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Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Concern has increased that widespread adverse effects are occurring in humans and wildlife populations as a result of exposure to environmental chemicals (mostly man-made) that disrupt the normal functioning of the endocrine system. Many pharmaceutical, agricultural and industrial chemicals, acting as endocrine modulating compounds (EDCs), have been detected in an accumulated form in food, in drinking water and in the environment. Although the levels of these chemicals can be measured analytically, it is important to evaluate biological activity. For this, animal models are used and relevant bioassays developed. These assays are based on biological markers, which are specific xenobiotically-induced physiological responses and are usually deviations in cellular or biochemical components, processes or structures. Vitellogenin is a large protein complex, produced in the liver under estrogen control and circulates in the plasma, destined for incorporation into the developing oocyte in female oviparous vertebrates. Since vitellogenin production is low or nonexistent in males, its presence may therefore be interpreted as evidence of exposure to estrogenic environmental contaminants. In this study the African Clawed Frog, Xenopus laevis was used as model to study the effects of estrogens on biochemical and physiological parameters associated with vitellogenesis. As a starting point the seasonal female reproductive cycle in a natural Xenopus laevis population in terms of ovarian state, plasma vitellogenin and plasma estrogen levels was studied. It was shown that plasma vitellogenin and estrogen levels were seasonal and correlated to a seasonal ovarian cycle, which peaked during spring. However, although seasonality existed, there were reproductively active individuals at any time during the year. Increases in plasma estrogen levels predated increases in plasma vitellogenin levels and ovarian maturation. Lipoprotein profiles, as well as plasma cholesterol, triglyceride and phospholipid concentrations were determined and it was shown that estrogen affected these in such a way that these parameters could be used as additional markers in estrogen contamination studies. In order to develop an in vitro bioassay to screen for estrogenic activity, the use of hepatic tissue cultures was investigated. Optimal culture conditions were established and increased sensitivity in the estrogenic response was obtained by using liver slices from male frogs that were pre-treated with estrogen. Validation studies proved that this bioassay could be employed for routine screening of water and chemical samples. In order to refine the Xenopus laevis vitellogenin ELISA and liver slice bioassay, existing polyclonal anti-vitellogenin antibodies were replaced by culturing monoclonal antibodies. Selected antibodies were characterised and ELISAs developed and validated. This study showed that the newly developed Xenopus laevis vitellogenin ELISA and liver slice bioassay have the potential to be employed in environmental monitoring programmes. / AFRIKAANSE OPSOMMING: Daar is toenemende besorgdheid dat afwykings in mens- en dierbevolkings voorkom as gevolg van blootstelling aan chemikalieë (hoofsaaklik mensgemaak) in die omgewing wat die normale werking van die endokrienstelsel versteur. Verskeie farmaseutiese, landbou- en industriële chemikalieë, wat as endokrienversteurders optree, is in die omgewing gevind. AI kan die vlakke van hierdie stowwe analities bepaal word, is dit belangrik om hulle biologiese aktiwiteit te evalueer. Hiervoor word diermodelle aangewend om toepaslike toetse daarvoor te ontwikkel. Hierdie toetse word baseer op biologiese merkers, spesifieke xenobioties-geïnduseerde fisiologiese reaksies, en is gewoonlik afwykings van sellulêre- of biochemiese komponente, -prosesse of - strukture. Vitellogeen ('n dooiervoorloper) is 'n lipoproteïenkompleks wat, onder estrogeenbeheer, in die lewer vervaardig word en in die plasma sirkuleer vir uiteindelike inkorporasie in ontwikkelende