were also identified as critical regulators of tumor cell growth and metastasis, effecting not only FGF2., but also 1-integrin signaling.
(cont.) Other GAGs, including dermatan sulfates, were characterized as modulators of FGFs and vascular endothelial growth factors. Finally, FGFs and HSGAGs were found to have important roles in maintaining epithelial monolayer integrity, with syndecan-l serving as a critical factor in inflammatory bowel disease. In addition to understanding HSGAGs in their normal physiological settings, techniques to internalize them were developed. Poly(3-amino ester)s were found to condense heparin and enable its endocytosis into cells. Internalized heparin is preferentially taken up by cancer cells, which often have a faster endocytic rate than non-transformed cells, and promotes apoptotic cell death. Internalized heparin can also be used as a tool to probe cell function. In Burkitt's lymphoma, poly(3-amino ester)-heparin conjugates served to identify cell surface HSGAGs as an important modulator of cell growth that can be harnessed to inhibit growth. Finally, studies that sought to broaden the scope of GAG biology were undertaken. Cell surface HSGA(:is were identified as mediators of vascular permeability. Furthermore a novel technique to immobilize GAGs was employed. The interactions between GAG and substrate were via hydrogen bonding. Immobilization of GAGs alters their properties, such that they can affect cells in ways distinct from GAGs free in the ECM.
(cont.) Furthermore, immobilized GAGs can regulate cancer cell adhesion, growth and progression, and may offer a new way to regulate the activity of cancer cells. In addition to directly providing new potential therapeutics and drug targets, these studies represent a foundation to enable additional studies of GAG function. Future work harnessing the techniques presented may open new avenues of research and facilitate the development of novel GAG-based therapeutics.
by David Berry.
Ph.D.
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Vespoli, Jessica L. "Genomic Regulation of Clock Function". Kent State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=kent1449500602.
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Tran, Stella Lê Minh. "Foxl2 regulation and function in gonadotropes". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116978.
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Pituitary follicle-stimulating hormone (FSH) is crucial for mammalian reproduction; it regulates gametogenesis and gonadal function. We know that impaired FSH production may leads to infertility, yet the mechanisms controlling FSH synthesis are poorly understood. Intra-pituitary activins stimulate FSHβ subunit (Fshb) gene transcription in gonadotrope cells, the rate-limiting step in FSH hormone synthesis. Studies conducted in immortalized gonadotrope cells indicate that activin A-stimulated Fshb gene transcription is dependent on homologs of Drosophila mothers against decapentaplegic (SMAD) proteins. More recently, the transcription factor forkhead box L2 (FOXL2) was reported to stimulate SMAD/activin A-induced Fshb transcription. Here, I focused on elucidating the mechanisms underlying Foxl2 regulation and function in gonadotrope cells. First, I demonstrated that overexpression of FOXL2 and SMAD 2, 3, and 4 confers activin-responsiveness to the murine Fshb promoter in heterologous cells. Upon activin A induction, FOXL2 synergizes with SMAD proteins to activate the Fshb promoter; this cooperation requires binding of both FOXL2 and SMAD3 (or SMAD4) to DNA. I showed that SMAD3-induction of the Fshb transcript is dependent on endogenous FOXL2 in homologous cells. I identified a proximal putative forkhead binding elements (FBE) and an adjacent SMAD binding element (SBE), that are crucial for FOXL2/SMAD3 induction of murine Fshb promoter-reporter activity. Based on previous data and my results, I propose a model where activins stimulate formation of FOXL2-SMAD2/3/4 complexes that drive murine Fshb transcription by binding to a conserved composite SBE/FBE element within the proximal promoter. FOXL2 plays a critical role in activin A-stimulated murine Fshb transcription in vitro. Next I tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, I generated a conditional knockout (cKO) mouse where Foxl2 is selectively ablated in the anterior pituitary gonadotrope cells. I observed that cKO mice are hypogonadal and sub-fertile in adulthood. I showed that cKO mice exhibit impaired spermatogenesis and folliculogenesis. Indeed, cKO males have decreased sperm counts, whereas cKO females ovulate fewer oocytes during natural estrous cycles. I demonstrated that both male and female cKO mice are FSH-deficient, secondary to diminished pituitary Fshb mRNA production. In Foxl2-depleted primary pituitary cultures, both basal and activin A-stimulated Fshb expression is impaired. These results indicate that gonadotrope-specific FOXL2 is required for selective induction of FSH production in vivo. Besides its role in gonadotrope cells, FOXL2 is also expressed in the pituitary thyrotropes, the perioptic mesenchyme of the developing eyelid and the ovarian granulosa cells. However the mechanisms governing this selective expression have not been described. In order to study Foxl2 transcription, I cloned the murine promoter region of Foxl2. Next, I demonstrated a correlation between promoter CpG methylation status and gene expression, whereby methylation may repress Foxl2 expression in some heterologous cell lines. Our results indicate that Foxl2 is not regulated by its promoter sequence alone and unveil a possible role for CpG methylation in mediating Foxl2 cell-specific gene expression in the mouse. In summary, my thesis work defined a necessary role for pituitary FOXL2 in FSH synthesis and reproduction in vivo. The FSH deficiency observed in our gonadotrope-specific cKO mice is in agreement with the mechanisms describing a role for endogenous FOXL2 in murine Fshb transcription. Collectively, my research on pituitary FOXL2 is part of our unrelenting efforts to investigate the many factors implicated in FSH regulation, and ultimately required for normal reproductive function. This, in turn, may help identify causes of idiopathic infertility or novel drug targets for infertility and contraceptive treatments.L'hormone folliculo-stimulante de l'hypophyse (FSH) est cruciale car elle régule gamétogenèse et fonction gonadique chez les mammifères. Une diminution dans la production de FSH peut mener à l'infertilité, mais les mécanismes contrôlant la synthèse de FSH demeurent incompris. Les activines de l'hypophyse stimulent la transcription du gène FSH sous-unité β (Fshb) dans les cellules gonadotropes; ceci est l'étape limitante dans la synthèse de l'hormone FSH. Les résults provenant de lignée gonadotrope immortalisée indiquent que la transcription de Fshb est stimulée par l'activine A; ceci est dépend des protéines homologues de Drosophila mothers against decapentaplegic (SMAD). Récemment, Forkhead box L2 (FOXL2) a été décrite comme étant un facteur-clé dans la stimulation de la transcription de Fshb induite par les SMADs et l'activine A. Ici, je dissèque les mécanismes expliquant la régulation et fonction de Foxl2 dans les cellules gonadotropes. Tout d'abord, la surexpression de FOXL2 et SMAD 2, 3, et 4 confère une réactivité à l'activine au promoteur du gène murin Fshb dans les cellules hétérologues. Sous stimulation par l'activine A, FOXL2 coopère de façon synergétique avec les SMADs pour activer le promoteur Fshb; cela nécessite que FOXL2 et SMAD3 (ou SMAD4) lient l'ADN. L'l'induction via SMAD3 du gène Fshb dépend de FOXL2 endogène dans les cellules homologues. Un élément proximal forkhead binding element (FBE) et un élément adjacent SMAD binding element (SBE) sont essentiels pour l'induction de l'activité