Profiles of plasma estradiol and testosterone concentrations, cathepsin D, L, and B activities, and quantitative and qualitative protein content were developed and evaluated in four commercial strains of channel catfish, Gold Kist (2), Thompson and NWAC-103 for one year (age 2 to age 3). Great variation between individuals of the same strain precluded the identification of any significant, strain-specific differences for the variables under investigation. When variables from fish of all strains were collectively evaluated over time, both estradiol and testosterone concentrations significantly increased in July and then later from February to April. The increase in hormone concentration was accompanied by oocyte growth and increases in proteolytic activity of cathepsins D, L, and B, supporting the role of estradiol in regulating vitellogenesis. Vitellogenin was enzymatically broken down into smaller protein units by cathepsins L, D, and B that were separately predominant at different stages of oocyte development. During oocyte development, there were sequential relationships among hormone concentration, cathepsin activity, protein content, and predominant oocyte proteins. This observation was associated with high levels of activity of cathepsin L in February, suggesting an important role in protein degradation during that time, while high activity of cathepsin B occurred, stimulating during November to January. Cathepsin B is more important in oogenesis or early vitellogenesis, and cathepsin L assumes a principal role during middle vitellogenesis. Twenty hours subsequent to the injection of fish with either carp pituitary hormone or luteinizing hormone releasing hormone, increases in the concentration of plasma estradiol and testosterone, activities of cathepsins L, D, and B, egg size, and egg protein content occurred, stimulating the process of oocyte maturation. The percentages of spawning obtained were 18.8% of LHRH injected fish, 12.4% of CPE injected fish, 9.4% of fish not injected, and 0% of saline injected fish. Injection of females with LHRH can potentially serve as a tool to increase spawning success in appropriate commercial settings, particularly for improving three year old catfish spawning success early in the spawning season. Low estradiol levels in all three-year-old fish suggest that insufficient stimulation of vitellogenin production by estradiol may underlie the lack of vitellogenin incorporation into developing oocytes. In the present study, the measurement of the activities of the cathepsins and their relationships to other parameters were evaluated for the first time. This is also the first study to report plasma estradiol and testosterone concentrations, protein content, and egg size in 2 to 3-year old channel catfish. All of the parameters collectively evaluated may serve to assist in the selection of the best 2- year old channel catfish female broodstock, and to determine the optimal timing of treatments of hormone injection to increase reproductive performance.

Three studies were conducted to determine the effects of ovarian steroids on bovine mammary epithelial cell proliferation. In a first study, estrogen (E), progesterone (P), or E+P were administered to prepubertal beef heifers and biopsied mammary parenchyma taken before and following treatment were compared for growth by evaluation of histoautoradiographic incorporation of thymidine. Estrogen increased epithelial cell growth by 24 h, and fibroblasts to a lesser magnitude by 48-96 h. Estrogen and P was less effective and P was ineffective in increasing proliferation in all cell types studied. Proliferation of adipocytes was not altered. A second study characterized hormone responsive proliferation of Mac-T cells, a recent clonal bovine mammary epithelial cell strain. Mac-T cells responded to all hormones tested as would be expected in vivo. Additionally, passage, harvesting, quantification, freezing, and co-culture techniques were modified to facilitate uncomplicated, timely, inexpensive, effective testing of growth responsiveness to hormones or growth factors. In a third study E and P alone, together, with or without serum were unable to increase Mac-T cell proliferation. Serum from prepubertal Holstein heifers after E treatment did not increase growth of Mac-T cells over serum before treatment. Conditioned media from Mac-T or Fib-T (mammary bovine fibroblast cell line) with or without steroids were tested for ability to increase Mac-T cell proliferation. Growth of Mac-T cells was greatest in Fib-T + E conditioned media followed by Fib-T, then Mac-T and lastly fresh media. Steroid exposure did not enhance the ability of Mac-T cell conditioned media to increase Mac-T cell proliferation. In conclusion, E appears to be the primary ovarian steroid involved in initiating bovine mammogenesis. However, estrogen’s action is not direct and may be caused by paracrine release of growth factors. / Master of Science

