Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin synthesis and release from the pituitary via binding to its specific receptor, known as the gonadotropin-releasing hormone receptor type I (GnRHR-I). The gonadotropins, luteinising hormone (LH) and follicle-stimulating hormone (FSH), bind to receptors in the gonads, leading to effects on steroidogenesis and gametogenesis. The recent finding of a second form of the GnRH receptor, known as the type II GnRHR or GnRHR-II, in non-mammalian vertebrates triggered the interest into the possible existence and function of a GnRHR-II in humans. The current study addressed this issue by investigating the presence of transcripts for a GnRHR-II in various human tissues and cells. While it was demonstrated that antisense transcripts for this receptor, containing sequence of only two of the three coding exons, are ubiquitously and abundantly expressed in all tissues examined, potentially full-length (containing all three exons), sense transcripts for a GnRHR-II were detected only in human ejaculate. Further analysis revealed that the subset of cells in the ejaculate expressing these transcripts is mature sperm. These findings, together with the reported role for GnRH in spermatogenesis and reproduction led to the further analysis of the presence of a local GnRH/GnRHR network in human and vervet monkey ejaculate or sperm. Indeed, such a network seems to be present in humans since transcripts for both forms of GnRH present in mammals, as well as transcripts for the GnRHR-I, are expressed in human ejaculate. Furthermore, transcripts for the GnRHR-II are expressed in both human and vervet monkey ejaculate. Thus, it would appear that locally produced GnRH-1 and/or GnRH-2 in the human male reproductive tract might mediate their effects on fertility via a local GnRHR-I, and possibly via GnRHR-II. Remarkably, in the pituitary, LH and FSH are present in the same gonadotropes, yet they are differentially regulated by GnRH under various physiological conditions. While it is well established that post-transcriptional regulatory mechanisms occur, the contribution of transcriptional regulation to the differential expression of the LHβ- and FSHβ-subunit genes is unclear. In this study, the role of GnRH-1 and GnRH-2 via the GnRHR-I and the GnRHR-II in transcriptional regulation of mammalian LHβ- and FSHβ genes was determined in the LβT2 mouse pituitary gonadotrope cell-line. It is demonstrated for the first time that GnRH-1 may affect gonadotropin subunit gene expression via GnRHR-II in addition to GnRHR-I, and that GnRH-2 also has the ability to regulate gonadotropin subunit gene expression via both receptors. Similar to other reports, it is shown that the transcriptional response to GnRH-1 of LHβ and FSHβ is low (about 1.4-fold for bLHβLuc and 1.2-fold for oFSHβLuc). In addition, evidence is supplied for the first time that GnRH-2 transcriptional regulation of the gonadotropin β subunits is also low (about 1.5-fold for bLHβLuc and 1.1-fold for oFSHβLuc). It is demonstrated that GnRH-1 is a more potent stimulator of bLHβ promoter activity as compared to GnRH-2 via the GnRHR-I, yet both hormones result in a similar maximum induction of bLHβ. However, GnRH-2 is a more efficacious stimulator of bLHβ transcription via the GnRHR-II than GnRH-1. No discriminatory effect of GnRH-1 vs. GnRH-2 was observed for oFSHβ promoter activity via GnRHR-I or GnRHR-II. By comparison of the ratio of expression of transfected oFSHβ- and bLHβ promoterreporters via GnRH-1 with that of GnRH-2, it is shown that GnRH-2 is a selective regulator of FSHβ gene transcription. This discriminatory effect of GnRH-2 is specific for GnRHR-I, as it is not observed for GnRHR-II, where GnRH-1 results in a greater oFSHβ- to-bLHβ ratio. These opposite selectivities for GnRHR-I and GnRHR-II on the ratios of oFSHβ:bLHβ promoter activity for GnRH-1 vs. GnRH-2 suggest a mechanism for fine control of gonadotropin regulation in the pituitary by variation of relative GnRHR-I vs. GnRHR-II levels. In addition, a concentration-dependent modulatory role for PACAP on GnRH-1- and GnRH-2-mediated regulation of bLHβ promoter activity, via both GnRHR-I and GnRHR-II, and of oFSHβ promoter activity, via GnRHR-I, is indicated. The concentration-dependent effects suggest the involvement of two different signalling pathways for the PACAP response. Together these findings suggest that transcription of the gonadotropin genes in vivo is under extensive hormonal control that can be finetuned in response to varying physiological conditions, which include changing levels of GnRH-1, GnRH-2, GnRHR-I and GnRHR-II as well as PACAP. / AFRIKAANSE OPSOMMING: Gonadotropien-vrystellingshormoon (GnRH) is bekend as die sentrale reguleerder van die voorplantingsisteem deur die stimulasie van gonadotropiensintese en - vrystelling vanaf die pituïtêre klier via binding aan ‘n spesifieke reseptor, die sogenaamde tipe I gonadotropien-vrystellingshormoonreseptor (GnRHR-I). Die gonadotropiene, lutineringshormoon (LH) en follikel-stimuleringshormoon (FSH), bind aan reseptore in die gonades waar dit steroïedogenese en gametogenese beïnvloed. Die onlangse ontdekking van ‘n tweede vorm van die GnRH-reseptor, bekend as die tipe II GnRHR of GnRHR-II, in nie-soogdier vertebrate het belangstelling in die moontlike bestaan en funksie van ‘n GnRHR-II in die mens gewek. Hierdie kwessie is aangeraak deur die teenwoordigheid van transkripte vir ‘n GnRHR-II in verskeie weefsel- en seltipes van die mens te ondersoek. Daar is aangetoon dat nie-sin transkripte vir hierdie reseptor, wat die DNA-opeenvolgings van slegs twee van die drie koderende eksons bevat het, oormatig uitgedruk word in al die weefseltipes wat ondersoek is. Daarteenoor is potensieel vollengte (bevattende al drie eksons) sin transkripte vir ‘n GnRHR-II in die mens slegs in semen gevind. Verdere analise het getoon dat dit volwasse sperma binne die semen is wat laasgenoemde transkripte uitdruk. Hierdie bevindinge, tesame met die aangetoonde rol vir GnRH in spermatogenese en reproduksie het gelei tot die verdere analise van die teenwoordigheid van ‘n lokale GnRH/GnRHR-netwerk in mens- en blouaapsemen of -sperm. So ‘n netwerk blyk om teenwoordig te wees in die mens, aangesien transkripte vir beide vorme van GnRH wat in soogdiere gevind word, asook transkripte vir die GnRHR-I, in menssemen uitgedruk word. Daarbenewens word transkripte vir die GnRHR-II uitgedruk in beide mens- en blouaapsemen. Dit wil dus voorkom asof lokaalgeproduseerde GnRH-1 en/of GnRH-2 in die manlike voortplantingstelsel van die mens hul effek op vrugbaarheid bemiddel via ‘n lokale GnRHR-I, en moontlik ook via GnRHR-II. Dit is opmerklik dat LH en FSH teenwoordig is in dieselfde gonadotroopselle van die pituïtêre klier en tog verskillend gereguleer word deur GnRH tydens verskeie fisiologiese kondisies. Terwyl dit bekend is dat post-transkripsionele reguleringsmeganismes teenwoordig is, is die bydrae van transkripsionele regulering tot die differensiële uitdrukking van die LHβ- en FSHβ-subeenheidgene minder duidelik. In hierdie studie is die rol van GnRH-1 en GnRH-2 via die GnRHR-I en die GnRHR-II in transkripsionele regulering van soogdier-LHβ- en -FSHβ-gene in die LβT2 muis pituïtêre gonadotroopsellyn bepaal. Dit is vir die eerste keer aangetoon dat GnRH-1 ‘n effek mag hê op gonadotropiensubeenheid-geenuitdrukking via GnRHR-II bykomend tot GnRHR-I, en dat GnRH-2 ook die vermoë besit om gonadotropiensubeenheid-geenuitdrukking via beide reseptore te reguleer. Soos deur ander studies aangetoon is die transkripsionele respons van LHβ en FSHβ tot GnRH-1 klein (ongeveer 1.4-voudig vir bLHβLuc en 1.2- voudig vir oFSHβLuc). Verder is daar vir die eerste keer bewys gelewer dat transkripsionele regulering van die gonadotropien β-subeenhede deur GnRH-2 ook gering is (ongeveer 1.5-voudig vir bLHβLuc en 1.1-voudig vir oFSHβLuc). Daar is aangetoon dat GnRH-1 ‘n sterker stimuleerder van bLHβ-promotoraktiwiteit is in vergelyking met GnRH-2 via die GnRHR-I, hoewel beide hormone tot ‘n soortgelyke maksimum induksie van bLHβ lei. GnRH-2 is egter ‘n meer effektiewe stimuleerder van bLHβ-transkripsie as GnRH-1 via die GnRHR-II. Geen verskille is gevind tussen die effekte van GnRH-1 en GnRH-2 op oFSHβ-promotoraktiwiteit via GnRHR-I of GnRHR-II nie. Wanneer die verhouding van uitdrukking van getransfekteerde oFSHβ- en bLHβ- promotor-verslaggewers via GnRH-1 met dié van GnRH-2 vergelyk is, is aangetoon dat GnRH-2 ‘n selektiewe reguleerder van FSHβ-geentranskripsie is. Hierdie diskriminasieeffek van GnRH-2 is spesifiek vir GnRHR-I aangesien dit nie vir GnRHR-II waargeneem word nie. GnRH-1 lei tot ‘n groter oFSHβ tot bLHβ-verhouding via GnRHR-II. Hierdie teenoorgestelde selektiwiteite van GnRHR-I en GnRHR-II op die verhoudings van oFSHβ tot bLHβ-promotoraktiwiteit vir GnRH-1 teenoor GnRH-2 suggereer dat daar ‘n meganisme bestaan vir die fyn regulering van gonadotropiene in die pituïtêre klier, deurdat die relatiewe vlakke van GnRHR-I teenoor GnRHR-II gevarieer word. Daarbenewens is ‘n konsentrasie-afhanklike moduleringsrol vir PACAP op GnRH-1- en GnRH-2-bemiddelde regulering van bLHβ-promotoraktiwiteit aangetoon, via beide GnRHR-I en GnRHR-II, asook op oFSHβ-promotoraktiwiteit via GnRHR-I. Hierdie konsentrasie-afhanklike effekte dui op die betrokkenheid van twee verskillende seinpadweë vir die PACAP-respons. Tesame suggereer hierdie bevindinge dat transkripsie van die gonadotropiengene in vivo onder ekstensiewe hormonale kontrole is wat verfyn kan word in respons to veranderlike fisiologiese kondisies. Laasgenoemde sluit veranderende vlakke van GnRH-1, GnRH-2, GnRHR-I en GnRHR-II asook PACAP in.