du promoteur-reporter Fshb par FOXL2/SMAD3. Basé sur ces résultats, je propose un modèle où les activines stimulent la formation de complexes FOXL2-SMAD2/3/4 induisant la transcription du gène murinFshb via liaison à un élément SBE/FBE conservé du promoteur proximal. J'ai ensuite testé l'hypothèse que FOXL2 est nécessaire pour la synthèse de FSH in vivo en utilisant une approche Cre/lox: j'ai généré une souris knock-out conditionnelle (cKO) où Foxl2 est sélectivement absent dans les cellules gonadotropes. J'ai observé que les souris cKO sont hypogonadiques et souffrent d'une baisse de fertilité à l'âge adulte. J'ai démontré que la spermatogenèse et la folliculogénèse sont altérées chez les souris cKO. En effet, les cKO mâles ont un nombre diminué de spermatozoïdes, tandis que femelles cKO ovulent moins d'ovocytes pendant leur cycle oestral. J'ai démontré que cKO mâles et femelles sont déficientes en FSH, découlant d'une diminution d'ARN Fshb dans l'hypophyse. Les cultures primaires de cellules hypophysaires où Foxl2 est absent ne réagissent pas à l'activine A : Fshb est diminuée, tant au niveau basal que dans sa réponse au ligand. Ces résultats indiquent que l'expression de Foxl2 dans les gonadotropes est requise pour l'induction sélective de la production de FSH in vivo. Outre son rôle dans les gonadotropes, FOXL2 est également détecté dans les cellules thyrotropes de l'hypophyse, dans la paupière en développement et les cellules granuleuses de l'ovaire. Cependant les mécanismes qui régissent cette expression sélective n'ont pas encore été décrits. Afin d'étudier ce qui contrôle la transcription de Foxl2, j'ai cloné la région du promoteur murin de Foxl2. Ensuite, j'ai démontré une corrélation entre l'état de méthylation des CpGs du promoteur et l'expression des gènes, par lequel la méthylation exerce une inhibition sur le gène Foxl2 dans certaines cellules hétérologues. Mes résultats indiquent que Foxl2 n'est pas seulement contrôlé par la séquence de son promoteur et dévoilent un rôle possible pour la méthylation des CpGs dans la régulation de l'expression spécifique de Foxl2 chez la souris. En résumé, ma thèse définit un rôle nécessaire pour FOXL2 exprimé dans les gonadotropes pour la synthèse de Fshb et FSH, ainsi que la reproduction in vivo. Collectivement, mes recherches sur FOXL2 hypophysaire contribueront à identifier des causes de l'infertilité idiopathique ou encore trouver de nouvelles cibles pharmacologiques.
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Cheng, C. W. "Regulation of endothelial and endometrial function". Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597573.
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Angiogenesis is highly regulated during reproduction, as vessel growth, maturation and regression are observed in cyclic endometrium. This thesis investigates the possible regulation of endothelial and endometrial function, mainly focused on Wnt signalling, in angiogenesis and the female reproductive tract. The transcript profiles in a murine model of menstruation were also studied using two independent microarray experiments and platforms, which provide a broader view of molecular processes during menstruation. The major findings of this thesis include: 1) mRNAs encoding many Wnt signalling-related molecules are present in human umbilical vein endothelial cells and female reproductive tissues. 2) Wnt signalling has a role in endothelial cell growth control and this is mediated through cell-cell contact. 3) Wnt signalling is tightly regulated in endothelial cells and endometrium through the balance of positive and negative regulators of Wnt signalling pathway. There is also a tight balance between the canonical pathway and the non-canonical pathway. 4) Transcript levels of genes involved in many molecular processes changed during the time-course of a murine model of menstruation, suggesting that these molecule processes, including immune response, cell growth and maintenance, metabolism, transport and cell-cell interaction, are regulated during menstruation and participate in regulating endometrial function. These results provide further insights into the complexity of endometrial function and offer new therapeutic possibilities for the treatment of gynaecological disease.
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English, Jane Louise. "Cellular regulation of matrix metalloproteinase function". Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247107.
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Raghavan, Srikala. "Connectin function and regulation in Drosophila". Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624670.
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BERCLAZ, PIERRE-YVES. "REGULATION OF ALVEOLAR MACROPHAGE IMMUNE FUNCTION". University of Cincinnati / OhioLINK, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1022869185.
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Roberts, Kate. "Regulation of neutrophil function by NAMPT". Thesis, University of Liverpool, 2012. http://livrepository.liverpool.ac.uk/8355/.
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Neutrophils have capacity to cause tissue damage in chronic inflammatory disease. In rheumatoid arthritis (RA) they infiltrate joints, secrete proteases and reactive oxygen species (ROS), and express cytokines. RA neutrophils express NAMPT which is a highly conserved, pleiotropic protein, that catalyses the rate-limiting step of the NAD salvage pathway, but also has cytokine-like activity. NAMPT is elevated in inflammation and exerts pro-inflammatory effects on neutrophils. The aim of this research was to determine the role of NAMPT, as a signalling molecule and as an enzyme, in regulating neutrophil activities of pathological importance. Neutrophils were isolated from the blood of healthy donors and either stimulated with NAMPT or else NAMPT was inhibited (with FK866), in the presence and absence of pro-inflammatory cytokines in vitro. Assays for specific neutrophil functions such as production of ROS, bacterial killing and apoptosis were performed, and expression of cytokines was also examined. Transcriptome sequencing of neutrophils treated with FK866 and TNFα in combination, was carried out to investigate the wider impact of NAMPT inhibition on neutrophil gene expression. NAMPT expression was also examined in control, cytokine treated and RA patient neutrophils. NAMPT had little capacity to prime neutrophils, although it did delay neutrophil apoptosis and stabilise the anti-apoptotic protein Mcl-1. NAMPT inhibition in neutrophils, depleted NAD(P)/H and had effects on neutrophil functions and gene expression. Notably, FK866 decreased ROS production but did not affect the ability of neutrophils to kill bacteria. NAMPT-inhibited neutrophils exhibited decreased activation of signalling pathways and a diminished response to cytokines; transcriptome analysis showed that FK866 decreased TNFα-induced expression of many genes. NAMPT expression was not dynamically regulated in control neutrophils, but in RA patient neutrophils, NAMPT mRNA correlated with TNFα expression in a cohort of patients, and NAMPT protein was elevated in synovial fluid neutrophils compared to those from paired blood. Thus, NAMPT is elevated in activated inflammatory neutrophils and correlates with other markers of inflammation in RA, suggesting that it may be a marker of inflammation in these cells. Also, as a NAD biosynthetic enzyme, NAMPT regulates neutrophil functions and gene products central to RA disease pathology, without affecting bacterial killing capacity. This suggests that inhibition of NAMPT may potentially have the capacity to decrease neutrophil mediated tissue-damage observed in chronic inflammation, without adversely compromising host defence, and thus may be a future treatment option for diseases such as RA.
