The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 6, 2010). Thesis advisor: M. Cathleen Kuehl-Kovarik. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 138 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 105-134).

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains xiv, 223 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 187-220).

One of the leading experts in the field of Neuroendocrinology and Neuroimmunmodulation, Samuel Mac Donald McCann, known by all his friends as ‘Don’, passed away in 2007. This article pays tribute to his outstanding scientific contribution and a glimpse on his fascinating personality. A member of the National Academy of Sciences of the United States and pioneer in the field of neuroendocrine regulation, he identified numerous hormones and peptides and set the stage for basic concepts in physiology and clinical medicine. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Lors d’un stress, l’activation de l’axe hypothalamo-hypophyso-surrénalien (HHS) conduit à une augmentation de la production de glucocorticoïdes (tel que la corticostérone) par les glandes surrénales. Le rôle de la corticotropin-releasing hormone (CRH), à l’origine de l’activation de l’axe HHS, est encore méconnu. En effet, les récepteurs à la CRH sont présents aussi bien au niveau du système nerveux central (SNC), notamment au niveau du cervelet, qu’au niveau du système immunitaire (SI). Cela suggère donc une action directe possible de cette hormone sur ces deux systèmes. Au cours de ce projet, nous avons étudié les régulations des SNC et SI lors d’un stress, et plus particulièrement le rôle de la CRH et de ses récepteurs dans ces régulations. Suite à des injections chroniques de corticostérone, mimant un stress, nous avons observé une altération des fonctions locomotrices qui semble être reversée lorsque le CRH-R1 est inhibé avec un antagoniste. Ces premiers résultats permettent de mettre en avant un éventuel rôle de la CRH dans la régulation des fonctions motrices au niveau du cervelet en conditions de stress. En parallèle, d’autres études in vitro réalisées sur des splénocytes murins stimulés avec de la CRH ont montré une diminution de la viabilité des lymphocytes B (LB). Suite à ces résultats, nous avons caractérisé pour la première fois la présence de récepteurs à la CRH sur cette population de LB murins. Ces résultats montrent l’importance de la CRH dans les régulations des SNC et SI en condition de stress et le rôle de cette hormone dans les interactions entre les deux systèmes / In stress conditions, the Hypothalamo-Pituitary-Adrenal (HPA) axis activation leads to an overproduction of glucocorticoïds (such as corticosterone in rodent) by adrenal glands and this activation is well characterized. However, various questions remain about the precise role of corticotropin-releasing hormone (CRH), which is at the beginning of the HPA activation. Indeed, CRH receptors are presents both in central nervous system (CNS), especially in cerebellum, and in immune system (IS). This suggest a possible direct action of this hormone on both system. In this project, we studied the regulations on CNS and IS in stress conditions and more particularly the CRH role and these receptors in these regulations. After chronic corticsterone injections, to mimic a stress, we observed a locomotor alteration which seems to be inverted when CRH-R1 were inhibited with an antagonist. These first results show an possible CRH role in locomotor regulation in cerebellum under stress condition. In parallel, others in vitro studies performed on murine splenocytes stimulated with CRH showed a B lymphocyte (LB) viability decrease. Furthermore, we are the first to characterise the CRH receptors on murine LB. This work show the CRH importance in CNS and IS regulations under stress conditions and its role in interactions between the two systems

[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.