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This study: 1. Describes the localization of CYP11B1 in the Cape baboon adrenal gland using Western blot analysis. CYP11B1 was localized to the adrenal cortex and medulla. 2. Describes the catalytic activity of CYP11B1 towards 11-deoxycorticosterone and corticosterone in adrenal cortical- and medullary tissue homogenates. Aldosterone formation in the adrenal medulla was identified using an atmospheric pressure chemical ionization-mass spectrometry method, which was developed in our department. 3. Compares the catalytic activity of three recombinant Cape baboon CYP11B1 cDNAs, expressed in COS-1 cells, towards 11-deoxycorticosterone and 11-deoxycortisol. 4. Describes the determination of the Michaelis-Menten constants and maximum reaction rates of 11-deoxycorticosterone and 11-deoxycortisol utilization by two functional recombinant Cape baboon CYP11B1 cDNAs, respectively. 11-Deoxycorticosterone metabolites were quantified using an enzyme immunoassay kit. 11-Deoxycortisol metabolites were quantified using a liquid chromatography-mass spectrometry method, which was developed in our department. 5. Describes the homology modeling of two isoforms of Cape baboon CYP11B1 using CYP102 and CYP2C5 as structural templates. The influence of three amino acid residue substitutions, located in the predicted D-E helix, on the catalytic activity of the two CYP11B1 isoforms was examined. / AFRIKAANSE OPSOMMING: Hierdie studie: 1. Beskryf die lokalisering van CYP11B1 in die bynier van die Kaapse bobbejaan deur gebruik te maak van die Western kladtegniek. CYP11B1 is gelokaliseer tot die adrenale korteks en medulla. 2. Beskryf die metabolisme van 11-deoksikortikosteroon en kortikosteroon in adrenale korteks- and medulla weefsel preparate, onderskeidelik. Die produksie van aldosteroon in die medulla is geïdentifiseer deur gebruik te maak van ‘n atmosferiese druk chemiese ionisasie-massa spektrometrie metode wat in ons departement ontwikkel is. 3. Vergelyk die katalitiese aktiwiteit van drie rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11- deoksikortisol metabolisme. 4. Beskryf die bepaling van die Michaelis-Menten konstantes en maksimum snelhede van twee funksionele rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11-deoksikortisol metabolisme. 11-Deoksikortikosteroon metaboliete is gekwantifiseer deur gebruik te maak van ‘n ensiem immunotoets. 11-Deoksikortisol metaboliete is gekwantifiseer deur middel van ‘n vloeistofchromatografie-massaspektrometrie metode, ontwikkel in ons departement. 5. Beskryf die modelering van drie-dimensionele strukture van twee funksionele Kaapse bobbejaan CYP11B1 isoensieme deur CYP102 en CYP2C5 as template te gebruik. Die effek van drie aminosuurresiduveranderinge in die voorspelde D-E heliks op die katalitiese aktiwiteit van die twee CYP11B1 isoforme is bepaal.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This study describes: 1. The isolation and the purification of tissue type plasminogen activator and urokinase plasminogen activator in bovine milk. 2. The biochemical characterisation of tissue type plasminogen activator in bovine milk. 3. An investigation of the influence of the addition of purified tissue type plasminogen activator to ultra high temperature milk, Gouda cheese and yoghurt. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. Die isolering en suiwering van weefseltipe-plasminogeenaktiveerder en urokinase-plasminogeenaktiveerder in beesmelk. 2. Die biochemiese karakterisering van weefseltipe-plasmingeenaktiveerder in beesmelk. 3. `n Ondersoek na die invloed van die byvoeging van gesuiwerde weefseltipe-plasminogeenaktiveerder by ultra hoë temperatuur melk, Gouda kaas en joghurt.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/- 236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence (TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been previously shown to be involved in regulation of transcription of the mouse GnRHR (mGnRHR) promoter in some cell lines, the role of site 3 has not been previously investigated. This study investigated whether transcription of the mGnRHR gene is regulated by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein- DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1 site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of investigation during this study. Luciferase reporter plasmids containing 600 bp of the mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site 3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into LβT2 cells was carried out to determine the effect of these mutations on the basal expression of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2 cells. This suggests that the AP-1 site is involved in positively mediating the basal transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins involved and the effect of varying doses for varying times of GnRH, as well as the overexpression of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it could be the c-Jun protein that binds to the AP-1 site under basal conditions. Another area that was investigated was whether the mGnRHR promoter can be regulated by dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1 site, as shown by the complete loss of response when these sites were individually mutated, consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells. / AFRIKAANSE OPSOMMING: Die gonadotropienvrystellings hormoon (GnRH) reseptor is ‘n G-proteïen-gekoppelde reseptor in die pituitêre gonadotrope en is ’n belangrike beheerpunt vir reproduksie. GnRH bind aan die GnRH reseptor (GnRHR) met die gevolg dat follikel stimulerende hormoon (FSH) en luteïeniserende (LH) gesintetiseer en vrygestel word. Die sensitiwiteit van die pituitêre klier vir GnRH kan direk met GnRHR vlakke gekorreleer word. Die muis GnRHR promotor bevat drie cis elemente met bindingssetels vir steroïedogeniese faktor 1 (SF1), naamlik setel 1 (-15/-7), setel 2 (-244/-236) en setel 3 (-304/-296) sowel as ’n aktiveerder proteïen 1 (AP-1) tipe konsensus sekwens (TGAGTCA) in posisie -336/-330. Terwyl setels 1 en 2 en die AP-1 setel voorheen getoon is om by die regulering van transkripsie van die muis GnRHR (mGnRHR) promotor in party sellyne betrokke te wees, is die rol van setel 3 nog nie vantevore bestudeer nie. In hierdie studie is ondersoek of die transkripsie van die mGnRHR geen deur GnRH en glukokortikoïede in die LβT2 gonadotroop pituitêre sellyn gereguleer word, en die rol van setel 3 en die AP-1 setel en hulle binders, deur gebruik te maak van in vitro proteïen-DNA bindings studies en promotor-verslaggewer essais. Die rol wat setel 3 en die AP-1 setel in basale transkripsie van die mGnRHR gene in LβT2 selle gespeel het, was die eerste onderwerp wat in hierdie studie bestudeer is. Lusiferase verslaggewer plasmiede wat die eerste 600 bp van die mGnRHR promotor bevat het en waarin setel 3 en die AP-1 setels óf wilde tipe óf gemuteer was, is gebruik. Two konstrukte is vanaf die wilde tipe konstruk berei, naamlik wilde tipe (LG), ’n setel 3 mutant (m3) en ’n AP-1 mutant (mAP-1). Transfeksie van LG, m3 en mAP-1 plasmiede in LβT2 selle is deurgevoer om te bepaal wat die effek van hierdie mutasies op die basale ekspressie van die mGnRHR gene was. Mutasie van setel 3 het ’n 1.5-voudige toename in die transkripsionele aktiwiteit van die mGnRHR promotor tot gevolg gehad. Dit suggereer dat setel 3 ’n rol in die inhibisie van die basale transkripsievlakke van die mGnRHR promotor in LβT2 selle speel. Mutasie van die AP-1 setel het tot ‘n 50% verlaging in basale transkripsievlakke van die mGnRHR promotor in LβT2 selle gelei. Dit suggereer dat die AP-1 setel betrokke is in die positiewe bemiddeling van die basale transkriptionele respons van die GnRHR promotor in LβT2 selle. Eksperimente wat gemik was om die meganisme van die cis-elemente (setel 3 en die AP-1 setel) op die mGnRHR promotor te verklaar, asook om die rol van proteïen kinase A (PKA) paaie, proteïene daarby betrokke en die effek van varieende dosisse vir verskillende tye van GnRH, sowel as die oorekspressie van PKA en die SF-1 proteïen, is deurgevoer. Dit is gevind dat setel 3 en die AP-1 setel nie betrokke by die GnRH respons is nie. Die resultate suggereer dat setel 3 gedeeltelik betrokke is by die PKA respons van LβT2 selle. Setel 3 kan SF-1 proteïen bind soos getoon deur kompeterence elektroforetiese mobiliteits verskuiwings essais (EMSA). As EMSA’s deurgevoer is op die AP-1 setel is bevind dat die c-Fos proteïen nie betrokke is in die aktivering van die AP-1 setel nie. ’n Faktor is gevind om aan die AP-1 setel te bind wat nie ’n intakte AP-1 setel vereis het nie, wat gesuggereer het dat dit die c-Jun proteïen kan wees wat aan die AP-1 setel onder basale omstandighede bind. ’n Ander area wat ondersoek is, is of die GnRHR promotor gereguleer kan word deur deksametasoon (dex) óf via die AP-1 setel óf via setel 3. ’n Dosis en tyds-afhanklike toename in promotor aktiwiteit is waargeneem met dex. ’n Vereiste vir hierdie effek blyk om die teenwoordigheid van setel 3 en die AP-1 setel te wees, soos aangetoon deur die totale verlies aan response as hierdie twee setels individueel gemuteer is, en wat weereens in ooreenstemming met die funksionele interaksie tussen setel 3 en die AP-1 setel in LβT2 selle is.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: • the comparison of the enzymatic activities of the two ovine cytochrome P450 17 - hydroxylase/17,20-lyase (CYP17) isoforms expressed in non-steroidogenic COS-1 cells. The Km and Vmax values for the metabolism of pregnenolone and progesterone were determined, while time-dependent metabolism of pregnenolone, 17-hydroxypregenolone, progesterone and 17-hydroxyprogesterone was also reported. The cloning and sequencing of ovine cytochrome b5 is reported and was co-expressed with CYP17. The results showed that the wild type 1 (WT1) isoform of ovine CYP17 produce more cortisol precursors than the wild type 2 (WT2) isoform; • the analysis of the frequency distribution of the CYP17 genotypes within a South African Merino population, which were divergently selected for (H-line) or against (L-line) the ability of a ewe to rear multiple offspring per birthing opportunity. It was observed that the CYP17 frequency distribution was the same within the H- and L-line, with 78.3 % heterozygous WT1/WT2 and 21.7 % homozygous WT1/WT1. No homozygous WT2/WT2 individuals were identified; • the development of a UPLC-MS/MS method for the separation and quantification of all thirteen adrenal steroids that are produced in the adrenal gland; • the relative contribution of the CYP17 genotypes in the total steroidogenic output in adult adrenocortical cells from the adrenal glands of H- and L-line sheep, with particular emphasis on cortisol production. The adrenocortical cells from the H-line sheep showed a marked higher cortisol production than the L-line, while adrenocortical cells from homozygous WT1/WT1 sheep also produced more cortisol than heterozygous WT1/WT2 sheep; • the blood cortisol responses upon the stimulation of the HPA axis by insulin induced hypoglycaemia of the H- and L-line sheep with known CYP17 genotypes. It was observed that the CYP17 genotype and selection line are important factors affecting the cortisol responses of sheep, where L-line heterozygous WT1/WT2 sheep showed the lowest cortisol response and glucose recovery; • the association of the CYP17 genotype with behavioural responses of H- and L-line sheep to flock isolation stress, as well as the association of the CYP17 genotype with ewe reproduction and lamb output. While reproduction seemed to be unaffected by the CYP17 genotype, the behavioural stress responses of sheep to flock isolation correlated with the CYP17 genotype, where the heterozygous WT1/WT2 genotype was associated with a wilder nature. / AFRIKAANSE OPSOMMING: Hierdie studie ondersoek: • die vergelyking van die ensiemaktiwiteite vir twee isoforme van skaap sitochroom P450 17 -hidroksilase/17,20-liase (CYP17), wat uitgedruk was in nie-steroïed genererende COS- 1 selle. Die Km and Vmax waardes was bepaal vir die metabolisme van pregnenoloon en progesteroon, terwyl die tyd-afhanklike metabolisme van pregnenoloon, 17- hidroksiepregnenoloon, progesteroon en 17-hidroksieprogesteroon ook gerapporteer word. Die klonering en volgorde bepaling van skaap sitochroom b5 was gedoen en gevolglik was sitochroom b5 saam met CYP17 uitgedruk in COS-1 selle. Die resultate het gewys dat wilde tipe 1 (WT1) meer voorlopers van kortisol produseer as wilde tipe 2 (WT2); • die frekwensie distrubusie van die CYP17 genotipes in ‘n Suid-Afrikaanse Merino populasie, waar skape in teenoorgestelde rigtings geselekteer was vir (H-lyn) of teen (L-lyn) die vermoë van ‘n ooi om geboorte te gee aan veelvoudige lammers per lamgeleentheid. Die frekwensie distrubusie van CYP17 was dieselfde in beide die H- en L-lyn, waar 78.3 % van die populasie heterosigoties WT1/WT2 en 21.7 % homosigoties WT1/WT1 was. Geen homosigote WT2/WT2 individue was geïdentifiseer nie; • die ontwikkeling van ‘n UPLC-MS/MS metode vir die skeiding en kwantifisering van al dertien steroïede wat natuurlik geproduseer word in die bynier van die skaap; • die relatiewe bydrae van die CYP17 isoforme tot die totale steroïedale uitsette vanuit die bynier kortex selle, vanaf die byniere van H- en L-lyn skape, waar klem geplaas word op die produksie van kortisol. Die bynierselle van die H-lyn skape het aansienlik meer kortisol produseer as die L-lyn, terwyl die bynierselle van die homosigotiese WT1/WT1 skape ook meer kortisol produseer het as heterosigotiese WT1/WT2 skape; • die bloed kortisol in reaksie tot die stimulering van die hipotalamus-hipofise-adrenale