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Troupiotis-Tsaïlaki, Anastassia. "Lipid-GPCR interactions: from activation of sphingosine-1-phosphate receptors to modulation of vasopressin V2 receptor function". Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/216727.
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GPCRs form the largest family of membrane proteins in human genome and mediate signal transmission in a wide panel of essential physiological processes, and they are thus a major source of pharmaceutical targets. Investigating GPCR interactions with their cognate ligands and their membrane environment is crucial to understand their function at a molecular level. While major breakthroughs in the determination of high resolution structures of GPCRs in inactive and active states have shed a new light on the structural basis of GPCR activation process, complementary approaches are needed to investigate its dynamic aspects in the context of a native lipid environment. Our research work falls within this scope and hinges on two main issues: on the one hand, understand which structural features of the agonist underlie the activation of S1P receptors; on the other hand determine if membrane lipids modulate the structure and the function of the vasopressin V2 receptor (V2R). First, we investigated the functional response of S1P1, S1P2, S1P4 and S1P5 receptors expressed in mammalian cells to a series of synthetic derivatives of the native ligand sphingosine-1-phosphate, of variable alkyl chain length. Our data demonstrated that the hydrophobic tail of the ligand is crucial to induce activation in S1P receptors family, and revealed subtype-specificities regarding the influence of the alkyl chain length. Our experimental results combined with molecular dynamics simulation lead us to propose an activation mechanism for S1P receptors family. In the second part of our work, we reconstituted purified V2R into systems of controlled lipid composition, mimicking the membrane bilayer. Structural and functional characterization of the receptor in different lipid environments, using infrared and fluorescence spectroscopy approaches, revealed that the lipid composition affects V2R conformation and its interaction with a specific ligand. Taken together, our research work contributes to a better understanding of GPCRs activation mechanism and its regulation by lipid environment.Les récepteurs couplés aux protéines G (GPCRs) forment la plus grande famille de protéines membranaires du génome humain et contribuent à une kyrielle de processus physiologiques essentiels, qui leur confèrent un intérêt pharmacologique majeur. Étudier l'interaction de ces protéines avec leurs ligands et leur environnement membranaire est primordial pour appréhender leur fonctionnement à l’échelle moléculaire. Bien que de remarquables avancées dans la détermination de structures à haute résolution de GPCRs à l'état inactif et actif aient permis de comprendre certaines bases structurales du fonctionnement des récepteurs, des approches complémentaires donnant un aperçu des aspects dynamiques et dans un environnement natif sont nécessaires pour cerner pleinement leur mécanisme d'activation. Notre travail de thèse s'inscrit dans cette problématique et s'articule autour de deux sujets: d'une part, comprendre quelles caractéristiques structurales du ligand sous-tendent l'activation de la famille des récepteurs au sphingosine-1-phosphate (S1P); d'autre part, déterminer si les lipides de la membrane plasmique modulent la structure et la fonction du récepteur à la vasopressine V2. Pour répondre à notre première question, nous avons étudié la réponse fonctionnelle en système cellulaire des récepteurs S1P1, S1P2, S1P4 et S1P5 à des composés synthétiques dérivés du S1P, portant des chaînes alkyles de longueur variable. Nos données mettent en évidence que la longueur de la chaîne hydrocarbonée du ligand est un paramètre crucial dans sa capacité d'induire l'activation du récepteur et ce pour l'ensemble des sous-types étudiés. De plus, nos résultats suggèrent que le comportement vis-à-vis de la longueur de chaîne dépend du sous-type de récepteur considéré. Nos résultats expérimentaux, combinés à une approche de modélisation dynamique, ont abouti à proposer un mécanisme d'activation pour la famille des récepteurs au S1P. Dans le second volet de notre travail, nous avons reconstitué le récepteur V2 purifié dans des systèmes de composition lipidique contrôlée, mimant la bicouche membranaire. Nous avons procédé à la caractérisation structurale et fonctionnelle du récepteur inséré dans différentes types de lipides, par des méthodes spectroscopiques infrarouge et de fluorescence. Les données obtenues suggèrent que la composition lipidique affecte la conformation et la fonction du récepteur. L'ensemble de nos travaux contribue ainsi à une meilleure compréhension du mécanisme d'activation des GPCRs et de leur régulation par l'environnement lipidique.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Sheffler, Douglas James. "The Regulation of G Protein-Coupled Receptor (GPCR) Signal Transduction by p90 Ribosomal S6 Kinase 2 (RSK2)". Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1130777469.
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Mukherjee, Abir. "ROLE OF LYSOPHOSPHATIDIC ACID IN REGULATION OF CANCER CELL METABOLISM". VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/391.
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The simplest phospholipid, lysophosphatidic acid (LPA), is a heat stable component of serum known for its proliferative and migratory activities in cancer cells. Strong evidence suggests that LPA production and expression of its receptors are dysregulated in multiple human malignancies. The mechanism behind LPA-mediated tumor cell growth and oncogenesis remains poorly understood. In this thesis project I used ovarian and other cancer cells as a model system to examine the hypothesis that LPA present in the tumor microenvironment is a pathophysiological determinant of hyperactive de novo lipogenesis and aerobic glycolysis, two hallmarks of cancer cells. We demonstrated that LPA induced proteolytic activation of sterol regulatory element binding proteins (SREBPs) in a cancer specific manner, leading to activation of the SREBP-FAS (fatty acid synthase) lipogenic pathway. Treatment of cancer cell lines with LPA also led to dephosphorylation and inhibition of AMP-activated kinase (AMPK), thereby activating acetyl CoA carboxylase (ACC). Moreover, these effects of LPA were mediated by LPA2, a receptor subtype overexpressed in multiple cancers, providing an explanation for the cancer specific regulation of FAS and ACC by LPA. Downstream of the LPA2 receptor, we identified the Gα12-Rho-Rock pathway to activate SREBPs and the Gαq-PLC (phospholipase C) pathway to inactivate AMPK. Consistent with LPA mediated activation of the key lipogenic enzymes FAS and ACC, LPA stimulated de novo lipid synthesis via LPA2, leading to accumulation of intracellular triacylglycerol and phospholipids. Pharmacological and molecular inhibition of LPA2, FAS or ACC attenuated LPA-dependent cell proliferation, indicating that upregulation of lipid synthesis is an integral component of the proliferative response to LPA. In further support of this, downregulation of LPA2 expression led to dramatic inhibition of anchorage-dependent and –independent growth of ovarian cancer cells. To support increased biomass generation, rapidly proliferating cancer cells enhance carbon influx by activating glycolysis. In the next part of the study, we investigated if LPA signaling was also involved in activating aerobic glycolysis in cancer cells. LPA indeed activated glycolysis in ovarian and other cancer cells but failed to elicit this response in non-transformed cells, suggesting a cancer specific role of LPA in regulation of glucose metabolism. While LPA had no effect on glucose uptake, we found that LPA altered expression of multiple genes involved in glucose metabolism. The most significant observation was that LPA treatment dramatically upregulated expression of HK-2, one of the rate-limiting glycolytic enzymes. We explored the underlying mechanism and found that LPA activates HK-2 transcription through LPA2-mediated activation of SREBP-1. Two sterol regulator elements (SREs) on the human HK-2 promoter were identified to be responsible for LPA activation of the promoter. DNA pulldown and chromatin immunoprecipitation assays confirmed that SREBP-1 bound to these SREs in LPA-treated cells. Although in ovarian cancer cells, LPA treatment also stabilized Hif-1α protein, an established activator of HK-2 and glycolysis, LPA-regulated HK-2 expression and glycolysis was largely independent of Hif-1α. These results established that LPA stimulates glycolysis via the LPA2-SREBP-HK-2 cascade in neoplastic cells. Taken together, this dissertation provides the first evidence for regulation of cancer cell metabolism by LPA. The results indicate that LPA signaling is causally linked to lipogenic and glycolytic phenotypes of cancer cells. Therefore, targeting the key LPA2 receptor could offer a novel and innovative approach to blocking tumor-specific metabolism.
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Maurel, Marion. "Les microARNs régulateurs de l’expression génique du Glypican-3 dans le Carcinome Hépatocellulaire". Thesis, Bordeaux 2, 2012. http://www.theses.fr/2012BOR21945/document.
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Le Glypican-3 (GPC3) est surexprimé dans 72% des carcinomes hépatocellulaire (CHC). C’est un co-récepteur membranaire du récepteur WNT, qui appartient à la famille des protéoglycanes à sulfates d'héparane. L'objectif général de ma thèse vise à étudier les mécanismes de régulation post-transcriptionnelle de l’expression du GPC3 dans le CHC. Pour cela, j’ai développé un test fonctionnel qui m’a permis de cribler une bibliothèque de 876 microARNs humains. Ceci a conduit à l’identification de 5 microARNs régulateurs de l’expression de l’ARNm codant pour le GPC3 via sa région 3’ non traduite (NT). Mon travail de thèse porte plus particulièrement sur le miR-1271 et le miR-1291 car ils sont dérégulés dans le CHC et sont respectivement inhibiteur et inducteur de l’expression du GPC3. Dans un premier projet, j’ai démontré que le miR-1271 cible directement la région 3’NT du GPC3 et diminue la stabilité de son ARNm. Ce microARN est sous-exprimé dans le CHC et son expression corrèle négativement avec celle de l'ARNm du GPC3 dans les CHC associés à une infection par le virus de l’hépatite B. Dans un deuxième projet, j’ai démontré que le miR-1291 régule positivement l’expression du GPC3 en inhibant un facteur intermédiaire. Une analyse in silico a permis d’identifier IRE1α comme candidat. IRE1α est une protéine transmembranaire du réticulum endoplasmique (RE) qui participe à « l’Unfolded Protein Response », une réponse adaptative activée lors de l’accumulation de protéines mal conformées dans le RE. J’ai démontré qu’IRE1α clive l’ARNm codant pour le GPC3 grâce à son activité endoribonucléase. D’autre part, le miR-1291 cible directement l’ARNm codant pour IRE1α dans sa région 5’NT ce qui inhibe son expression et induit une surexpression du GPC3. Le miR-1291 est surexprimé dans le CHC et son expression corrèle positivement avec celle de l’ARNm du GPC3. En conclusion, mon travail de thèse m’a permis de mettre en évidence et de caractériser deux nouveaux microARNs (miR-1271 et miR-1291) contrôlant l’expression du GPC3 par des mécanismes directs ou indirects. La pertinence physiopathologique de ces régulations dans le CHC est en accord avec les niveaux d’expression respectifs de ces microARNs, qui pourraient contribuer à la surexpression du GPC3 dans ces tumeursGlypican-3 (GPC3) is overexpressed in 72% of hepatocellular carcinoma (HCC). It is a co-receptor for WNT receptor and belongs to the heparan sulfate proteoglycans family. The general objective of my PhD thesis was to study the mechanisms by which GPC3 is post-transcriptionnally regulated in HCC. To this end, I developed a functional test that allowed me to screen a library of 876 human microRNAs. This led me to identify 5 microRNAs that regulate the expression of GPC3 mRNA through its 3’Untranslated Region (UTR). The work presented in this thesis particulary focuses on miR-1271 and miR-1291 as both microRNAs present a deregulated expression in HCC and are respectively inhibitor and activator of GPC3 mRNA expression. In a first project, I demonstrated that miR-1271 directly binds to GPC3 mRNA 3’UTR and affects its stability. This microRNA is underexpressed in HCC and its expression negatively correlates with that of GPC3 mRNA in a subgroup of HCC corresponding to those associated with hepatitis B virus infection. In a second project, I demonstrated that miR-1291 postively regulates the expression of GPC3 mRNA by targeting an intermediate factor. An in silico analysis led to the identification of the Inositol Requiring Enzyme 1 alpha (IRE1α) as a potential candidate. IRE1α is an endoplasmic reticulum (ER) resident type I transmembrane protein and contributes to the signaling of the Unfolded Protein Response (UPR). The UPR is an adaptive response activated upon accumulation of improperly folded proteins in the ER. I showed that IRE1α cleaves GPC3 mRNA through its endoribonuclease activity. Moreover I demonstrated that miR-1291 directly targets IRE1α mRNA through its 5’UTR, thereby decreasing its expression and contributing to GPC3 mRNA overexpression. MiR-1291 is overexpressed in HCC and its expression positively correlates with that of GPC3 mRNA. In summary, the work carried out during my PhD allowed the identification and the characterization of two new microRNAs (miR-1271 and miR-1291) that control the expression of GPC3 mRNA through direct or indirect mechanisms. The pathophysiological relevance of these regulatory mechanisms is in agreement with the respective expression levels of these microRNAs in HCC, which could therefore contribute to the overexpression of GPC3 in those tumors
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Roux, Benoit Thomas. "Characterisation of the molecular mechanisms regulating the signalling and post-endocytic sorting of the receptors for calcitonin gene-related peptide and adrenomedullin". Thesis, University of Bath, 2013. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604647.
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Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) receptors are heterodimeric complexes composed of the calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP). Association with RAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 or RAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributed throughout the body and play important roles and are implicated in many diseases including migraine, heart failure and sepsis. Recently, CGRP has been shown to promote nitric oxide (NO) production and inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells via ERK activation. CGRP is known to induce iNOS/NO production in thoracic artery smooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, the molecular mechanism of CGRP-induced iNOS/NO production in TA-SMC is unknown. Therefore, in order to determine if CGRP induces iNOS/NO production via ERK activation, I first investigated the exact mechanisms through which CGRP activates ERK1-2 in HEK cells. By using different inhibitors I showed that CGRPinduced ERK activation is mainly activated through two major pathways. I showed for the first time that CGRP induces ERK activation through transactivation of ErbB1 and as expected through the cAMP/PKA pathway. Then, in order to characterise a suitable model to study CGRP-induced iNOS expression, I used primary TA-SMC and I showed that CGRP induces iNOS upregulation, which is reduced when cells are incubated with U0126, a MEK inhibitor. Thus, these results suggest that CGRP induces iNOS expression via ERK activation in TA-SMC. However, further experimentation is required to determine the exact ERK pathway responsible for iNOS induction. Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressing CLR•RAMP2, I investigated the molecular mechanisms regulating the ADM receptor. I first showed that, unlike CLR•RAMP1, even transient stimulation of CLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicating that this ADM receptor does not recycle to the cell-surface. Moreover, I showed that CLR, not RAMP2, is constitutively ubiquitinated, which was further enhanced upon ADM stimulation. In order to elucidate the role of ADM-mediated ubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. I showed that ubiquitination of CLR did not affect ADM-induced trafficking of CLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signalling of CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rate of degradation of the receptor. Together, these results indicate that CLR•RAMP2 does not recycle and is degraded via a molecular mechanism that is accelerated by ADM-induced ubiquitination of CLR.
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Runne, Caitlin M. "Function and Activation Mechanism of PLEKHG2, A Novel G Beta Gamma-Activated RhoGEF in Leukemia Cells". Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/4907.
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The Rho family of GTPases plays a crucial role in the regulation of diverse cellular processes, including proliferation and actin cytoskeletal rearrangement to promote cell migration. However, dysregulation of RhoGTPases has been associated with disease, particularly cancers such as leukemia. Despite this, RhoGTPases are rarely mutated in cancer. Rather, dysregulation of their regulatory proteins through mutation or overexpression contributes to disease pathogenesis. RhoGTPases are activated through Rho guanine nucleotide exchange factors (GEFs). Although over eighty RhoGEFs have been identified that activate the 25 RhoGTPases, the pathological role of the majority of these proteins remains unclear. Further, whereas the majority of RhoGEFs are activated through tyrosine phosphorylation, a small subset can be activated through heterotrimeric G proteins, including through GΒ;Γ; subunits. However, the mechanism by which GΒ;Γ; induces RhoGEF activation remains unclear.
PLEKHG2 is a Dbl family RhoGEF that was originally identified as a gene upregulated in a leukemia mouse model, and later shown to be activated by heterotrimeric G protein Β;Γ; subunits. However, its function and activation mechanisms remain elusive. Here we show that, as compared to primary human T cells, the expression of PLEKHG2 is upregulated in leukemia cell lines. Downregulation of PLEKHG2 by siRNAs specifically inhibited GΒ;Γ;-stimulated Rac and Cdc42, but not RhoA activation. Consequently, inhibition of PLEKHG2 blocked actin polymerization, protrusion formation, and leukemia cell migration in response to SDF1alpha;. Additional studies indicate that GΒ;Γ; likely activates PLEKHG2 by binding the N-terminus of PLEKHG2. This interaction results in the release of autoinhibition imposed by the C-terminus within a region encompassing the catalytic DH domain. As a result, overexpressing either the N-terminus of PLEKHG2 that binds GΒ;Γ; or the C-terminus that autoinhibits PLEKHG2 blocked GΒ;Γ;-stimulated Rac and Cdc42 activation and the ability of leukemia cell to form membrane protrusions and to migrate. Together, our results have demonstrated that PLEKHG2 functions as a novel GΒ;Γ; -stimulated RhoGEF that could contribute to chemokine-induced leukemia cell dissemination and leukemia pathogenesis.
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Jelic, Marko. "The function of Toc34 and its regulation". Diss., lmu, 2003. http://nbn-resolving.de/urn:nbn:de:bvb:19-14213.
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Li, Cathy Shije 1974. "Function and regulation of histone deacetylase 4". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98750.
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Histone acetyltransferases and histone deacetylases (HDACs) maintain dynamic acetylation and deacetylation of histories and other proteins in vivo, and are actively involved in the control of gene transcription and other nuclear processes. One mechanism by which functions of these enzymes are regulated operates through differential intracellular compartmentalization. HDAC4, -5, -7 and -9, the four members of class IIa, shuttle between the nucleus and the cytoplasm in a manner dependent on specific phosphorylation stimulated by several known kinases, and these deacetylases possess intrinsic nuclear import and export signals for dynamic nucleocytoplasmic trafficking. The ability to change their intracellular localization implies that class IIa HDACs have some potential functions in different subcellular compartments. To gain additional insights into this, I first focused on studying the function and regulation of HDAC4. As a result, I identified protein kinase D3 as a novel kinase for HDAC4 and found that this kinase physically interacts with HDAC4 and stimulates its nuclear export. Then I tried to purify protein complexes of RFXANK and ANKRA2, two homologous ankyrin-repeat proteins that are known to associate with HDCA4, using the tandem affinity purification (TAP) strategy. The results that I have obtained reveal a novel mechanism for regulating the nuclear export of HDAC4 and suggest that its cytoplasmic localization may also be indicative of potential cytoplasmic functions rather than just for simple sequestration from its nuclear targets.
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Jelić, Marko. "The function of Toc34 and its regulation". [S.l.] : [s.n.], 2003. http://edoc.ub.uni-muenchen.de/archive/00001421.
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Wu, Qun Garris Paul A. "Function and regulation of the dopamine transporter". Normal, Ill. Illinois State University, 1999. http://wwwlib.umi.com/cr/ilstu/fullcit?p9960430.
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Thesis (Ph. D.)--Illinois State University, 1999.Title from title page screen, viewed July 31, 2006. Dissertation Committee: Paul A. Garris (chair), Maarten E.A. Reith, Anthony J. Otsuka, Robert L. Preston, David L. Williams. Includes bibliographical references (leaves 221-242) and abstract. Also available in print.
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Wang, Chunbo. "Structure, function and regulation of TRP channels". Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1148676549.