oësiete van vroulike, ovipare werweldiere. Aangesien daar min of geen vitellogeen in manlike diere geproduseer word, is die teenwoordigheid daarvan 'n aanduiding dat die dier aan estrogeniese omgewingsbesoedeling blootgestel is. In hierdie studie is die Platanna, Xenopus laevis, as model gebruik om die gevolge van estrogene op biochemiese en fisiologiese veranderlikes, wat met vitellogenese geassosieer word, te bestudeer. As vertrekpunt is die seisoenale voortplantingsiklus van die wyfie, in terme van vitellogeen en -estrogeen vlakke in die plasma en aktiwiteit van die ovaria bepaal. Daar is aangetoon dat die estrogeen- en vitellogeenvlakke in die plasma met die ovariumsiklus, wat gedurende die lente hoogtepunte bereik, korreleer. Alhoewel daar seisoenaliteit bestaan, was daar dwarsdeur die jaar wyfies wat ovarium dooierneerlegging getoon het. Verhoging in estrogeenvlakke het vitellogeenpieke en rypwording van die ovaria voorafgegaan. Lipoproteïenprofiele, sowel as die cholesterol- , trigliseried- en fosfolipiedkonsentrasies in die plasma is bepaal en daar is aangetoon dat estrogeen hierdie medeveranderlikes in só 'n mate affekteer dat hulle as bykomende biomerkers vir estrogeenblootstelling in besoedelingstudies gebruik kan word. In vitro Xenopus laevis lewersnitte in die weefselkultuur omgewing is ondersoek om 'n biotoets te onwikkel vir die gebruik in vinnige estrogenisiteits-toetsing van watermonsters en chemise stowwe. Die mees gunstige kultuurtoestande is bepaal en die sensitiwiteit van estrogeenreaksies is verhoog deur lewer van mannetjies, wat vooraf met estrogeen behandel is, te gebruik. Hierdie biotoets se geldigheid is gestaaf en kan in roetine eerstevlaktoetsing van watermonsters gebruik word. Die gebruik van poliklonale teenliggaampies in 'n bestaande enzyme-linked immunosorbent assay (ELISA) is vervang deur spesiaal-ontwikkelde monoklonale anti-Xenopus laevis vitellogeen teenliggaampies. Uitgesoekte teenliggaampies, spesifiek teen die estrogeengeïnduseerde proteïene, is gekarakteriseer en ELISAs saamgestel en die geldigheid gestaaf. Hierdie studie het aangetoon dat die nuut-onwikkelde Xenopus laevis vitellogeen-ELISA en lewerkultuurbiotoets die potensiaal het om In omgewingsmoniteringprogramme gebruik te word.

[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.

Congenital hypothyroidism (CH) is the most frequent of the rare endocrine diseases (e.g. Addison's disease, Cushing's syndrome, Congenital adrenal hyperplasia.), which affects 1:2000 – 4000 newborns. If not immediately diagnosed after birth, thyroid hormones deficiency causes severe defects in brain and skeletal development leading to a complex clinical scenario called cretinism. CH can be due to a defective synthesis of thyroid hormones (dyshormonogenesis) or an abnormal embryonic development of the gland. Data obtained using knockout mouse models have shown the pivotal role of four specific transcription factors (NKX2.1, PAX8, FOXE1 and HHEX) for the correct organogenesis or function of the gland. Although mutations in those genes have been identified in few cases of CH patients, the pathogenetic mechanisms remain still elusive in the vast majority of CH cases (95%).<p>For the identification of new genes and molecular events controlling thyroid organogenesis it would be useful to develop an in vitro cellular model to recapitulate thyroid embryogenesis in a dish. Embryonic Stem Cells (ESCs) have recently emerged as system model to recapitulate the embryogenesis of several tissues in vitro.<p>Induced overexpression of defined transcription factors has been shown to have a directing effect on the differentiation of pluripotent stem cells into specific cell types. In this thesis I show that a transient overexpression of the transcription factors NKX2.1 and PAX8 is sufficient to direct the differentiation of murine ESCs into thyroid follicular cells (TFC) and promotes in vitro self- assembly of TFC into three-dimensional follicular structures, when associated to a subsequent thyrotropin (TSH) treatment. Cells differentiated by this protocol showed significant iodide organification activity, a hallmark of thyroid tissue function. Importantly, athyroid mice grafted with mESC-derived thyroid follicles show normalization of plasma T4 levels with concomitant decrease of plasma TSH. In addition, a full normalization of body temperature at 4 weeks after transplantation was observed. Together, these data clearly demonstrate that grafting of our mESC-derived thyroid cells rescues the hypothyroid state and triggers symptomatic recovery along with the normalization of plasma hormone concentrations. The high efficiency of TFC differentiation and follicle morphogenesis in our system will provide an unprecedented opportunity for future studies to decipher regulatory mechanisms involved in embryonic thyroid development, a major research need towards an improved understanding of the molecular mechanisms underlying congenital hypothyroidism, the most common congenital endocrine disorder in humans. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Water quality analysis forms the basis in assessing and monitoring catchments. As urban development continuously increase, pollution sources increase in either point source (wastewater treatment works, industrial effluents) and/or non-point source origin (storm water discharge, domestic pollutants), accumulating pollutants in the environment. It was only recently discovered that certain pollutants have subtle disrupting effects on the endocrine system resulting in health related problems associated with the reproductive system and thyroid system (growth and development) of animals and potentially humans. Natural water resource management proves to include limited biological assays measuring endpoints for cytoxicity, inflammatory activity and endocrine disruption. The broad objective of this study was therefore to include several bioassays, not normally used in municipal (City of Cape Town) monitoring programmes, along with water quality data collected by the City of Cape Town. The Eerste/Kuils River catchment system, Western Cape, under the auspices of the City of Cape Town was chosen, and although this catchment does not contribute to drinking water resources, is subjected to a range of anthropogenic influences (industrial effluents, household wastewater, agricultural runoff). Within the short time-frame available for this study (six months) two months, July (following a dry summer and autumn season) and October (following a wet winter and early spring season) were selected for water quality monitoring. Spatial variation (with relevance to specific point and non-point contamination) among sampling sites were also obtained by choosing several (n=10) along the catchment. Specific aim of the study therefore included: Firstly (Chapter 2), the use of in vitro bioassays, lactate-dehydrolises assay (LDH) for cytotoxic activity, pro-inflammatory hormone Interleukin-6 (IL-6) secretion by human blood cells and a specific Salmonella ELISA for faecal contamination, in conjunction with routine chemical and biological (mostly microbiological) monitoring activities. The study indicated significant variation among sites in all microbiological measures as well in IL-6 secretion and Salmonella presence. Between months, variations were also evident in certain variables. Secondly (Chapter 3), two bioassays using the yolk precursor protein, vitellogenin (Vtg) as endpoint was implemented in a) an in vitro Xenopus laevis liver slice assay (five day exposure) and b) an in vivo Zebrafish (Danio rerio) bioassay (seven day exposure) assessing estrogenic activity in the Eerste/Kuils River catchment. Although estrogen spiked positive control water samples stimulated Vtg production in vitro as well as in vivo, no dramatic estrogenic activity was measured at any of the selected sites. Thirdly (Chapter 4), a bioassay using the thyroid controlled metamorphosis in Xenopus laevis tadpoles to assess effects on the thyroid hormonal system was implemented. Thyroid stimulatory activity, compared with a negative control sample, was measured at two sites along the catchment. Although the practical implementation of the tadpole semi-static