aksis, deur insulien geïnduseerde hipoglisemiese stress, in skape van die H- en L-lyne met bekende CYP17 genotipes. Dit was gevind dat die kortisol reaksie geaffekteer word deur beide die CYP17 genotipe en seleksie lyn, waar L-lyn heterosigotiese WT1/WT2 skape die minste kortisol geproduseer het en die stadigste herstel van glukose vlakke getoon het; • die assosiasie tussen die CYP17 genotipe en die gedrags reaksies op trop-isolasie, sowel as ooi-reproduksie en lamuitset, van die H- en L-lyn skape. Die reproduksie parameters was onafhanklik van die CYP17 genotipe, terwyl ‘n sterk assosiasie gevind was tussen die CYP17 genotipe en gedrags reaksies op trop-isolasie. Die heterosigotiese WT1/WT2 skape het ‘n wilder natuur getoon gedurende trop-isolasie in vergelyking met homosigotiese WT1/WT1 skape.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: · Development of analytical tools to investigate the light struck flavour (LSF) in beer by Gas chromatography mass spectrometry (GCMS) and by liquid chromatography mass spectrometry/mass spectrometry (LCMS/MS). Development of a high performance liquid chromatography (HPLC) method to analyse carbohydrates in beer. · The efficiency a pilot scale ultraviolet (UV-C) system at 254 nm to inactivate spoilage microorganisms spiked in commercial beer. Bacteria test were Lactobacillus brevis, Acetobacter pasteurianus and Saccharomyces cerevisiae · A pilot scale UV treatment of commercial and non-commercial lager beers at UV dosage of 1000 J/L. Following the UV treatment, the correlation between chemical analyses and sensory tests conducted by consumers’ tasters were investigated. · A pilot scale UV treatment of non-commercial beer brewed with reduced hops iso-α-acids (tetrahydro-iso-α-acids) at UV dosage of 1000 J/L. Sensory changes and chemical properties were investigated. · The development and optimisation of an UV light emitting diodes (UV-LED) bench scale apparatus. Chemical and microbiological tests were conducted to investigate the effect of UV-LEDs on beer at 250 nm and 275 nm wavelengths. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: · Die ontwikkeling van analitiese toerusting om die invloed van lig op die smaakontwikkeling in bier te bestudeer m.b.v gaschromatografie massa spektrometrie (GCMS) en vloeistofchromatografie massa spektrometrie/massa spektrometrie, asook die ontwikkeling van ‘n hoë druk vloeistofchromatografiese metode vir die analise van koolhidrate in bier. · Die doeltreffendheid van ‘n toetsskaal ultraviolet (UV-C) sisteem om die nadelige mikroorganismes waarmee die bier geïnnokuleer was, by 254 nm te inaktiveer.. Toetse is uitgevoer met die volgende bakterieë, Lactobacillus brevis, Acetobacter pasteuriants en Saccharomyces cerevisiae. · ‘n Toetsskaal UV behandeling van kommersiële en nie-kommersiële lager biere by ‘n UV dosering van 1000 J/L. Na UV behandeling is die verwantskap tussen chemiese analises en ‘n reeks sensoriese toetse deur vebruikers proeërs ondersoek.. · ‘n Toetsskaal UV behandeling van ‘n nie-kommersiële bier gebrou met verlaagde hops-iso-α-sure (tetrahidro-iso-α -sure) by UV dosering van 1000 J/L. Sensoriese veranderinge asook chemiese eienskappe is ondersoek. · Die ontwikkeling en optimalisering van ‘n UV-lig emissie diodes bankskaal apparaat. Chemiese en mikrobiologiese toetse is uitgevoer om die effek van UV lig op bier by 250 nm en 275 nm te ondersoek.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NETA)), are used by millions of women as contraceptives and in hormone replacement therapy (HRT). Although both progestins are widely used, very little is known about their mechanism of action at the molecular level. In this thesis, the differential regulation of gene expression and molecular mechanism of action via different steroid receptors by these synthetic progestons, as compared to progesterone (Prog) was investigated in human cell lines. In the first part of the study, the effect of Prog, MPA and NET-A on the expression of endogenous cytokine genes was investigated in two epithelial cell lines of the human female genital tract, Ect1/E6E7 (an ectocervical cell line) and Vk2/E6E7 (a vaginal cell line). Quantitative realtime RT-PCR (QPCR) showed ligand-specific and cell-specific regulation of the interleukin (IL)-6, IL-8 and RANTES (Regulated-upon-Activation, Normal T cell Expressed and Secreted) genes with Prog, MPA and NET-A. Moreover, the repression of the TNF -induced RANTES gene by MPA in the Ect1/E6E7 cell line was found to be mediated by the androgen receptor (AR). The second part of the study focused on elucidating the androgenic activities of these two progestins, in comparison to Prog. Competitive binding