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Pyszniak, Andrew M. "Regulation of LFA-1 (CD11a/CD18) function". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25139.pdf.
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Lindebro, Maria. "Mechanisms of regulation of dioxin receptor function /". Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-231-0/.
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Bahram, Fuad. "Post-translational regulation of Myc oncoprotein function /". Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/a467.pdf.
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Lundgren, Josefin. "Studies of metazoan proteasome function and regulation". Doctoral thesis, Stockholm : Department of molecular biology and functional genomics, Stockholm university, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-496.
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Burnett, Amanda. "Regulation of Neutrophil Function by the Angiopoietins". Thesis, University of Sheffield, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522497.
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Savage, Joshua S. "Protein kinase-dependent regulation of platelet function". Thesis, University of Bristol, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559232.
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Platelets are essential in the initiation, growth and stabilisation of thrombi in normal haemostasis upon injury to the vasculature, or in cardiovascular disease upon rupture of an atherosclerotic plaque. Protein kinases are a crucial component in platelet signalling pathways, playing a combination of positive and negative regulatory roles. The AGC kinase family contains several known key regulatory kinases in platelet signalling, most notably PKC. PKC is largely considered a positive regulator of platelet function, although recent data suggests that some PKC isoforms play minor negative regulatory roles. Secretion of pro-aggregatory and pro-inflammatory mediators from platelet granules, a process previously shown to be PKC-dependent, is necessary for propagation of activation and for thrombus growth and stability. For this reason, in this study a clinically relevant PKCP inhibitor ruboxistaurin, used in the treatment of diabetic retinopathy, has been demonstrated to be an effective anti-platelet compound acting primarily through the inhibition of secretion. Ruboxistaurin ablated platelet aggregation which could be restored by exogenous ADP, unlike secretion which remained strongly inhibited. Additionally, thrombus formation under flow was significantly reduced in the presence of ruboxistaurin. This promising data suggests there may be potential clinical uses of ruboxistaurin as an anti-thrombotic agent. The mechanism by which PKC regulates granule secretion is yet to be elucidated. Unc13d'inx mice lack Munc13-4, a protein responsible for coordinating components of the secretory complex, possibly directly regulated by or downstream of PKC. Munc13-4 was shown to be absolutely essential for dense granule secretion, and knockdown of the gene caused a reduction in aggregation and thrombus formation, both of which were rescued by exogenous ADP. However, this study also reveals the existence of a Munc13-4 independent pathway, and a novel positive feedback mechanism for u granule release via ADP and P2Y12. The role of another AGC kinase, PKN, had not previously been studied in platelets. It was , hypothesised that, like PKC, PKNl and PKN3, the isoforms identified in platelets, would act as regulatory proteins via secretion and cytoskeletal reorganisation. Using PKNl/3-I- mice, PKN was revealed to be a negative regulator of aggregation, integrin UlIbP3 activation and spreading. The work presented here demonstrates the varied positive and negative roles that protein kinases play in platelet function, including, but by no means exclusively, in secretion. It also reveals a novel ADP-dependent pathway in platelet secretion and demonstrates that a clinically tested PKC inhibitor has potential as a novel anti-thrombotic therapy.
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Witzel, Ini-Isabee. "Investigating the function and regulation of SNIP1". Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529875.
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Janas, Maja. "Novel Regulation of MicroRNA Biogenesis and Function". Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10121.
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MicroRNAs are small noncoding RNAs that post-transcriptionally reduce protein output from most human mRNAs by mechanisms that are still obscure. This thesis provides insights into three aspects of microRNA biogenesis and function described below. MicroRNA precursors are excised from primary transcripts by the Microprocessor complex containing Drosha and DGCR8. Although most microRNAs are located in introns of protein-coding and noncoding genes, the mechanisms coordinating microprocessing and splicing are unclear. MiR-211 is a microRNA expressed from intron 6 of melastatin, a suspected melanoma tumor suppressor. We demonstrate that miR-211, and not melastatin, is responsible for the tumor suppressive function of this locus, that Drosha-mediated processing of the miR-211 precursor promotes splicing of melastatin exon 6-exon 7 junctions, and that perturbing 5' splice site recognition by the U1 snRNP reduces Drosha recruitment to intron 6 specifically and intronic microRNA levels globally. Thus we identify a novel physical and functional coupling between microprocessing and splicing. Typically, Agos stabilize mature microRNAs and as a complex stoichiometrically bind to complementary mRNAs. We demonstrate an alternative order of events in which Agos bind and repress pre-formed imperfect microRNA-mRNA duplexes in processing bodies of live cells, and cleave pre-formed perfect microRNA-mRNA duplexes in vitro. Our data support a novel catalytic model whereby Agos first deposit microRNAs onto mRNAs and dissociate, thus priming multiple microRNA-mRNA duplexes for concurrent repression by a single Ago. Despite key roles in development and pathogenesis, effectors and regulators of microRNA-mediated repression are still poorly characterized. An RNAi screen revealed that depletion of ribosomal proteins of either small or large ribosomal subunit dissociates microRNA-containing complexes from mRNAs repressed at translation initiation, increasing their polysome association, translation, and stability relative to untargeted mRNAs. Thus ribosomal proteins globally regulate microRNA function. Another RNAi screen revealed that Akt3 phosphorylates Ago2, which negatively regulates cleavage and positively regulates translational repression of microRNA-targeted mRNAs. Thus Ago2 phosphorylation is a molecular switch between its mRNA cleavage and translational repression activities. The following pages will place these novel insights into biological and disease-relevant context, will describe what was known prior to these studies, and will provide perspectives for future studies.
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Cadman, Chris. "Regulation of the helicase function of PriA". Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428956.
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Clemett, Delyth A. "5-HTâ†7 receptor regulation and function". Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262781.
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McMullan, Rachel Jane. "Regulation of keratinocyte function by Rho kinase". Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275126.
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Higham, Andrew Damian. "Neuroendocrine regulation of gastric endocrine cell function". Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266054.
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Finkelstein, Erik I. "Regulation of neutrophil function by toxic aldehydes /". For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Hillwig, Melissa S. "Regulation, function, and evolution of T2 RNases". [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3389103.
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Meng, Meng. "Plant UDP-glucose Pyrophosphorylase : Function and Regulation". Doctoral thesis, Umeå : Department of Plant Physiology, Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1796.
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Glanz, Anna Nicole. "Regulation of the Antiviral Function of IRF3". University of Toledo Health Science Campus / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=mco1596792904962609.
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Lindås, Ann-Christin. "Tripeptidyl-Peptidase II : Structure, Function and Gene Regulation". Doctoral thesis, Uppsala University, Department of Biochemistry, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7345.
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The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.
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Holmqvist, Marie. "The Cyanobacterial Uptake Hydrogenase : Regulation, Maturation and Function". Doctoral thesis, Uppsala universitet, Mikrobiell Kemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-129223.
