Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 183-273).

Thesis (doctoral)--Universität, Würzburg, 2000.

Neuropeptide Y (NPY) has been shown to play a critical role in the regulation of bone metabolism by signalling via Y1 and Y2 receptors. Centrally, hypothalamic Y2 but not Y1 receptors are important for the action of NPY on bone formation and osteoblast activity. This project investigates how the NPY system, in particular, the Y1 receptor, influences bone metabolism peripherally by examining a range of conditional and germline knockout mice using in vitro and in vivo techniques. Revealing the possibility of a direct role for the Y1 receptor on bone cells, we demonstrate the presence of Y1 but not Y2 receptor mRNA in osteoblasts by in situ hybridisation on femur sections and RT-PCR on bone marrow stromal cells (BMSCs). In addition, we show that NPY mRNA is also expressed by osteoblasts suggesting that locally produced NPY may directly influence bone cell activity. In order to investigate the role of osteoblastic Y1 receptors, mice with selective osteoblastic deletion of the Y1 receptor under control of the osteoblast-specific α1(I)-collagen promotor were generated. In male mice, osteoblast-specific Y1 receptor deletion resulted in a marked increase in femoral trabecular bone volume, trabecular number, cortical bone volume and cortical thickness due to elevated osteoblast activity as shown by increased mineral apposition rate and bone formation rate. Further in vitro investigations using cells isolated from germline and conditional knockout mice demonstrated that NPY inhibits the proliferation of BMSCs via the Y1 receptor and that mineralisation is enhanced in vitro in the absence of NPY or osteoblastic Y1 receptors. Moreover, this study reveals a critical role for the NPY system via Y1 receptors in the proliferation and differentiation of mesenchymal stem cells and osteoprogenitor cells. Together these data demonstrate a direct role for the NPY system via Y1 receptors on the proliferation and differentiation of osteoblastic progenitor cells as well as on regulating the activity of mature osteoblasts, thereby altering bone formation both in vitro and in vivo. Understanding the action of NPY on osteoblasts to regulate bone metabolism could have powerful therapeutic implications for stimulating bone accrual in diseases such as osteoporosis.

München, Techn. Universiẗat, Diss., 2007.

Thèses (Ph.D.)--Université de Sherbrooke (Canada), 2001. / Titre de l'écran-titre (visionné le 15 août 2006). Publié aussi en version papier.

Thesis (Ph.D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 183-273) Also available in print.

At the hypothalamo-pituitary interface, multiple neurotransmitters and neuropeptides interact to control luteinizing hormone and follicle stimulating hormone release from gonadotrophs. The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. While it is well-established that gonadotropin-releasing hormone and/or kisspeptin are required for pulsatile and surge release of luteinizing hormone in mammalian species, their essentiality is challenged by studies showing knockouts in zebrafish and medaka do not block reproduction. In goldfish, secretoneurin-a, a neuropeptide derived from secretogranin-IIa processing, stimulates luteinizing hormone release in vivo and from dispersed pituitary cells in vitro. Secretoneurin does not bind to the human gonadotropin releasing hormone receptor and can enhance luteinizing hormone release when applied directly to mouse LbetaT2 cells. Our study indicates the presence of secretogranin-IIa and secretogranin-IIb mRNA in specific regions of the zebrafish brain and pituitary that have been implicated in the control of reproductive processes. I also observed that secretogranin-II knockout disrupts normal morphology of the pectoral fins by reducing the number of breeding tubercule rows, breeding tubercule width and breeding tubercule cluster length which could potentially lead to a reduced spawning success. Knocking out the secretogranin-IIa and secretogranin-IIb genes using TALENs in zebrafish indicates that these genes are required for optimal reproduction. Rates of oviposition for double secretogranin-II knockout females are 6% and 11% when crossed with double secretogranin-II knockout and wild-type males, respectively, compared to 62% in virgin wild-type pairings. Comprehensive video analysis demonstrates that secretogranin-II knockout reduces all stereotypical male courtship behaviours. Severe reductions in the expression of gonadotropin releasing hormone 3 in the hypothalamus and luteinizing hormone in the pituitary suggest that secretogranin-II-derived peptides drive the gonadotropin releasing hormone-luteinizing hormone control system. Spawning success is rescued in double secretogranin-II knockouts following one injection of synthetic secretoneurin-a in which it increases from 11% to 30% thereby supporting the hypothesis that secretoneurin-a is a key reproductive hormone. However, embryo survival rate of secretoneurin-a injected double secretogranin-II knockout was lower than saline-injected wild-type pairings. Injection of human chorionic gonadotropin, a hormone that mimics the action of luteinizing hormone on binding and activating the luteinizing hormone receptor in fish, increased double secretogranin-II knockout spawning success to 38%, thus comparing favourably to 47% in saline-injected wild-type controls. My data provides support that secretogranin-II is required for optimal reproductive functions although the precise mechanisms behind this reduced capacity in zebrafish lacking the secretogranin-II genes remain to be investigated. Moreover, the high conservation of secretoneurin from lamprey to human suggests a broader importance of this emerging peptide family.