in whole cells revealed that Prog, MPA and NET-A have a similar binding affinity for the hAR as the natural androgen dihydrotestosterone (DHT). Both transactivation and transrepression transcriptional assays demonstrate that, unlike Prog, MPA and NET-A are efficacious AR agonists, with activities comparable to DHT. Using a mammalian two-hydrid assay, it was shown that MPA and NET-A exert their androgenic actions by different mechanisms. NET-A, like DHT and other well-characterised androgens, induces the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C-interaction) of the AR independent of promoter-context, while MPA does this in a promoterdependent manner. In the third part of this study, competitive binding revealed that MPA and NET-A have a similar binding affinity to each other, but about a 100-fold lower affinity than Prog for the human mineralocorticoid receptor (hMR), while RU486 has an even lower affinity for the hMR. Promoter-reporter assays showed that MPA, NET-A and RU486 are all antagonists of the hMR, but unlike Prog, they have weak antagonistic activity. However, on the endogenous MR-regulated Orm-1 (a-glycolytic protein or orosomucoid-1) gene expressed in a rat cardiomyocyte cell line, NET-A and RU486, but not MPA, has similar antagonistic activity as Prog. This study is the first to show that, NET-A and RU486, but not MPA, can dissociate between transrepression and transactivation via the hMR. Taken together, these results show that natural Prog and the synthetic progestins, MPA and NET-A display differential promoter-, cell- and receptor-specific effects on gene expression. Furthermore they may have important implications for cervicovaginal immune function, cardiovascular and other physiological functions. / AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan (noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), word deur miljoene vroue gebruik as voorbehoedmiddels en vir hormoon vervangingsterapie (HVT). Tenspyte daarvan dat beide hierdie progestiene algemeen gebruik word, is min bekend oor hulle meganisme van werking op molekulêre vlak. In hierdie proefskrif word die differensiële regulering van geenuitdrukking asook die molekulêre meganisme van werking deur middel van steroïedreseptore van beide hierdie sintetiese progestiene, ondersoek, en vergelyk met progesteroon (Prog), in menslike sellyne. In die eerste deel van die studie is die effek van Prog, MPA en NET-A op die uitdrukking van endogene sitokinien gene ondersoek in twee epiteel sellyne van die menslike vroulike geslagskanaal, Ect1/E6E7 (‘n ektoservikale sellyn) en Vk2/E6E7 (‘n vaginale sellyn). Kwantitatiewe intydse RT-PKR het ligand-spesifieke en selspesifieke regulering van interleukien (IL)-6, IL-8 en RANTES (Regulering-na- Aktivering, Normale T-sel Uitgedrukte en Afgeskei) gene getoon met Prog, MPA en NET-A. Verder is gevind dat die onderdrukking van die TNF- - geïnduseerde RANTES geen deur MPA in die Ect1/E6E7 sellyn bemiddel word deur die androgeen reseptor (AR). Die tweede deel van die studie het gefokus op die toeligting van die androgeniese aktiwiteit van die twee progestiene in vergelyking met Prog. Kompeterende binding in volselle het getoon dat Prog, MPA en NET-A ‘n soortelyke bindings affiniteit vir die menslike AR as die natuurlike androgeen dehidrotestosteroon (DHT) vir die menslike AR het. Beide transaktiverings en transonderdrukkings transkripsionele analieses toon dat, anders as Prog, MPA en NET-A effektiewe AR agoniste is met aktiwiteite wat vergelykbaar is met die van DHT. Deur die gebruik van ‘n soogdier twee-hibried toets, kon gewys word dat MPA en NET-A hul androgeniese effekte uitoefen deur verskillende meganismes. NET-A, soos DHT en ander goed gekarakteriseerde androgene, induseer die ligand-afhanklike interaksie tussen die NH2- en COOH-terminale domeine (N/C-interaksie) van die AR, onafhanklik van die promoter-konteks. MPA, aan die ander kant, doen dit op ‘n promoter-afhanklike manier. In die derde deel van die studie het kompeterende binding getoon dat MPA en NETA soortelyke relatiewe bindings affiniteite vir die menslike mineralokortikoïed reseptor (hMR) het, maar dat hierdie affiniteit ongeveer 100-voud laer is as die van Prog en dat die affiniteit van RU486 vir hMR selfs nog laer is. Promoter-rapporteerder toetse het getoon dat MPA, NET-A en RU486 almal antagoniste van die hMR is, maar anders as Prog, is hierdie ‘n swak antagonistiese aktiwiteit. Nietemin, op die endogene MR-gereguleerde Orm-1 ( -glikolitiese proteïen of orosomukoïed-1) geen, uitgedruk in ‘n rot kardiomiosiet sellyn, het NET-A en RU486, maar nie MPA nie, ‘n soortgelyke antagonistiese aktiwiteit as Prog. Hierdie studie is die eerste om te wys dat NET-A en RU486, maar nie MPA nie, kan onderskei tussen transrepressie en transaktivering deur middel van die hMR. Samevattend toon die resultate dat natuurlike Prog en die sintetiese progestiene, MPA en NET-A, ‘n differentiële promoter-, sel- en reseptor-spesifieke effek op geenuitdrukking het. Verder mag die resultate belangrike implikasies vir servikovaginale immuunfunksie, asook kardiovaskulêre en ander fisiologiese funksies, inhou.