exposure protocol (water replacement) proved to be labour intensive, all the added bioassays proved to be valuable tools to add valuable information regarding water quality. It is clear that more research related to anthropogenic influences along the Eerste/Kuils River catchment system are needed, specifically in monitoring monthly variations to better understand annual variation in several of the endpoints studied. / AFRIKAANSE OPSOMMING: Waterkwaliteit vorm die basis vir die evaluering en monitering van opvangsgebiede. Voortdurende stedelike ontwikkeling gee aanleiding tot ‘n toename in die voorkoms van besoedelstowwe in die natuurlike omgewing deur gelokaliseerde (punt) bronne (rioolwerke/industriële uitvloeisel) en/of nie gelokaliseerde (nie punt) bronne (vloed uitlaat/huishoudelike uitvloeisels) van besoedeling. Dit het onlangs aan die lig gekom dat van hierdie chemise besoedelstowwe subtiel die endokriene sisteem versteur en so aanleiding gee tot gesondheidsprobleme in terme van die voortplantingsisteem en tiroied sisteem (groei en ontwikkeling) by diere en moontlik ook die mens. Daar is beperkte gebruik van biologiese toetse wat inligting verskaf oor sitotoksiteit, inflammatoriese aktiwiteit en endokriene versteuring. Die doel van hierdie studie was dus om van hierdie biologiese toetse, wat normaalweg nie deel uitmaak van die roetine munisipale (Stad van Kaapstad) opvangsgebied monitering nie, gebruik te maak. Die Eerste-, Kuilsrivier, Wes Kaap, onder beheer van Stad Kaapstad is gekies en alhoewel die opvangsgebied nie water bydra tot drinkwaterbronne nie, word die opvangsgebied beïnvloed deur verskeie mensgemaakte bronne van besoedeling (afloop vanuit omliggende lanbougebiede). Binne die kort tydsraamwerk van die projek (ses maande) is besluit om twee maande, Julie (volg ‘n droë somer en herfs seisoen) en Oktober (volg ‘n nat winter en vroeë lente seisoen) vir water kwaliteit monitering te kies. Ruimtelike variasie langs die loop van die opvangsgebied is ingesluit deur moniteringspunte (n=10), met in ag name van die potensiële besoedelingsbronne. Spesifieke doelwitte van die projek sluit in: Eerstens (Hoofstuk 2), om die in vitro biotoetse, laktaat hidrolise (LDH) vir sitotoksisiteit, proinflammatoriese hormoon Interleuikin-6 (IL-6) vir inflammatoriese aktiwiteit, vrygestel deur menslike bloedselle en ‘n Salmonella ELISA vir ontlasting besoedeling saam met bestaande chemise en biologiese (hoofsaaklik migrobiologiese) veranderlikes te gebruik. Die studie het getoon dat beduidende variasie in alle mikrobiologiese toetse asook IL-6 vrystelling en Salmonella voorkoms bestaan het tussen versamelpunte. Maandelikse variasie in sekere van die veranderlikes het ook voorgekom. Tweedens (Hoofstuk 3), is twee biotoetse wat die dooiervoorloperproteïen, vitellogeen (Vtg) as eindpunt gebruik geimplimenteer in a) ‘n in vitro Xenopus laevis lewersnit biotoets (vyf dag blootstelling) en b) ‘n in vivo Zebravis (Danio rerio) biotoets (sewe dag blootstelling) om estrogenisiteit in die Eerste-, Kuilsrivier opvangsgebied te evalueer. Alhoewel, die estrogeen behandelde positiewe kontrole water monsters Vtg produksie veroorsaak het in beide die in vitro lewer-kulture en in vivo vistoets, is geen dramatiess estrogeniese aktiwiteit by enige van die moniteringspunte gevind nie. Derdens (Hoofstuk 4), is ‘n biotoets wat die tiroïedbeheerde metamorfose in Xenopus laevis paddavisse gebruik om effekte op die tiroïedsisteem te evalueer. Die differensiële stimulering (versnelling), in vergelyking met ‘n negatiewe kontrole watermonster, van die tiroied sisteem is by twee moniteringspunte in die opvangsgebied waargeneem. Alhoewel die praktiese implementering van die paddavis semi-statiese (water word gereeld vervang) biotoets arbeidsintensief is, het alle bykomstige biotoetse waardevolle bykomstige inligting oor water kwaliteit verskaf wat ‘n belangrike bydrae tot ingeligte bestuursbesluite kan verleen. Dit is duidelik dat meer navorsing int verband met die menslike invloed langs die Eerste-, Kuilsrivier opvangsgebied nodig is, veral met maandlikse monitering vir seisoenale veranderinge.