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / Gonadotropin-releasing hormone (GnRH) acting through the cognate GnRH receptor (GnRH-R) plays an important role in the regulation of mammalian reproductive function by regulating the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of pituitary gonadotropes to GnRH depends on the number of GnRH receptors present on the gonadotrope cell surface. GnRH-R is regulated at a transcriptional, post-transcriptional and post-translational level. Hormones such as GnRH and glucocorticoids (GCs) regulate GnRH-Rs in a time- and dose-dependent manner. Previous studies have shown that the GnRH-R promoter confers glucocorticoid-dependent activation via the activating protein 1 (AP-1) site in the nongonadotrope GGH3 cell line. The mechanism by which GCs regulate the GnRH-R promoter is not precisely known as the literature is contradictory. Therefore this study investigates the mechanism of transcriptional regulation of the mouse GnRH-R promoter in the mouse gonadotrope cell line LβT2, treated with the synthetic GC dexamethasone (dex). Assays used include promoter-reporter studies, Western blotting, endogenous mRNA expression studies, electrophoretic mobility shift assay (EMSA) as well as the in vivo chromatin immunoprecipitation (ChIP) assay. A transfected promoter-reporter plasmid containing 600 bp of the mouse GnRH-R promoter was used to investigate the effect of dex on transcriptional regulation. Previously it was determined in our laboratory that the GnRH-R promoter is activated via an AP-1 binding site in the LβT2 cell line, and is regulated in a time- and dose-dependent manner by dex. In the present study in the LβT2 cell line a small induction was indeed seen upon dex treatment. Cotransfecting a expression vector for rat GR succeeded in inducing a 2 fold positive dex response. Western blot analysis revealed that GR levels remain consistent even after 8 hours dex induction. The effect of dex on the endogenous GnRH-R gene was investigated by means of real-time RT-PCR. Dex did indeed upregulate the gene in a time-dependant manner. Maximal induction (7.4 fold) was obtained after at least 12 hours of dex treatment. Untreated LβT2 nuclear extracts were investigated using EMSA, for protein binding to the mouse GnRH-R promoter AP-1 binding site, and these proteins were identified as c- Fos and GR. This suggests that the GR interacts with the AP-1 transcription factor via a tethering mechanism to mediate the positive dex response. The results of an in vivo ChIP assay were consistent with this hypothesis, showing that the GR interacted with a genomic fragment containingthe AP-1 site, in response to dex. The transactivation of the GnRH-R promoter by means of the GR tethering to AP-1 has not been shown before in the LβT2 cell line.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The regulation of gonadotropin releasing hormone (GnRH) receptor numbers in the pituitary is a crucial control point in reproduction. Pituitary sensitivity to GnRH can be directly correlated with GnRH receptor levels, which can be regulated at transcriptional and post-transcriptional level. The proximal promoter of the mouse GnRH receptor gene contains two cis elements bearing the consensus sequence for a Steroidogenic Factor-l (SF -1) binding site. The distal site has previously been shown to be involved in basal and tissue-specific transcriptional regulation, whereas the function of the proximal site was not established. SF-I, a member of the nuclear receptor superfamily of transcription factors, is involved in the transcriptional regulation of a large number of genes involved in steroidogenesis and reproduction. The consensus SF-I binding site can serve as a binding site for several members of the nuclear receptor superfamily. The aim of this study was to investigate the binding of SF-I protein from the aT3-1 gonadotrope cell line to the two putative SF-I binding sites in the mouse GnRH receptor promoter in vitro, in order to provide supporting evidence for their functional roles in GnRH receptor gene regulation. It was shown by Western blotting that SF-I and Nur77, another nuclear receptor transcription factor, are both expressed in aT3-1 cells, in a manner that is influenced by cell culture conditions. Gel mobility shift assays using specific antibodies showed that both SF-I and Nur77 protein in aT3-1 nuclear extracts bind to both sites in a mutually exclusive fashion. As shown by competition assays using mutated versions of the two sites, Nur77 protein had different base pair requirements than that of SF-I protein for binding to the sites. Additionally, SF-I mRNA was shown by Northern blotting to be increased in aT3-1 cells in response to stimulation of the Protein Kinase A (PKA) pathway by forskolin. These results highlight unexpected degeneracy in so-called "consensus" nuclear receptor binding sites. Furthermore, since Nur77 protein is involved in the stress response of the hypothalamic-pituitary-adrenal (HPA) axis, the unexpected presence of Nur77 protein in a gonadotrope cell line has potentially important implications for cross-talk between the HPA and hypothalamic-pituitary-gonadal (HPG) axes. / AFRIKAANSE OPSOMMING: Daar bestaan 'n direkte verband tussen pituïtêre sensitiwiteit vir gonadotropien-vrystellingshormoon (GnRH) en GnRH-reseptorvlakke Die regulering van GnRH-reseptorvlakke op transkripsionele en post-transkripsionele vlak in die pituïtêre klier is belangrik by die beheer van voortplantingsfunksies. Die proksimale promotor van die GnRH-reseptorgeen in die muis bevat twee cis elemente met die konsensus volgorde vir 'n Steroidogenic Factor-l (SF-I) bindingsetel. Die distale element is betrokke by basale en weefsel-spesifieke transkripsionele regulering, maar die funksie van die proksimale element is nog nie vasgestel nie. SF-1 is 'n lid van die superfamilie van selkernreseptore en is betrokke by die transkripsionele regulering van gene verantwoordelik vir steroïedogenese en voortplanting. Die konsensus SF-I bindingsvolgorde kan dien as bindingsetel vir verskeie selkernreseptore. Ten einde 'n beter insig ten opsigte van die regulering van die GnRH reseptorgeen te verkry, is ondersoek ingestel na die binding van SF-I-proteïen, afkomstig van die aT3-1 pituïtêre gonadotroopsellyn, aan die twee moontlike SF-l bindingsetels in die GnRH-reseptor promotor, in vitro. Die Western-klad metode het getoon dat beide SF-l en Nur77, 'n ander selkernreseptor-transkripsiefaktor, in die aT3-1 sellyn uitgedruk word. Die uitdrukking is afhanklik van selkultuurtoestande. Elektroforetiese mobiliteitsessais met spesifieke antiliggame het getoon dat SF-l en Nur77 proteïene in aT3-1 selkernproteïenekstraksies eksklusief aan beide bindingsetels bind. Nur77 proteïen benodig ander basispare as SF-l proteïen om aan die bindingsetels te bind. Hierdie resultate dui op onverwagse degenerasie in sogenaamde "konsensus" selkernreseptor-bindingsvolgordes. Die Northern-kladmetode het ook getoon dat SF-l mRNA vlakke in aT3-1 selle styg wanneer die proteïenkinase A (PKA) pad gestimuleer word met forskolin. Aangesien Nur77 proteïen betrokke is by die stres-respons van die hipotalamus-pituïtêre klier-adrenale (HP A) aksis, hou die onverwagse teenwoordigheid van Nur77 proteïen in 'n gonadotroop-sellyn potensieel belangrike inplikasies in vir kommunikasie tussen die HPA-aksis en die hipotalamus-pituïtêre klier-gonadale (HPG) aksis.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The GnRH receptor is a G-protein-coupled receptor in pituitary gonadotrope cells. Binding of its ligand, GnRH, results in synthesis and release of gonadotropin hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH). Steroidogenic factor 1 (SF-1), a transcription factor, binds to specific sites in the promoter region of gonadotropin genes, and thus regulates transcription of these genes. The promoter region of the GnRHreceptor gene contains two SF-1-like binding sites, one at -14 to -8 (site 1) and another at -247 to -239 (site 2), relative to the methionine start codon. The role played by these two SF-1-like sites in basal transcription of the mouse GnRH receptor (mGnRH-R) gene in a pituitary precursor gonadotrope cell line, aT3 cells, was the first area of investigation during this study. Luciferase reporter constructs containing 580 bp of mGnRH-R gene promoter were prepared, where SF-1-like sites were either wildtype or mutated. Four such constructs were made, i.e. wildtype (LG), site 1 mutant (LGM1), site 2 mutant (LGM2) and mutated site 1 plus site 2 (LGM1/2). These constructs were transfected into aT3 cells to determine the effect of mutations of sites 1 and/or 2 on the basal expression of the mGnRH-R gene. Mutation of either site 1 or site 2 had no effect on basal expression of the mGnRH-R gene. It was found that only upon simultaneous mutation of both sites 1 and 2, a 50% reduction in basal transcription took place. The implications of this is that SF-1 protein seems to only require one intact DNA-binding site, to mediate basal transcription of the mGnRH-R gene, suggesting that these two sites lie in close proximity during basal transcription. The effect of the protein