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With accellerating global warming and pollution problems a change of energy regime is necessary. Solar energy offers a clean and unlimited energy source of enormous potential. Due to it’s intermittenet nature solar energy must be stored - ideally in the chemical bond of a carrier molecule. Hydrogen gas, H2, an energy carrier with water as only emission when used in a fuel cell, is considered to be the choise for the future. In this context cyanobacteria show promising potential as future H2 factories since they can produce H2 from solar energy and water. The main enzymes directly involved in cyanobacterial hydrogen metabolism are nitrogenases and hydrogenases. Cyanobacterial hydrogenases are either uptake hydrogenases or bidirectional hydrogenases and their maturation requires assistance of six maturation proteins and two hydrogenase specific proteases. In this thesis the transcriptional regulation, maturation and function of the cyanobacterial uptake hydrogenases were investigated in the filamentous, heterocyst forming strains Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120. Five genes, encoding proteins putatively involved in the maturation of the uptake hydrogenase were identified upstream the known maturation genes. Two transcription factors, CalA and CalB, were found interacting with the stretch of DNA forming the upstream regions of the uptake hydrogenase structural genes and the novel maturation genes. The expression of the uptake hydrogenase were heterocysts specific and the specificity mapped to a short promoter region starting -57 bp upstream the transcription start point. In addition, the function of the uptake hydrogenase was inserted in a metabolic context. Among the proteases, a conserved region was discovered possibly involved in determining the hydrogenase specificity. This thesis has given valuable information about the transcriptional regulation, maturation and function of the uptake hydrogenase in filamentous, heterocystous cyanobacteria and identified new targets for bioengineering of mutant strains with higher H2 production rates.
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Eriksson, Anna S. "Syndecan - Regulation and Function of its Glycosaminoglycan Chains". Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-197691.
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The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility. Regulation of intracellular signaling is crucial to prevent abnormal cellular behavior. In the second part of this thesis, the aim was to see how the presentation of glycosaminoglycan chains to the FGF signaling complex could affect the cellular response. When attached to the plasma membrane via syndecan-1, CS chains could support the intracellular signaling, although not promoting as strong signals as HS. When glycosaminoglycans were attached to free ectodomains of syndecan-1, both types of chains sequestered FGF2 from the receptors to the same extent, pointing towards functional overlap between CS and HS. To further study the interplay between HS and CS, their roles in the formation of pharyngeal cartilage in zebrafish were established. HS was important during chondrocyte intercalation and CS in the formation of the surrounding extracellular matrix. Further, the balance between the biosynthetic enzymes determined the ratio of HS and CS, and HS biosynthesis was prioritized over CS biosynthesis. The results presented in this thesis provide further insight into the regulation of HS biosynthesis, as well as the roles of both HS and CS on the cell surface. It is evident, that in certain situations there is a strict requirement for a certain HS structure, albeit in other situations there is a functional overlap between HS and CS.
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Afar, Ronith. "Regulation and function of neuronal nicotinic acetylcholine receptors". Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41288.
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Two major subtypes of nicotinic acetylcholine receptors (nAChRs) have been identified in the nervous system. One site is bound by $ alpha$-bungarotoxin ($ alpha$-BGT), while the other has a higher affinity for agonists and does not bind the $ alpha$-toxin. Muscle nAChRs, which also bind $ alpha$-BGT, had been reported to be sensitive to a thymus-derived polypeptide, thymopoietin (TPO). The present work was therefore done to determine whether this agent might also interact with the different nAChR populations in neuronal cells.Initial studies involved thymopentin (TP-5), a 5 amino-acid peptide which may represent the active site of TPO. TP-5 inhibited nicotinic receptor-induced release of catecholamines in bovine adrenal medullary cells in culture, a function mediated through the $ alpha$-BGT-insensitive nAChR. On the other hand, TP-5 did not inhibit either ($ sp3$H) (-)nicotine or ($ sp{125}$I) $ alpha$-BGT binding to rat brain membranes. These results suggested that TP-5 interacted in a non-competitive manner with the $ alpha$-BGT-insensitive neuronal nAChR.
Studies were subsequently done with thymic preparations presumed to be purified TPO ('TPO'), the native polypeptide containing the TP-5 amino acid sequence. In contrast to the effect of TP-5, 'TPO' preparations did not alter nicotinic receptor mediated catecholamine release from neuronal cells in culture. However, 'TPO' preparations selectively decreased ($ sp{125}$I) $ alpha$-BGT binding to brain membranes suggesting an interaction between this polypeptide and $ alpha$-BGT receptors. Quantitative autoradiography revealed that 'TPO' inhibited specific ($ sp{125}$I) $ alpha$-BGT binding uniformly and with similar potency in different brain regions. As $ alpha$-BGT binding sites are highly expressed in the hippocampal formation, primary cultures of fetal rat hippocampal cells were used next to investigate regulation of the $ alpha$-BGT receptor by 'TPO'. 'TPO' caused a dose-dependent and slowly reversible decrease in the density of $ alpha$-BGT receptors. After completion of this work with 'TPO', studies by Quik and coworkers (1993) showed that $ alpha$-Naja toxin (or $ alpha$-cobratoxin) from Naja naja siamensis snake venom was present in the 'TPO' preparations; furthermore, this toxin component appeared to be responsible for the reported effects of 'TPO' on $ alpha$-BGT receptors. Therefore, the above results which had initially been interpreted to occur as a consequence of the interaction of the thymic polypeptide TPO at the nicotinic $ alpha$-BGT site, must now be attributed to the presence of $ alpha$-Naja toxin contaminant in the 'TPO' preparations.
Studies were also undertaken to identify a nicotinic function for $ alpha$-BGT receptors in neuronal cells. Intracellular calcium levels were measured in response to nicotine as recent work using parasympathetic neurons showed that this may represent an $ alpha$-BGT-sensitive response. In contrast to earlier findings, the present work indicates that nAChR-mediated calcium fluxes in cultured chromaffin cells do not reveal an $ alpha$-BGT-sensitive component. These results thus suggest that nicotinic $ alpha$-BGT receptors mediate their response by altering intracellular calcium levels in some but not all neuronal preparations.
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Unterberger, Alexander. "The role of DNMT1 regulation in cellular function". Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92154.
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Disruption of the epigenome and its components is a hallmark of all forms of cancer. Typically observed in cancer is an alteration of the DNA methylation pattern, with silencing of tumour suppressor genes, as well as an increase in DNA methyltransferase 1 (activity or expression). However it has yet to be determined exactly how DNMT1 increases in cancer and how this increase might serve as therapeutic target. This thesis focuses on the regulation of DNMT1 in the cell cycle and the consequences of depleting DNMT1 in cancer cells.During the cell cycle DNMT1 levels increase as the cell enters into S-phase. It has previously been shown that this cyclical regulation of DNMT1 occurs by destabilization of DNMT1 mRNA in G0/G1 through the action of a protein, identified to be the mRNA binding protein AUF1. AUF1 binds a regulator element located in the 3'UTR of DNMT1 mRNA and recruits the exosome, the RNA degradation complex, to degrade it.
