• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 1
  • 1
  • Tagged with
  • 2
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • 1
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Desensitisation of the Pituitary Vasopressin Receptor: Development and Use of a Stably-Transfected Model Cell System to Assess the Role of G Protein-Coupled Receptor Kinases

Cummings, Siobhan Anne January 2011 (has links)
Stress impacts upon all organisms and a robust stress response is required for adaptive interactions of the organism with the environment. In most higher organisms, an individual’s response to stress is mediated by the hypothalamic pituitary adrenal (HPA) axis. Inappropriate regulation of this axis can cause debilitating mental health disorders including depression and anxiety. These disorders can affect an individual’s ability to interact and respond appropriately as different situations arise. An important component of this axis is the vasopressin V1b receptor (V1bR), which mediates adrenocorticotropin (ACTH) secretion from the anterior pituitary in response to stimulation by arginine vasopressin (AVP). AVP also potentiates the ACTH secretion mediated by corticotropin-releasing hormone type 1 receptor (CRH-R1) in response to corticotropin- releasing hormone (CRH) stimulation. Both the V1bR and CRH-R1 are G protein coupled receptors (GPCRs). A common feature of GPCR signalling is desensitisation of the response following prolonged or repeated exposure to an agonist. Phosphorylation of the receptor is one of the mechanisms of desensitisation. This directly, or indirectly, results in rapid and reversible uncoupling of the receptor from its heterotrimeric guanine nucleotide binding protein (G-protein). Previous research has shown that G protein coupled receptor kinases (GRKs) are key phosphorylators involved in the molecular mechanism of GPCR desensitisation. One of the mains goals of the research carried out in the Mason laboratory is to examine the molecular mechanisms of V1bR desensitisation. The current short term aim is to examine the potential role for GRKs in this mechanism. It is difficult to study a single receptor type and the molecular mechanisms involved in its regulation in a system larger than a cell based assay. As the proposed method of assessing the involvement of GRKs in desensitisation of the V1bR is to use RNA interference (RNAi) to knock down the expression of the GRKs, primary cell cultures of pituitary corticotrophs are an inappropriate choice. This is due to a number of factors, including the difficulty involved in transfecting primary cells, and the difficulty involved in interpreting the results from primary cell culture experiments as these cultures are composed heterogenous population of cells. Therefore, the main aim of this research was to develop a model cell system from an immortalised cell line, stably-transfected with the V1bR, in which the involvement of GRKs in the molecular mechanism of V1bR desensitisation could be studied. Development of stably-transfected cell lines requires substantial preliminary work and planning in order to produce a successful outcome. Once developed, characterisation of the clonal cell lines is required. The preliminary work involved determining the cell proliferation rate of the parental cell line, plasmid sub-cloning and production of a large quantity of plasmid DNA, optimisation of the antibiotic selection conditions, and optimisation of the transfection protocol, as well as modification of the inositol phosphate (IP) assay protocol. The V1bR activates the phospholipase Cβ (PLCβ) second messenger signalling pathway in response to stimulation with AVP. This results in the production of IPs and therefore, measurement of IPs in response to AVP stimulation of cells labelled with myo-[³H]inositol can be used as an indicator of functional V1bR expression. In this research a total of nine clonal cell lines resistant to the antibiotic G418 were generated. Initial testing of these lines indicated that four probably expressed the V1bR and these were selected for characterisation in greater detail. All of these four lines showed significantly increased IP production in response to AVP stimulation (P<0.05; t-test). A significant decrease in IP production in response to AVP stimulation following an AVP pre-treatment was also seen with all four lines (P<0.05; t-test). Current evidence therefore suggests that the V1bR in these clonal cell lines signals and desensitises in the normal way. Although further characterisation of the clonal cell line is desirable, the data to date indicate that these lines should be considered to provide an appropriate model system for examining the molecular mechanisms involved in the regulation of the V1bR. It appears that there are some minor differences in signalling between the clonal cell lines and therefore this should be a consideration when deciding which line is most appropriate to use for investigating a particular question.
2

Recherche ou développement, et caractérisation fonctionnelle et structurale d'effecteurs peptidiques de deux récepteurs membranaires à incidences physiopathologiques / Research or development, and functional and structural characterization of peptidic effectors of two membrane receptors with pathophysiological incidences

Mebarki, Lamia 03 October 2017 (has links)
Les récepteurs à la vasopressine V1bR et à la sérotonine 5HT3R jouent des rôles physiologiques importants dans la détection des signaux extracellulaires, les mécanismes de transmission nerveuse et diverses pathologies dont le cancer, le diabète et des maladies des SNC et SNP. Mes études avaient pour but de générer ou trouver des modulateurs peptidiques de ces deux récepteurs. Pour le V1bR, j’ai développé plusieurs anticorps de type VHH et les ai caractérisés aux plans biochimique et fonctionnel. L’un de ces VHHs agit comme un agoniste allostérique complet et spécifique du V1bR humain (hV1bR). In vitro ce VHH est capable d’activer les voies de signalisation de l’inositol phosphate et des MAP kinases et d’induire l'internalisation du hV1bR. Dans des îlots pancréatiques surexprimant le hV1bR, il induit une augmentation du Ca2+ intracellulaire et une sécrétion d'insuline. Pour le 5HT3R, j’ai criblé par SPR 31 venins de serpents sur des récepteurs recombinants immobilisés et mis en évidence une interaction à partir d’un de ces venins. Suite à purification par chromatographie liquide et identification par spectrométrie de masse, j’ai identifié une toxine préalablement caractérisée comme une enzyme à activité Ca2+-dépendante. Cette toxine interagit avec les 5HT3R A et AB indépendamment du Ca2+ et avec des valeurs de Kd ≤ 10 nM. L’analyse fonctionnelle par électrophysiologie suggère qu’elle agit comme un PAM de l’activité canal du 5HT3R. Des images de ME en coloration négative montrent la toxine fixée sur le domaine extracellulaire du 5HT3R, à distance du site pour la 5HT. Le VHH et la toxine pourraient être utilisés comme outils pharmacologiques et/ou agents thérapeutiques. / The vasopressin V1bR and serotonin 5HT3R receptors play important physiological roles in the detection of extracellular signals, in the mechanisms for neuronal transmission, and in various pathologies including cancer, diabetes, and CNS and PNS diseases. My studies were aimed at generating or finding peptidic modulators of these two receptors. For the V1bR, I developed several antibodies of the VHH type and characterized them biochemically and functionally. One of these VHHs acts as a complete allosteric agonist specific for the human V1bR (hV1bR). In vitro this VHH is able to activate the signaling pathways of inositol phosphate and MAP kinases and to induce the internalization of hV1bR. In pancreatic islets overexpressing hV1bR, it induces an increase in intracellular Ca2+ and a secretion of insulin. For the 5HT3R, using SPR I screened 31 snake venoms on immobilized recombinant receptors and for one of these venoms, evidenced an interaction. Following purification by liquid chromatography and identification by mass spectrometry, I identified a toxin previously characterized as an enzyme with Ca2+-dependent activity. This toxin interacts with the 5HT3R A and AB independently of Ca2+ and with Kd values ≤ 10 nM. Functional analysis by electrophysiology suggests that it acts as a PAM of the 5HT3R channel activity. Images recorded by negative staining EM show that the toxin binds to the 5HT3R extracellular domain, at a distance from the 5HT binding site. Both this VHH and this toxin could be used as pharmacological tools and / or therapeutic agents.

Page generated in 0.0186 seconds