Thesis (MSc (Biochemistry))--University of Stellenbosch, 2007. / A detailed mathematical description of all the processes in a cell could be an informative tool for investigating biological function. Detailed kinetic models could be built either by obtaining enzyme kinetic parameters in vitro, or by obtaining them from time series analyses of metabolite data from rapid pulse experiments. A genome scale in vitro enzyme kinetic assay project would be prohibitively laborious with the current technologies. Further, there are still uncertainties about the importance of in vivo effects such as metabolite channelling, spatial effects and molecular crowding which could make in vitro determined parameters invalid. Accordingly, there is much interest in in vivo experiments for kinetic modelling. In vivo experimental methods suffer from a number of technical and even fundamental problems. Technical problems are being solved by more sensitive metabolomics tools and rapid sampling technologies. However, the large number of effectors of each enzyme reaction makes it impossible to obtain models at the level of detail possible with the in vitro method. Ultimately, the solution to building a genome scale Silicon Cell is to make use of both strategies. As metabolomics technologies are rapidly improving, it would thus make sense to follow the parts-based in vitro kinetics methodology, and carry out a detailed accuracy assessment of the model with in vivo experiments. To address the problem of the fundamental limit of information from concentration time-series, other in vivo experiments will have to be carried out as well. 13C-metabolic flux analysis has recently undergone vast improvements with the use of better experimental protocols and powerful algorithms for flux calculation. Incorporation of this type of experiment in the validation protocol is the aim of this thesis, which represents an intermediary step towards using the genome-scale stoichiometric models as platforms for building genome-scale kinetic models. It is illustrated here how kinetic models can be combined with metabolic flux data in a special way which allows correct modelling of boundary conditions and validation using novel concepts. We used 13C-metabolic flux analysis and gas chromatography-mass-spectrometry to measure metabolic fluxes through the central metabolic pathways of the yeast Saccharomyces cerevisiae. This data was integrated with a previously constructed detailed kinetic model of fermentative glycolysis in the yeast to illustrate our approach. Various implications for such data integration with kinetic models were identified and a software program was designed for this purpose.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Presently, the observed variation in symptoms of Potato leafroll virus (PLRV) infection in potato cultivars in South Africa cannot be reconciled with PLRV symptoms obtained 10-15 years ago, even if the different interactions between the pathogen and the cultivar are taken into account. In an effort to analyze this variation, mutations in the coat protein (CP) gene of South African isolates of PLRV were assessed. The CP gene of PLRV isolates from different areas within South Africa was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Significant sequence variation in the CP gene was found within the analyzed South African isolates of PLRV. Phylogenetic analysis revealed two major clades with most South African isolates and an Australian and North American isolate grouped together and the remainder grouped with isolates from diverse countries worldwide. The deduced amino acid sequences from representatives of these two clades indicated differences in CP threedimensional structure. In an effort to produce recombinant PLRV CP for the production of antibodies specific for South African isolates of PLRV for use in enzyme-linked immunosorbent assay (ELISA), the CP gene of a South African isolate of PLRV was subcloned into a bacterial expression vector (pET14-b). Expression of full length recombinant PLRV CP was attempted in Escherichia coli strains BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS and Rosetta-2(DE3)pLysS. As this was not successful, the PLRV CP gene was subcloned in another expression vector (pGEX) for expression as an N-terminal fusion protein