Acute Myeloid Leukemia (AML), the most common form of acute leukemia in adults, is only curable in approximately 30% of all cases. Despite prognostic risk stratification using sub-typing and cytogenetic analysis to direct therapy, the mortality and relapse rate remains high. AML patients with normal karyotypes are defined as intermediate risk and are the most challenging to treat. Somatic mutations may be the key in refining prognostic stratification and providing useful therapeutic targets. The FMS-like tyrosine kinase 3 (FLT3) and Nucleophosmin (NPM1) genes have common mutated forms that are associated with overall survival and response to therapy. We assessed mutations in the FLT3 and NPM1 genes and their levels of expression in twenty eight AML patients in the presence and absence of HIV and their response to induction therapy. Furthermore, we used a novel technique, High Resolution Melting (HRM) Analysis to detect FLT3 Internal Tandem Duplications (ITD) and NPM1 exon 12 mutations. Five of the patients in this study were HIV positive, three of whom did not survive post-induction therapy. Of the AML patients, 17.9% were positive for the NPM1 mutation and 21% had mutated FLT3. Interestingly, the presence of the FLT3 and NPM1 mutations were coupled with an increase in expression levels of FLT3 and NPM1 from presentation to post-induction respectively and the loss of these mutations were coupled with a decrease in levels of expression from presentation to post-induction. However, an increase/decrease from presentation to post-induction did not necessarily denote the presence/absence of a mutation. Therefore, while mutational status of genes may generally confer mRNA levels, our results showed that there existed no definitive trend between mRNA levels of NPM1 and FLT3 expression and mutational status. We found that the HRM method was definitive for the simpler NPM1 mutation however detection of the FLT3-ITD mutation was challenging. There isn’t a clear distinction between mutated and non-mutated FLT3 due to the formation of hetero-duplexes during analysis, making detection highly subjective and error-prone. Sequencing allowed confirmation of mutated FLT3 and non-mutated FLT3 which were not in all instances in concordance with HRM analysis. The prognostic value in terms of overall survival of NPM1 and FLT3 mutations in this study is indefinite. Furthermore, the analysis of the HIV positive AML patients revealed no clear correlation between NPM1 and FLT3 levels of mRNA expression and mutational status. Also, the small number of HIV positive AML patients did not allow for conclusions to be made regarding HIV status and survival when affected with AML. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

ESF1 is a 2.052 kDa antimicrobial peptide, mimicking the charge distribution and amphipathic alpha-helical structure of magainin, pGLa, a naturally occurring antimicrobial peptide. ESF1 has been reported to display high activity against Fusarium oxysporum f. sp lycopersici and F. oxysporum f. sp cubense race 4, the tomato and banana crop plant, wilt-causing pathogens, respectively. To assess whether this synthetic peptide can be heterologously expressed in yeast in significant quantities, and still retain full bioactivity, within a eukaryotic system, the ESF1 gene was designed and synthesized from five oligonucleotides, and cloned into pUC18. From the pUC18/ESF1 recombinant plasmid, the ESF1 gene sequence was amplified and cloned into the pBluescript-based vector, pVD4, downstream of the yeast pheromone mating factor alpha (MFa1) promoter, and in frame with the MFa1 signal peptide sequence for expression and secretion in yeast. The expression cassette comprising the MFa1 promoter and signal peptide sequence, and ESF1 gene was subsequently cloned into the yeast/ E. coli shuttle vector, pTG3828 and transformed into Saccharomyces cerevisiae. Chicken IgY antibodies against ESF1 peptide were raised and immunoaffinity purified. Following this, western dot blot analysis and mass spectrometry confirmed the presence of ESF1 in partial HPLC purified fractions of the recombinant yeast culture supernatant. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.