Un développement normal et optimal du système nerveux central durant l'embryogenèse est crucial pour l'organisation des circuits nociceptifs chez le nouveau-né. Durant le développement de l'enfant, les mécanismes impliqués dans la perception de la douleur maturent et sont influencés par l'exposition aux stress environnants. En effet, le stress peut induire un phénomène de l'analgésie liée au stress (stress induced analgesia,SIA). Ce phénomène peut impliquer les opioïdes et/ou la neurotensine (NT). D'ailleurs, il existe de nombreuses évidences que la NT endogène joue un rôle primordial dans le SIA de type naloxone-indépendant. La NT exerce ses effets analgésiques par sa liaison aux récepteurs NTS1 et NTS2. Nous proposons donc de déterminer le rôle du récepteur NTS2 dans le SIA chez le rat issu d'une colonie non stressée ainsi que chez la souris de type sauvage (WT) et la souris ayant été délétée du gène responsable de l'expression du récepteur NTS2 (KO NTS2). Nous avons évalué l'influence d'un stress sévère de type non-opioïdergique en utilisant le test de la nage en eau froide (15[degré]C) durant trois minutes, sur la modification de la perception de la douleur. Les expérimentations chez les rats mâles ont été effectuées aux jours postnataux 25 et 60 (P25 et P60). Les agonistes neurotensinergiques utilisés sont le JMV-431(30[mu]g/kg) et [bêta]-lactotensine (200[mu]g/kg), deux agonistes sélectifs du récepteur NTS2. Le NT69L (5?g/kg) a été utilisé comme agoniste spécifique des récepteurs neurotensinergiques (NTS1 et NTS2). Les agonistes neurotensinergiques ont été administrés de façon individuelle par voie intrathécale et la nage en eau froide a été effectuée 10 minutes suivant l'administration du composé en question. Les comportements douloureux ont été évalués grâce au test plantaire (Hargreaves test) 20, 30 et 40 minutes suivant l'administration du composé. Les résultats de la modification de la perception de la douleur suite à l'administration de chacun des agonistes à P25 ont révélé une augmentation du temps de retrait des pattes arrières au test plantaire au temps 20 minutes après l'injection. Les agonistes neurotensinergiques NTS2 amplifient l'effet du SIA en période infantile et cet effet est soutenu jusqu'au temps 30. Chez l'adulte (P60), les agonistes potentialisent également le SIA. Le SIA est aussi présent chez la souris. La modification de la perception de douleur a été évaluée au test plantaire et au test à la formaline (douleur tonique). Suite au stress de nage en eau froide, les souris WT étaient moins sensibles à la douleur que les souris KO NTS2 à 30 minutes. De plus, les résultats du test à la formaline ont aussi révélé, suite au stress sévère, que les souris KO NTS2 étaient plus sensibles à la douleur que les souris WT au cours de la phase inflammatoire. Afin de supporter ces résultats, nous avons récolté des échantillons de plasma pour dosage de corticostérone par radioimmunoassay (RIA). En condition basale, les souris KO NTS2 ont des concentrations plus basses que les souris WT. Les résultats ont aussi démontré des concentrations plus élevées de corticostérone en condition de stress en comparaison avec les individus non stressés. Également, on peut constater que les souris KO NTS2 stressées ont des niveaux de corticostérone plus bas que les individus de type sauvage ayant subi un stress. En utilisant une approche pharmacologique et génétique, nous avons ainsi démontré que le récepteur NTS2 était impliqué dans le SIA. Ces études ciblant le stress permettront d'améliorer notre compréhension entourant les facteurs externes pouvant influer sur le développement optimal des circuits nociceptifs au cours de l'enfance.

Alzheimer’s disease (AD) is a neurodegenerative disease which affects over 500,000 people in the UK. Worldwide 44 million people are affected by AD and other dementias. Most cases occur over the age of 65 and is characterised by gradual and increasing loss of cognitive function and behavioural abnormalities. The main causes are a build-up of the toxic protein amyloid-β (Aβ) and hyperphosphorylation of the microtubule stabilising protein: tau, leading to neurofibrillary tangles (NFT). These two hallmarks of disease result in neuronal damage and cell death causing associated symptoms and eventually death. Orexins (OX) are neuropeptides which function to regulate the sleep-wake cycle and feeding behaviour. They are produced from a prepro-orexin (PPO) molecule and cleaved into two isoforms: orexin-A (OXA) and orexin-B (OXB). OXA and OXB are the ligands for two G-protein coupled receptors (GPCR): orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). 50-80,000 OX producing neurons project to many areas of the brain including the lateral hypothalamus (LHA), locus coeruleus (LC), tuberomammillary nucleus (TMN), paraventricular nucleus (PVN) and raphe nuclei and from these areas regulate feeding and appetite and the sleep wake cycle through their receptors. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity including the control of feeding behaviour. It is the ligand for the GPCR GPR103, both of which are widely expressed in the brain and also in the retina, testes, thyroid, pituitary and prostate. GPR103 also shares 48 and 47% protein sequence homology with OX1R and OX2R respectively. It is in these tissues where it can exert other physiological functions including regulation of feeding, control of the gonadotropic axis and bone formation. The exact expression and signalling characteristics and physiological actions of QRFP/GPR103 are still poorly understood. It is through the physiological functions of the orexigenic system and the clinical symptoms observed in AD which suggests a possible link between the two. For example, in AD one of the main reasons for institutionalisation is the severely dysregulated sleep pattern that is experienced by sufferers. They experience increased nocturnal activity and early awakenings as well as hypersomnia and excessive daytime sleepiness; all of which is beyond what someone of the same age experiences. As well as this AD patients suffer from significant weight loss and a significant negative correlation has been identified between progression of disease and appetite. All of this points towards an involvement of the orexigenic system in AD. AD patients have been found to have a 40% loss of immunoreactive OX neurons and have severe reductions in circulating OXA. This led us to believe that the OX system is of vital importance in AD and could be targeted to ameliorate symptoms. Studies have implicated OX and OXR in memory processes, appetite regulation, and severe disturbances of the sleep-wake cycle all of which are phenotypes of AD. Given that they play a key role in energy homeostasis and physiological behaviour, we hypothesise that OXs and their receptors are implicated in the pathophysiology of AD. Therefore, in this study we will investigate the detailed expression and signalling characteristics of OXR and GPR103 in vitro and in clinical samples In this study we neuronally differentiated two human neuroblastoma cell lines: IMR32 and SH-SY5Y. Neuronally acquired phenotype was confirmed through increased neurite length, increased expression of key neuronal proteins and increases in microtubule-associated protein tau (MAPT), neurogenin1 (NG1) and neuron-specific enolase (NSE) as well as a reduction in the neuronal marker of immaturity; nestin (NES). OXR and GPR103 were confirmed in both cell lines after differentiation at mRNA and protein level and were shown to be fully functional through phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). We also identified possible cross talk of GPR103 with the OXR though addition of selective OXR antagonists, which blocked QRFP induced ERK1/2 phosphorylation. We show for the first time that addition of Aβ42 and zinc sulphate to mimic AD in vitro, results in a significant reduction of OX1R and GPR103 in the cell lines SH-SY5Y and we have performed the first comprehensive study in clinical AD patients which demonstrate a loss of OX1R, OX2R and GPR103 at mRNA and protein level compared to age matched controls in the hippocampus. We performed microarray analysis which identified many genes and pathways regulated by the OXA, OXB and QRFP; including corticotropin-releasing hormone receptor (CRHR1), regulated in development and DNA damage responses 1 (REDD1), erythropoietin (EPO), Bcl-2-like protein 1 (BCL2L11), myb proto-oncogene protein (c-myb), vasoactive intestinal peptide (VIP), endothelin 1 (EDN1) as well as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and hypoxia-inducible factor-1α (HIF-1α) pathways. These genes are all implicated in neuroprotection, particularly in AD. This represents the first comprehensive gene expression data in a neuroblastoma cell line for these orexigenic proteins. Collectively these data suggest a potential role of the orexigenic system in neuroprotection and a functional loss of the receptors in AD patients which could confer a loss of neuroprotection through the orexigenic system. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways.

Endocrine and metabolic disease or dysfunctions are of growing concern in modern societies across the globe, underlining the need for continued focus on the development of pharmaceuticals. Subsequent scientific research has revealed a trend in the increase of such abnormalities and expansion of chemical industries, highlighting concerns that these disorders may, in part, be caused by exposure to environmental pollutants. This has led to changes in legislation concerning chemicals safety testing involving an increasing number of vertebrate animal tests as a part of environmental risk assessment process, at significant financial and ethical costs. A solution that is appropriate and aligned with the three R’s (reduction, refinement and replacement) in relation to animal research is to exploit the use of small invertebrate organisms as possible replacements for mammals. In line with the above approach/solution, this thesis is based on the null hypothesis that common genes, proteins and processes in gastropod molluscs and humans underlie the response of male reproductive organs to androgenic chemicals. Using a freshwater pulmonate snail, Biomphalaria glabrata, physiological effects of two steroid androgens on the development of mollusc secondary sexual organs were studied. Furthermore, an exhaustive investigation on the mollusc nuclear receptor repertoire and reproductive type neuropeptides was conducted. This also included the study of the evolutionary degree of conservation of these genes in non-model molluscs. The results obtained suggest that the snails did not respond to, and were not affected by exposure to the androgens. These results were supported by the absence of the members of subfamily 3C of nuclear receptors, which includes some of the “vertebrate” steroid hormone targets, suggesting that this mollusc may be an inappropriate model for steroid hormone mediated mammalian endocrine function. The nuclear receptor (NR) repertoire of B. glabrata comprised of 39 nuclear receptors representing all the known subfamilies of the NR superfamily. 21 reproductive type neuropeptide genes were identified encoding precursors that are predicted to release over 124 bioactive cleavage products. The consequence of these findings is significant in the context of the development of alternative model organisms for chemical testing as well as elucidating the taxonomic scope of nuclear receptor mediated endocrine disruption.