with glutathione-S-transferase (GST) in E. coli strains BL21(DE3)pLysS and Rosetta-2(DE3)pLysS. The recombinant GST-PLRV CP fusion protein was purified and used for antibody production in rabbits. Using western blots, the effectiveness of antibodies produced to recombinant GST-PLRV CP fusion protein was assessed for PLRV recognition. It was found that antibodies to the recombinant GST-PLRV CP fusion protein were more effective for the detection of GST than PLRV CP and that production of antibodies to the cleaved PLRV CP product would be necessary if antibodies are required for ELISA applications. / AFRIKAANSE OPSOMMING: Huidiglik kan die waargeneemde simptome van infeksie met aartappelrolbladvirus (Potato leafroll virus, PLRV) in aartappelkultivars in Suid-Afrika nie vereenselwig word met PLRV simptome wat 10-15 jaar gelede verkry was nie, selfs al word die verskillende interaksies tussen die patogeen en kultivar in ag geneem. In ‘n poging om hierdie variasie te analiseer, was mutasies in die mantelproteïen (CP) geen van Suid-Afrikaanse isolate van PLRV bepaal. Die CP geen van PLRV isolate van verskillende areas in Suid-Afrika was ge-amplifiseer met behulp van die tru transkripsie-polimerase ketting reaksie (RT-PCR), gekloneer en die nukleotiedvolgorde bepaal. Noemenswaardige nukleotied variasie is in die CP gene van die ge-analiseerde Suid-Afrikaanse isolate van PLRV gevind. Filogenetiese analises het gedui op twee hoof klades met die meeste van die Suid-Afrikaanse isolate wat saam met ‘n Australiese en Noord-Amerikaanse isolaat gegroepeer en die res wat met isolate van verskillende lande wêreldwyd gegroepeer. Die afgeleide aminosuurvolgordes van verteenwoordigers van bogenoemde twee klades het gedui op verskille in die CP driedimensionele struktuur. In ‘n poging om rekombinante PLRV CP te produseer vir die produksie van antiliggame spesifiek teen Suid-Afrikaanse isolate van PLRV om in “enzyme-linked immunosorbent assay” (ELISA) te gebruik, was die CP geen van ‘n Suid-Afrikaanse isolaat van PLRV gesubkloneer in ‘n bakteriële ekspressie vektor (pET14-b). Daar was gepoog om vollengte rekombinante PLRV CP in die Escherichia coli rasse BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS en Rosetta- 2(DE3)pLysS te produseer. Aangesien dit nie suksesvol was nie, was die PLRV CP gesubkloneer in ‘n ander ekspressie vektor (pGEX) sodat die proteïen as ‘n N-terminale fusie proteïen met “glutathione-S-transferase” (GST) in E. coli rasse BL21(DE3)pLysS en Rosetta- 2(DE3)pLysS geproduseer kon word. Die rekombinante GST-PLRV CP fusie proteïen was gesuiwer en gebruik vir antiliggaam produksie in konyne. Die effektiwiteit van die antiliggame wat teen rekombinante GST-PLRV CP fusie proteïen geproduseer was vir PLRV herkenning is deur middel van “western blots” geanaliseer. Dit was gevind dat antiliggame teen die rekombinante GST-PLRV CP fusie proteïen meer effektief was vir die herkenning van GST as PLRV CP. Gevolglik sal dit nodig wees om antiliggame teen die gesnyde PLRV CP produk te maak vir gebruik in ELISA.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2004. / This study describes: 1. Investigation of product inhibition regarding the metabolism of progesterone in ovine adrenal micosomes. 2. The employment of novel cell culture techniques to study the effect of CYP17 and CYP21 concentration on adrenal progesterone metabolism. 3. The formulation of a mathematical model describing the behaviour of the observed results in point 2.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2006. / The phytoestrogenic activity of Cyclopia, used to prepare honeybush tea, was evaluated and compared with that of the endogenous estrogen, 17-β-estradiol (E2) and the known phytoestrogen, genistein. Phytoestrogens are plant polyphenols much in demand in the nutraceutical market as they mediate an estrogenic effect through binding to estrogen receptor (ER) subtypes, ERα and ERβ.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This study: 1. describes the preparation of a methanol extract of Sutherlandia frutescens and the HPLC fractionation of the methanol extract. 2. investigates the influence of S. frutescens on the binding properties of mitochondrial cytochrome 11 -hydroxylase (CYP11B1) to deoxycorticosterone (DOC) and deoxycortisol, demonstrating that methanol extracts of S. frutescens inhibit the Type I substrate-induced difference spectra. 