kinase A (PKA) pathway on the endogenous mGnRH-R gene was also investigated by incubating non- , transfected aT3 cells with the PKA activators, forskolin and 8-Br-cAMP. Similar incubations were also performed on the wild type and mutated site 1 constructs transfected into pituitary gonadotrope aT3 cells. It was found that forskolin and 8-Br-cAMP were able to increase endogenous mGnRH-R mRNA levels in a concentration-dependent fashion, showing that endogenous GnRH receptor gene expression is stimulated via a protein kinase A pathway. Similar results were obtained with the wildtype promoter construct, showing that the protein kinase A pathway stimulates transcription of the promoter. This effect was only seen with wild type and not with the mutated site 1. These results are consistent with a role for a SF-1-like transcription factor in mediating the protein kinase A effect via binding to the site 1 at position -14 in the GnRH receptor gene. A separate investigation was performed to determine whether 25-hydroxycholesterol (25-0HC) is a ligand for SF-1, by incubating aT3 cells transfected with the various constructs with 25-0HC. Results show a dose-dependant response, with an increase in gene expression at 1 μM and a decrease at higher concentrations, for both mutant and wild type constructs. This suggests that, if SF-1 is indeed the protein binding to sites 1 and 2, then 25-0HC is not a ligand for SF-1 protein in aT3 cells and that the effect of 25-0HC on the mGnRH-R gene is not mediated via site 1. The results indicate that these decreases of expression at the higher concentrations may be due to cytotoxic effects. Towards the end of the study the laboratory obtained a luminoskan instrument with automatic dispensing features. Optimisation studies on the luciferase and β-Gal assays were performed on the luminoskan in a bid to decrease experimental error. It was found that automation of these assays resulted in a decrease in experimental error, showing that future researchers could benefit substantially from these optimisation studies. / AFRIKAANSE OPSOMMING: Die GnRH reseptor is 'n G proteïen-gekoppelde reseptor in pituitêre gonadotroopselle. Binding van die ligand, GnRH, lei tot die sintese en vrystelling van die gonadotropien hormone, luteïniserende hormoon (LH) en follikel stimulerende hormoon (FSH). Steroidogeniese faktor-t (SF-1) is 'n transkripsie faktor wat aan spesifieke areas in die promotergebied van die gonadotropien hormone bind, en dus transkripsie van hierdie gene reguleer. Die promotergebied van die GnRH reseptor geen bevat twee SF-1 bindings areas, een by -14 to -8 (area 1) asook by -247 to -239 (area 2), relatief to die metionien beginkodon. Die rol wat hierdie twee SF-1 areas speel in basale transkripsie van die muis GnRH reseptor (mGnRH-R) geen in 'n pituïtêre voorloper gonadotroop sellyn, aT3 selle, was die eerste gebied van ondersoek gedurende hierdie studie. Plasmiede bestaande uit die 580 basispaar mGnRH-R promoter verbind aan 'n lusiferase geen is vervaardig, waar SF-1-soortige areas enersyds onveranderd gelaat is, of gemuteer is. Vier sulke plasmiede is vervaardig, nl. onveranderd (LG), area 1 mutant (LGM1), area 2 mutant (LGM2) en gemuteerde area 1 plus area 2 (LGM1/2). Hierdie plasmiede is gebruik om aT3 selle te transfekteer om die effek van mutasies van areas 1 en/of 2 op die basale ekspressie van die mGnRH-R geen te ondersoek. Daar is gevind dat mutasies van areas 1 of 2 geen effek op basale ekspressie op die bogenoemde geen gehad het nie. Slegs tydens gelyktydige mutasie van areas 1 en 2 het 'n 50% vermindering in basale transkripsie plaasgevind. Die implikasies hiervan is dat die SF-1 proteïen blykbaar slegs een volledige DNA-bindingsarea benodig om basale transkripsie van die mGnRH-R geen te reguleer. Dit wil dus voorkom of hierdie twee areas baie na aan mekaar geposisioneer is tydens basale transkripsie. Die effek van die proteïen kinase A (PKA) roete op die natuurlike mGnRH-R geen is ook ondersoek tydens inkubasie van nie-getransfekteerde aT3 selle met die PKA akiveerders, forskolin en 8-Br-cAMP. Soortgelyke inkubasie is ook gedoen op die onveranderde en gemuteerde area 1 plasmiede wat in aT3 selle getransfekteer is. Daar is gevind dat forskolin en 8-Br-cAMP daarin geslaag het om die natuurlike mGnRH-R geen mRNA vlakke op 'n konsentrasie-afhanklike wyse te vermeerder. Hierdie resultaat dui daarop aan dat die natuurlike mGnRH-R geen se ekspressie gestimuleer kan word via 'n proteïen kinase A roete. Soortgelyke resultate is verkry met die onveranderde promoter plasmied en dit wys ook daarop dat proteïen kinase A transkripsie deur die promoter kan stimuleer. Hierdie effek was slegs aanwesig met die onveranderde en nie met die gemuteerde area 1 plasmied nie. Die resultate stem ooreen met 'n rol vir SF-1 transkripsie faktor in die regulering van proteren kinase A effek deur middel van binding aan die area 1 by posisie -14 in die GnRH-R geen. 'n Afsonderlike ondersoek is gedoen om vas te stel of 25-hidroksiecholesterol (25-0HC) 'n ligand vir SF-1 is deur getransfekteerde aT3 selle met 25-0HC te inkubeer. Resultate toon 'n dosis-afhanklike respons met 'n verhoging in geen ekspressie by 1 μM en 'n verlaging met hoër konsentrasies vir beide onveranderde en gemuteerde plasmiede. Dit impliseer dat, indien SF-1 wel die faktor is wat aan areas 1 en 2 bind, 25-0HC nie die ligand vir SF-1 proteren in aT3 selle is nie en dat die effek van 25-0HC op die mGnRH-R geen nie gereguleer word via area 1 nie. Die verlaging in ekspressie gevind by die hoër konsentrasies is dalk die gevolg van sitotoksiese effekte. Teen die einde van die studie het die laboratorium luminoskan toerusting met outomatiese pipettering verkry. Optimiseringstudies van die lusifirase en β-Galtoetse is met die luminoskan gedoen in 'n poging om eksperimentele foute te minimaliseer. Daar is gevind dat outomatisering van hierdie toetse wel gelei het tot 'n verlaging in eksperimentele foute. Toekomstige navorsers kan dus grootliks voordeel trek uit hierdie optimiseringstudies.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2006. / Gonadotropin-releasing hormone (GnRH), acting via its cognate receptor (GnRHR) is the primary regulator of mammalian reproductive function. Pituitary sensitivity to GnRH can be directly correlated with GnRHR levels on the surface of the pituitary gonadotrope cells, which can be regulated at transcriptional, post-transcriptional and post-translational levels. This study investigated mechanisms of transcriptional regulation of mouse GnRHR expression in two mouse gonadotrope cell lines, αT3-1 and LβT2, using a combination of endogenous mRNA expression studies, promoter-reporter studies, a two-hybrid protein-protein interaction assay, Western blotting, and in vitro protein-DNA binding studies. In the first part of the study, the role of two GnRHR promoter nuclear receptor binding sites (NRSs) and their cognate transcription factors in basal and Protein Kinase A (PKA)-stimulated regulation of GnRHR promoter activity was investigated in αT3-1 cells. The distal NRS was found to be crucial for basal promoter activity in these cells. While the NRSs were not required for the PKA response in these cells, results indicate a modulatory role for the transcription factors Steroidogenic Factor-1 (SF-1) and Nur77 via these promoter elements. The second part of the study focused on elucidating the mechanism of homologous regulation of GnRHR transcription in LβT2 cells, with a view to defining the respective roles of PKA and Protein Kinase C (PKC) in the transcriptional response to GnRH. In addition, the respective roles of the NRSs, the cyclic AMP response element (CRE) and the Activator Protein-1 (AP-1) promoter cis elements, together with their cognate transcription factors, in basal and GnRH-stimulated GnRHR promoter activity, were investigated. Homologous upregulation of transcription of the endogenous gene was confirmed, and was quantified by means of real-time RTPCR. The GnRH response of the endogenous gene and of the transfected promoter-reporter construct required PKA and PKC activity, and the GnRH response of the promoter-reporter construct was found to be dependent on a functional AP-1 site. Furthermore, GnRH treatment resulted in increased binding of phosphorylated cAMP-response element binding protein (phospho-CREB) and decreased expression and binding of SF-1 to their cognate cis elements in vitro, and stimulated a direct interaction between SF-1 and CREB, suggesting that these events are also required for the full transcriptional response to GnRH. This study is the first providing detail regarding the mechanism of transcriptional regulation of GnRHR expression in LβT2 cells by GnRH. Based on results from this study, a model has been proposed which outlines for the first time the kinase pathways, the promoter cis elements and the cognate transcription factors involved in homologous regulation of GnRHR transcription in the LβT2 cell line. As certain aspects of this model have been confirmed for the endogenous GnRHR gene, the model is likely to be physiologically relevant, and provides new ideas and hypotheses to be tested in future studies.