When AUF1 is depleted in these cells, DNMT1 mRNA is stabilized which leads to increased DNMT1 protein levels, methyltransferase activity and genomic methylation. The changes of DNMT1 mRNA levels in the cell cycle were determined to occur as an inverse function of AUF1 protein levels. AUF1 levels were observed to decrease in S-phase which lead to increased stability in DNMT1 mRNA. This cell cycle regulation of AUF1 was determined to occur as a function of Rb. Rb actively stabilizes AUF1 protein. Indeed, upon elimination of Rb, AUF1 is degraded through the function of Hsp70 and the proteasome. This consequently leads to an elevation in DNMT1 protein levels which in turn increases genomic methylation levels. Elevated DNMT1 levels resulted in greater association with EZH2, which in turn leads to increased methylation of EZH2 targeted promoters, including p16 and CNR1. This promoter hypermethylation occurred as a function of DNMT1 and EZH2.These observations indicate that regulation of DNMT1 is tied into the cell cycle function of Rb and upon disruption of this system, a characteristic of cancer, site-specific methylation occurs at tumour suppressors, another characteristic of cancer.
Furthermore, we examined the effect of depleting DNMT1 in cancer cells. Upon depletion of DNMT1, a signaling pathway known as the replication arrest/DNA damage checkpoint was induced. Activation of this pathway results in arrest of cell growth and cell cycle blockage and occurred independently of the catalytic activity of DNMT1 and instead responded to the absence of DNMT1. This supports a role for DMNT1 as a negative regulator of the replication arrest/DNA damage checkpoint through the action of interaction with an unknown protein. Moreover, suppression of the replication arrest/DNA damage checkpoint has been determined to be a necessary step in the proliferation of cancer cells. Taken together, the data from this thesis determined that common events in cancer, such as inactivation of Rb, lead to deregulation of DNMT1 mRNA, through AUF1, leading to site-specific methylation of tumour suppressors and could potentially serve to block growth arresting checkpoints like the replication arrest/DNA damage checkpoint. The novel functions of DNMT1, such as cell cycle regulation, site-specific methylation and role in the replication arrest/DNA damage checkpoint discovered in this thesis could serve to help better understand how cancer develops. The results of this thesis could serve to develop novel strategies to target these events and better treat cancer.
L'altération de l'épigénome et de ses composants est une marque caractéristique de tous types de cancer. Une altération des profils de méthylation de l'ADN, associée à une inactivation de gènes suppresseurs de tumeurs ainsi qu'une augmentation de l'(activité/expression) de la méthyltransférase de l'ADN (DNMT1) sont largement observés dans les cancers. Cependant, les causes de cette augmentation de DNMT1 (expression/activité) dans le cancer et l'utilisation potentielle de cette augmentation comme cible thérapeutique n'ont pas encore été déterminées.
Au cours du cycle cellulaire, le niveau de DNMT1 augmente dès lors que la cellule entre en phase S. Il a été montré précédemment qu'une régulation cyclique de DNMT1 se met en place grâce à une déstabilisation de son ARN messager en phase G0/G1 sous l'action d'une protéine non identifiée. Cette protéine a été identifié comme AUF1. AUF1 interagit avec un élément régulateur situé dans la partie 3'-UTR de l'ARNm de DNMT1 et entraîne la dégradation de cet ARNm en recrutant l'exosome, un complexe de dégradation de l'ARN. La déplétion d'AUF1 stabilise l'ARNm de DNMT1 ce qui conduit à une augmentation de l'expression de cette protéine, de son activité méthyltransférase ainsi que de la méthylation du génome. Il a été également montré que le niveau d'expression de l'ARNm de DNMT1 au cours du cycle cellulaire est inversement corrélé à celui de la protéine AUF1. Ce niveau d'AUF1 est diminué en phase S ce qui traduit par une stabilité accrue de l'ARNm de DNMT1. Il a été montré que cette régulation d'AUF1 au cours du cycle cellulaire est fonction de la protéine Rb. Rb stabilise activement la protéine AUF1. En effet, AUF1 est dégradée par l'intermédiaire de la protéine Hsp70 et du protéasome. Cette dégradation a pour conséquence une augmentation du niveau d'expression de DNMT1 lequel conduit à une augmentation du niveau de méthylation du génome. De plus, cette augmentation de DNMT1 résulte en une plus grande association avec la protéine EZH2 entraînant une hyperméthylation de promoteurs de gènes ciblés par EZH2 (ex : p16, CNR1 et PCNA). Ces observations démontrent que la régulation de DNMT1 est étroitement liée aux fonctions de Rb dans le cycle cellulaire. Caractéristique dans les cancers, une rupture de cette relation DNMT1-Rb, entraîne ainsi une méthylation site-spécifique de gènes suppresseurs de tumeurs, une autre caractéristique des cancers.
En parallèle, nous avons étudié l'effet d'une déplétion de DNMT1 dans des cellules cancéreuses. Suite à une déplétion de DNMT1, une voie de signalisation connue comme un point de contrôle de l'arrêt de la réplication/lésions de l'ADN est induite. L'activation de cette voie de signalisation entraîne l'arrêt de la croissance cellulaire et le blocage du cycle cellulaire. L'activation de cette voie répond à l'absence de DNMT1 et de façon indépendante de son activité catalytique. Ceci est en faveur d'un rôle pour DNMT1 de régulateur négatif du contrôle de l'arrêt de la réplication/lésions de l'ADN via l'interaction avec une protéine qui reste encore à identifier. De plus, la suppression des points de contrôle de l'arrêt de la réplication/lésion de l'ADN a été montré comme étant une étape nécessaire à la prolifération des cellules cancéreuses. L'ensemble des données de cette thèse démontre que des événements communs aux cancers, telle que l'inactivation de Rb, peuvent conduire à la dérégulation, via AUF1, de l'ARNm de DNMT1, laquelle entraîne la méthylation site-spécifique de gènes suppresseurs de tumeurs. Cette dérégulation de DNMT1 pourrait potentiellement servir à bloquer les points de contrôle d'arrêt du cycle cellulaire/lésions de l'ADN.
Les nouvelles fonctions de DNMT1, telles que la régulation du cycle cellulaire, la méthylation site-spécifique et le contrôle de la réplication/lésions de l'ADN découverts dans cette thèse devraient permettre de mieux comprendre le développement cancéreux et de développer de nouvelles stratégies thérapeutiques.