3. investigates the influence of S. frutescens on the catalytic activity of CYP11B1 expressed in COS1 cells, demonstrating that the methanol extract of S. frutescens inhibits the conversion of DOC and deoxycortisol. 4. describes the sequential extraction of the methanol extract of S. frutescens using organic solvents and the inhibition of the conversion of DOC by CYP11B1 expressed in COS1 cells in the presence of these extracts. 5. describes the inhibition of the binding of DOC to CYP11B1 in ovine adrenal mitochondria, and the conversion of DOC by CYP11B1 expressed in COS1 cells by these fractions. 6. identifies the presence of the flavonoid compounds, orientin vitexin and rutin, in S. frutescens. 7. investigates the influence of the flavonoid compounds on the binding of DOC to CYP11B1 and on the catalytic activity of DOC by CYP11B1 expressed in COS1 cells. 8. identifies the presence of the triterpenoid, sutherlandioside A (SU1), in S. frutescens extracts and investigates its effect on the binding of DOC to CYP11B1. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. die voorbereiding van ‘n metanol ekstraksie van Sutherlandia frutescens en die HPLC fraksionering van die metanol ekstrakte. 2. ‘n ondersoek na die invloed van S. frutescens op die bindingseienskappe van sitochroom P450 11 -hidroksilase (CYP11B1) in skaap bynier mitochondria en demonstreer dat S. frutescens metanol ekstrakte die vorming van steroïed-geinduseerde tipe I verskil spektra van deoksiekortisol en deoksikortikosteroon (DOC) inhibeer. 3. ‘n ondersoek na die invloed van S. frutescens op die katalitiese aktiwiteit van CYP11B1 in COS1 selle en demonstreer die inhibisie van DOC en deoksikortisol omsetting na hul produkte deur die methanol ekstrakte. 4. die opeenvolgende ekstraksie van methanol extrakte van S. frutescens met organiese oplosmiddels en beskryf die inhibisie van die CYP11B1 gekataliseerde omsetting van DOC in COS1selle in die teenwoordigheid van die ekstrakte. 5. die inhibeerende effek op die binding van DOC aan CYP11B1 in skaap bynier mitochondria en die inhibisie van die CYP11B1 gekataliseerde omsetting van DOC in COS1selle. 6. die identifisering van flavonoïed verbindings, orientin vitexin en rutin in S. frutescens. 7. ‘n ondersoek na die invloed van die flavonoïed verbindings op die binding van DOC aan CYP11B1 en op die katalitiese aktiwiteit van CYP11B1 in COS1 selle. 8. die indentifisering van die triterpenoïed, sutherlandiosied A (SU1), in S. frutescens en ondersoek die invloed van SU1 op die binding van DOC aan CYP11B1.

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / This study describes: 1. The preparation of chloroform, methanol and aqueous extracts of unfermented and fermented rooibos (Aspalathus linearis). 2. The chromatographic fractionation of aqueous rooibos extracts and an investigation into the polyphenol content and antioxidant activity of the fractions. 3. The preparation of ovine adrenal microsomes containing active steroidogenic P450 enzymes, including cytochrome P450 17a-hydroxylase, CYP17, and cytochrome P450 steroid 21-hydroxylase, CYP21. 4. An investigation into the influence of chloroform and methanol extracts of rooibos on the binding of steroid substrates, progesterone and 17-hydroxyprogesterone, to CYP17 and CYP21.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs on immune function. The effect of the immune system on GC action, especially in the periphery, is not as well studied. The liver plays an important role in inflammation and stress in producing the acute phase proteins (APPs) required for the resolution of inflammation as well as in producing systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding effects of stress and inflammation and their interplay in the liver is thus not only useful to expand our understanding of these systems but could also have clinical applications in understanding the side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be developed as an anti-inflammatory drug that displays fewer side effects. The current study investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects at the protein level, using Western blotting and ELISA assays, the protein activity level, using enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs on immune function. The effect of the immune system on GC action, especially in the periphery, is not as well studied. The liver plays an important role in inflammation and stress in producing the acute phase proteins (APPs) required for the resolution of inflammation as well as in producing systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding effects of stress and inflammation and their interplay in the liver is thus not only useful to expand our understanding of these systems but could also have clinical applications in understanding the side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be developed as an anti-inflammatory drug that displays fewer side effects. The current study investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects at the protein level, using Western blotting and ELISA assays, the protein activity level, using enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes / AFRIKAANSE OPSOMMING: Kruiskommunikasie bestaan tussen die stres– en die immuunsisteem en hierdie kruiskommunikasie is van farmakologiese belang vir die gebruik van glukokortikoïede (GKe) as anti-inflammatoriese medikasie vir siektes soos asma en artritis. Tot dusver was die fokus van studies oor hierdie kruiskommunikasie hoofsaaklik op die effek van GKe op immuunfunksie. Die effek van die immuunsisteem op GK werking, veral in die periferie, is nie so goed bestudeer nie. Die lewer speel ʼn belangrike rol in inflammasie en stres deurdat dit die akute fase proteïene (AFPs) produseer wat benodig word vir die resolusie van inflammasie en omdat dit ook sistemiese glukose produseer, d.m.v. glukoneogenese, wat benodig word om die stres reaksie te dryf. ’n Beter insig in die effek van stres en inflammasie sowel as hul interaksie in die lewer is dus handig, nie net om ons begrip van hierdie sisteme te verbeter nie, maar ook omdat dit kliniese toepassing kan hê deurdat dit ons begrip van die newe-effekte wat gepaard gaan met die farmakologiese gebruik van GKe verbeter. Verbinding A (CpdA) is geïdentifiseer as ʼn selektiewe glukokortikoïed reseptor (GR) moderator (SERGM) omdat dit die vermoë het om gene te onderdruk maar nie te aktiveer d.m.v. die GR. Hierdie eienskap dui op die potensiaal van CpdA om ontwikkel te word as ʼn anti-inflammatoriese middel met minder newe-effekte. Die huidige studie het die effekte van dexamethasone, ʼn sterk GR agonis, en CpdA, beide in die teenwoordigheid en afwesigheid van interleukin 6 (IL6), op die GR, drie metaboliese ensiem gene wat betrokke is by glukoneogenese, sowel as drie APP gene, ondersoek en vergelyk. Die metaboliese ensiem gene wat ondersoek is, is tirosien aminotransferase (TAT), fosfoenolpirovaat karboksikinase (PEPCK), en gamma glutamieltransferase (GGT), terwyl die APP gene serum amiloïede A (SAA), C-reaktiewe proteïen (CRP), en kortikosteroïed bindings globien (CBG) was. Die studie het die effekte in ʼn muis hepatoma sellyn (BWTG3) op die proteïen vlak, deur van Western blotting en ELISA essays gebruik te maak, die proteïen aktiwiteits vlak, deur van ensiem aktiwiteits essays en vol-sel binding gebruik te maak, sowel as op die mRNA vlak, deur van kwantitatiewe polimerase ketting reaksie (qPCR) gebruik te maak, ondersoek. Die studie toon dat dexamethasone (Dex) en IL6 in die algemeen divergente effekte het op die GR en metaboliese ensieme deurdat Dex GR af-reguleer en die metaboliese ensieme op-reguleer, terwyl IL6 die GR op-reguleer en die metaboliese ensieme af-reguleer, en dat hulle funksies konvergerend is vir die APPs deurdat beide positiewe APPs opreguleer en negatiewe APPs afreguleer. In teenstelling met Dex het CpdA die GR op-gereguleer en die metaboliese ensieme af-gereguleer terwyl dit, soos Dex, die positiewe APPs op-gereguleer en die negatiewe APPs af-gereguleer het. Ons resultate vir Dex en IL6 word ondersteun deur vorige werk in die literatuur. Ons studie is wel uniek omdat dit die ondersoek van drie metaboliese ensieme kombineer met die ondersoek van drie APPs, sowel as GR vlakke in ʼn enkele sisteem onder dieselfde eksperimentele kondisies. Verder het ons resultate met CpdA verskeie nuwe aspekte, soos die af-regulering van metaboliese gene, opgelewer wat bydra tot die groeiende poel van kennis oor hierdie ongewone GR ligand en die moontlike farmakologiese gebruik daarvan.

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study: I. Investigates the influence of S. frutescens on the binding properties of cytochrome P450-dependent enzymes in ovine adrenocortical mitochondria and microsomes, demonstrating that S. frutescens extracts elicit difference spectra and inhibit the Type 1 difference spectra induced by natural steroids. II. Indicates inhibition by S. frutescens extracts of the catalytic activity of cytochrome P450-dependent-17a-hydroylase and cytochrome P450-dependentsteroid- 21-hydroxylase enzymes in ovine adrenocortical microsomes. III. Describes an assay determining the inhibitory effects of S. frutescens, in COS L cells, on individual cytochrome P450-dependent enzymes - ovine, baboon and human cytochrome P450-dependent- L7a-hydroylase, and bovine cytochrome P450-dependent-21-hydroxylase enzymes. IV. Demonstrates that the inhibition of elevated plasma glucocorticoid levels in rats exposed to chronic immobilization stress could possibly be attributed to the influence of hydrophilic and hydrophobic compounds in S. frutescens on cytochromes P450-depndent enzymes. / AFRIKAANSE OPSOMMING: Hierdie studie: I. Ondersoek die invloed van S. frutescens op die bindingseienskappe van sitochroom P450-afhanklike ensieme in skaapbynier mitokondriale en - mikrosomale preparate en toon aan dat komponente in S. frutescens ekstrakte verskil spektra induseer en tipe I verskil spektra van natuurlike steroïede inhibeer. II. Dui die inhiberende effek van S. frutescens ekstrakte op die katalitiese aktiwiteit van sitochroom P450-afhanklike-17a-hidroksilase en sitochroom P450- afhanklike-steroïed- 21-hidroksilase ensieme in skaapbyniermikrosome aan. III. Beskryf 'n tegniek om die inhiberende effek van S. frutescens op individuele sitochroom P450-afhankilike ensieme - bobbejaan, skaap en mens sitochroom P450-afhanklike-17a-hidroksilase, en bees sitochroom P450-afhanklike-steroïed- 21-hidroksilase - in COS 1 selle te bepaal. IV. Demonstreer dat inhibisie van verhoogde glukokortikoïed plasma konsentrasies waargeneem in rotte blootgestel aan kroniese immobiliserende stress, moontlik toegeskryf kan word aan die effek van S. frutescens op die sitochroom P450- afhanklike ensieme.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: This study describes: 1. The isolation of total RNA and mRNA from Angora goat adrenals. 2. Synthesis and nucleotide sequence alignment of Angora goat CYPI7 cDNA. Two DNA sequences were produced, identifying two CVP 17 alleles in an Angora goat from the Swartland district. 3. The development of a CYPI7 genotype test for Angora goats. 4. Genotyping of Angora goats and Boer goats with the developed genotype test. S. Mapping of the substituted amino acids in the amino terminal of CVP 17 to a specific CYPI7 genotype. 6. Partial synthesis and alignment of Angora goat genomic nucleotide CYPI7 sequences. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. Die isolering van totale RNA en mRNA van Angorabok byniere. 2. Sintese en nukleotied volgorde oplyning van Angorabok CYP17 eDNA. Twee DNA volgordes is geproduseer, en so is twee CYP17 allele in 'n Angorabok van die Swartland omgewing geïdentifiseer. 3. Die ontwikkeling van 'n CYP17 genotipe toets vir Angorabokke. 4. Genotipering van Angorabokke en Boerbokke met die ontwikkelde genotipe toets. 5. Korrelering van die omgeruilde aminosure in die aminoterminaal van CYPl7 met 'n spesifieke genotipe. 6. Gedeeltelike sintese en oplyning van Angorabok genomiese CYPl7 nukleotied volgordes.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) the optimization of the current industrial-scale extraction and purification of Horseradish peroxidase from horseradish at BBI Enzymes, focussing on: a. Raw material quality, b. Extraction, c. Ultra-filtration, d. Salt fractionation, e. Diafiltration, f. Ion Exchange Chromatography, b) developing an new in-process microtitre plate calorimetric assay, c) characterization of main groups of HRP relevant to BBI Enzymes by SDS-PAGE- and HPLC analysis. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: a) die optimisering van die huidige industriële-skaal ekstraksie en suiwering van peperwortelperoksidase vanuit peperwortel by BBI Enzymes, deur te focus op: a. Rou material kwaliteit, b. Ekstraksie, c. Ultra-filtrasie, d. Sout fraksionering, e. Diafiltrasie, f. Ioon-uitruilchromatografie b) Ontwikkeling van ‘n nuwe in-proses mikro-titer gebaseerde kalorimetriese toetsmetode c) die karakterisering van die hoof groepe peperwortel-peroksidase belangrik vir BBI Enzymes.