Chan Oi Chi. / "May 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 259-310) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1998. / Typescript. Bibliography: leaves 191-201.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The aim of this study was to build a mathematical model that describes the binding of 17- -estradiol (E2) to estrogen receptor (ER- ) and the influence the sex hormone binding globulin (SHBG) has on this interaction. The influence of SHBG on the transactivation of an estrogen response element, via ligand bound ER- , was also studied. COS-1 cells, derived from the kidney of a green african monkey, were used to study the binding of E2 to ER- in the absence of SHBG. The influence of SHBG on the binding of E2 to ER- was studied using Hep89 cells, human hepatacoma carcinoma, which express SHBG endogenously and are stably transfected with the ER- gene. Human pregnancy plasma was used to study the interaction of E2 with SHBG in the absence of ER- . The results of this study have shown that the Kd (E2) for ER- was determined as between 3.4nM and 4.4nM in the absence of SHBG. With respect to the binding of E2 to ER- it was not possible to determine the Kd app and Bmax for ER- using the Hep89 experimental system. The Kd (E2) for SHBG was not determined using the human pregnancy plasma experimental system. With the aid of mathematical modelling, a model of the Hep89 and human pregnancy plasma experimental systems, was built. The results of the numerical modelling, using mathematical modelling, showed that the presence of albumin together with SHBG was the reason that the Kd app (E2) could not be determined in the Hep89 experimental system. With respect to the use of human pregnancy plasma to determine the Kd (E2) for SHBG it was shown that if the plasma was diluted 200 times it would have been possible to determine the Kd app (E2) for SHBG, in the presence of albumin. Ligand independent transactivation of an estrogen response element was shown to be a problem in the COS-1 cell system when promoter reporter gene assays were undertaken. As COS-1 cells were used as a control for the absence of SHBG no further promoter reporter gene assays were undertaken using the Hep89 experimental system. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was die bou van ‘n wiskundige model wat die verbinding van E2 met die estrogeenreseptor (ER- ) en die invloed wat die geslagshormoon-verbindingglobulien (SHBG) op hierdie interaksie het, beskryf. Die effek van SHBG op die transaktivering van ‘n estrogeen responselement, via die ligandverbonde ER- , is ook bestudeer. COS-1-selle uit die nier van ‘n groen afrika-aap is gebruik om die verbinding van E2 met ER- in die afwesigheid van SHBG te bestudeer. Die invloed van SHBG op die verbinding van E2 met ER- , is bestudeer deur gebruik te maak van Hep89-selle, die menslike lewergeswelkarsinoom, wat SHBG uitwendig afgee en wat stabiel getransfesteer kan word met die ER- geen. Menslike swangerskapplasma is gebruik om die interaksie van E2 met SHBG in die afwesigheid van ER- te bestudeer. Die uitslag van hierdie studie toon aan dat die Kd (E2) vir ER- vasgestel tussen 3.4nM en 4.4nM in die afwesigheid van SHBG. Met betrekking tot die verbinding van E2 met ER- , was dit nie moontlik om die Kd (E2) en Bmax app vir ER- met die gebruik van die Hep89 eksperimentele stelsel vas te stel nie. Die Kd (E2) vir SHBG is nie vasgestel deur die gebruik van die menslike swangerskapplasma eksperimentele stelsel nie. ‘n Model van die Hep89 en menslike swangerskapplasma eksperimentele stelsels is met behulp van wiskundige modellering gebou. Die uitslag van die numeriese modellering, met gebruik van wiskundige modellering, toon dat die teenwoordigheid van albumien, saam met SHBG, die rede was dat die Kd app (E2) nie in die Hep89 eksperimentele stelsel vasgestel kon word nie. Wat betref die gebruik van menslike swangerskapplasma om die Kd (E2) vir SHBG vas te stel, is daar aangetoon dat, indien die plasma 200 maal verdun was, dit moontlik sou gewees het om die Kd app (E2) vir SHBG in die teenwoordigheid van albumien vas te stel. Promotor verkilkkergeen toetse het ligandonafhanklike transaktiveering van ‘n estrogeen responselement aangetoon as ‘n probleem in die COS-1-selle stelsel. Omdat COS-1-selle gebruik is as ‘n kontrole vir die afwesigheid van SHBG, is geen verdere promotor verkilkkergeen toetse onderneem met die gebruik van die Hep89 eksperimentele stelsel nie.

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 (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography. / AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: - the characterization and comparison of the enzymatic activity of both Angora and ovine 3βHSD expressed in non-steroidogenic COS-1 cells. The apparent Km and Vmax values for the metabolism of PREG, 17-OHPREG and DHEA were determined; - the characterization of steroid metabolites produced by COS-1 cells coexpressing either Angora or ovine 3βHSD together with Angora CYP17, in the presence and absence of overexpressed Cyt-b5, following the metabolism of PREG and 17-OHPREG. 3βHSD was identified as an additional factor in causing hypocortisolism in the South African Angora goat; - the influence of Cyt-b5 on the enzymatic activity of both Angora and ovine 3βHSD coexpressed in non-steroidogenic COS-1 cells; - the influence of purified ovine live Cyt-b5 and anti-Cyt-b5 IgG on adrenal microsomal 3βHSD activity. Cyt-b5 was shown to specifically augment 3βHSD activity which represents the first documentation of such augmentation in any species; - the overexpression and purification of Angora 3βHSD using a baculovirus expression system coupled with a detergent based enzyme purification method; - the characterization of both substrate and co-factor kinetics for the individual dehydrogenase and isomerase activities of purified 3βHSD, in the presence and absence of purified ovine liver Cyt-b5. Cyt-b5 was shown to increase the affinity of 3βHSD towards NAD+ during the dehydrogenase reaction whilst having no significant influence on the isomerase reaction. This represents the first documentation of Cyt-b5 influencing co-factor binding in any member of the -ydroxysteroid dehydrogenases; - the FRET analysis of COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP fusion proteins, suggesting an allosteric interaction between 3βHSD and Cyt-b5. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: - die karakterisering en vergelyking van die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD, wat uitgedruk was in nie-steroïed genererende COS-1 selle. Die Km en Vmax waardes tydens die metabolisme van PREG, 17-OHPREG en DHEA was bepaal; - die karakterisering van steroïed metaboliete gegenereer deur COS-1 selle wat Angora of skaap 3βHSD uitdruk saam met Angora CYP17, in die aanwesigheid of afwesigheid van sitochroom b5, na die metaboliseering van PREG en 17-OHPREG. 3βHSD was geïdentifiseer as ‘n bydraende faktor in die oorsaak van hipokortisolisme in die Suid-Afrikaanse Angorabok; - die invloed van sitochroom b5 op die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD wat saam uitgedruk was in nie-steroïed genererende COS-1 selle; - die invloed van gesuiwerde skaap lewer sitochroom b5 en sitochroom b5 teenstof op mikrosomale 3βHSD aktiwiteit. Dit is getoon dat sitochroom b5 die aktiwiteit van 3βHSD spesifiek verhoog. Hierdie studie verteenwoordig die eerste dokumentasie van so ‘n verhoging in enige spesie; - die uitdrukking en suiwering van Angora 3βHSD deur middel van ‘n bakulo-virus sisteem gekoppel aan ‘n detergent gebaseerde ensiem suiwerings metode; - die karakterisering van beide substraat en ko-faktor kinetika vir die afsonderlike dehidrogenase en isomerase aktiwiteite van gesuiwerde 3βHSD, in die aanwesigheid of afwesigheid van gesuiwerde sitochroom b5. Dit is getoon dat sitochroom b5 die affiniteit van 3βHSD teenoor NAD+ tydens die dehidrogenase reaksie verhoog sonder om ‘n beduidende invloed op die isomerase reaksie te hê. Hierdie studie verteenwoordig die eerste dokumentasie van sitochroom b5 wat ko-faktor binding beïnvloed in enige lid van die hidroksisteroïed dehidrogenase familie van ensieme; - die analise van FRET sein in COS-1 selle wat beide 3βHSD-eCFP en Cyt-b5- eYFP fusie proteïene uitdruk. Die resultate stel voor dat sitochroom b5 3βHSD aktiwiteit beïnvloed deur middel van ‘n allosteriese meganisme.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • the influence of a methanolic extract of unfermented Rooibos and five major Rooibos flavonoids, aspalathin, nothofagin, rutin, orientin and vitexin, on the activities of key adrenal steroidogenic enzymes - cytochrome P450 17β- hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase • the development of a novel UPLC-MS/MS method for the separation and quantification of 21 adrenal steroid metabolites; • the influence of Rooibos and aforementioned flavonoids on adrenal steroid hormone production in H295R cells - a human adrenal carcinoma cell line expressing the enzymes catalysing the production of mineralocorticoids, glucocorticoids and adrenal androgens, assayed under both basal (normal) and forskolin-stimulated (stressed) conditions; • the influence of Rooibos on the inter-conversion between cortisol and cortisone by 11βHSD1 and 11βHSD2 expressed in CHO-K1 cells; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male Wistar rats; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male and female human test subjects at risk for developing cardiovascular disease. (3βHSD2), cytochrome P450 21-hydroxylase (CYP21A2) and cytochrome P450 11β-hydroxylase (CYP11B1), expressed in non-steroidogenic COS-1 cells; / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: • die invloed van metanoliese ekstrakte van ongefermenteerde Rooibos en vyf van die hoof flavonoïedverbindings in Rooibos, aspalatien, notofagien, rutien, oriëntien en viteksien, op die aktiwiteite van ensieme wat steroïedbiosintese in die bynier kataliseer – sitochroom P450 17α-hidroksilase/17,20-liase (CYP17A1), 3β-hidroksisteroïed dehidrogenase (3βHSD2), sitochroom P450 21-hidroksilase (CYP21A2) en sitochroom P450 11β-hidroksilase (CYP11B1), uitgedruk in nie-steroïed produserende COS-1 selle; • die ontwikkeling van ‘n geskikte UPLC-MS/MS metode vir die skeiding en kwantifisering van 21 steroïedmetaboliete in die bynier; • die invloed van Rooibos en die bg. flavonoïede op steroïedproduksie in H295R selle – ‘n menslike bynier kanker sellyn gekenmerk deur die ekspressie van die steroidogeniese ensieme wat die produksie van mineralokortikoïede, glukokortikoïede en bynierandrogene kataliseer, geanaliseer onder beide basale (normale) en forskoliengestimuleerde (gestresde) kondisies; • die invloed van Rooibos op die omeenskakeling tussen kortisol en kortisoon deur 11βHSD1 and 11βHSD2 in CHO-K1 selle; • die invloed van Rooibosinname op vlakke van sirkulerende steroïed hormone en relatiewe verhoudings in die bloed van manlike Wistarrotte; • die invloed van Rooibosinname op sirkulerende steroïed hormoon vlakke en relatiewe verhoudings in die bloed van mans en vrouens met ‘n hoë risiko vir die ontwikkeling van kardiovaskulêre siektes.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy