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, 2002. / ENGLISH ABSTRACT: The aim of this thesis was to define the interactions of the androgen receptor (AR) with an analog of a non-steroidal plant compound, Compound A (CpdA), as well as two synthetic progestins, medroxyprogesterone acetate (MPA) and norethindrone acetate (NET-A). The data presented indicates that CpdA has antiandrogenic properties, as it represses androgen-induced activation of both specific and non-specific androgen-responsive reporter constructs. It was found that CpdA exerts these effects by a mechanism other than competition with androgen for binding to the ligand-binding domain (LBD) of the receptor. On the other hand, it is demonstrated that both MPA and NET-A compete with androgen for binding to the AR and induce partial agonist activity via the receptor. Using mammalian two-hybrid assays it was revealed that CpdA, similar to anti-androgenic compounds that are able to compete with androgens for binding to the receptor, represses the androgen-induced interaction between the NH2- and COOH-terminals of the AR (N/C-interaction) without competing for binding to the LBD. Furthermore, it was shown that CpdA slightly represses the androgen-dependent recruitment of steroid receptor co-activator 1 (SRC1) to the activation function (AF2) domain of the AR. When the effects of MPA and NET-A on the N/C-interaction were studied, intriguing results were obtained. NET-A, as expected, induced this AR agonist-induced interaction. MPA, however, repressed this AR agonist-induced interaction, an effect previously associated with anti-androgenic activity, despite displaying partial agonist activity in transctivation experiments. On the other hand, both MPA and NET-A induced the interaction between SRC1 and the AF2 domain. In additional experiments with CpdA, it was found that CpdA did not affect the recruitment of SRC1 to the AF1 domain of the receptor; neither did it influence the constitutive activity of the NH2-terminal domain. The anti-androgenic activities of CpdA were confirmed by the toxic effect that this compound had on the androgen-dependent lymph node carcinoma of the prostate (LNCaP) cell-line as well as its ability to repress the androgen-induced expression of the prostate specific antigen (PSA) protein. Taken together, the results presented in this thesis, in combination with the knowledge available on AR function, contribute to an improved understanding of AR function. Furthermore, the importance of defining the precise mechanism by which individual compounds exert their effects is highlighted. In this regard it is demonstrated that two compounds (MPA and NET-A) that display partial agonist activity, can exert their effects via different mechanisms at the molecular level. Detecting such differences in the molecular mechanisms of action could facilitate the improved design of progestins as well as aid clinicians and their patients in selecting the best method of contraception. Lastly, the insights gained into the mechanisms of the anti-androgenic action of CpdA could be useful in therapeutic drug design for diseases, such as prostate cancer, that have an androgen-dependent etiology. / AFRIKAANSE OPSOMMING: Die doel van hierdie tesis was om die interaksies van die androgeen reseptor (AR) met ‘n analoog van ‘n nie-steroiediese plant verbinding, Verbinding A (VbgA), sowel as met twee sintetiese progestogene, medroksiprogesteroon asetaat (MPA) en noretiendroon asetaat (NET-A), te definieer. Die data verskaf dui daarop dat VbgA anti-androgeniese eienskappe besit deurdat dit androgeen-gei'nduseerde aktivering van beide spesifieke- en nie-spesifieke androgeen-responsiewe rapporteerderkonstrukte onderdruk. VbgA veroorsaak hierdie effekte deur ‘n meganisme wat nie kompetisie met androgeen vir binding aan die ligand-bindingsdomein (LBD) van die reseptor behels nie. In teenstelling hiermee word getoon dat beide MPA en NET-A kompeteer met androgeen vir binding aan die AR en gedeeltelike agonistiese aktiwiteit induseer via hierdie reseptor. Deur gebruik to maak van ‘n soogdier twee-hibried essai word getoon dat VbgA, soos ander anti-androgeniese verbindings wat kompeteer met androgeen vir binding aan die reseptor, die androgeen-gei'nduseerde interaksies tussen die NH2- en COOH-terminale van die AR (N/C-interaksie) onderdruk, sonder om te kompeteer vir binding aan die LBD. Daarby is dit bewys dat VbgA die androgeenafhanklike werwing van steroied reseptor ko-aktiveerde 1 (SRC1) na die aktiverings funksie (AF2) domein van die AR gedeeltelik onderdruk. Die studie van die effekte van MPA en NET-A op die N/C-interaksie het interessante resultate opgelewer. NETA, soos verwag, het hierdie AR agonis-gei'nduseerde interaksie geinduseer. MPA, aan die ander kant, het hierdie AR agonis-gei'nduseerde interaksie onderdruk, ‘n effek wat tevore met anti-androgeniese aktiwiteit geassosieer is, al het die transaktiveringseksperimente daarop gedui dat MPA ‘n AR agonis is. Aan die ander kant, het beide MPA en NET-A die interaksie tussen SRC1 en die AF2 domein geinduseer. In addisionele eksperimente met VbgA is gevind dat VbgA geen effek het op die werwing van SRC1 na die AF1 domein van die reseptor nie en ook geen invloed het op die konstitutiewe aktiwiteit van die NHh-terminaal domein nie. VbgA se antiandrogeniese eienskappe is bevestig deur die toksiese effekte op die androgeenafhanklike limfknoop karsinoom van die prostaat (LNCaP) sellyn sowel as deur sy vermoe om die androgen-gei'nduseerde uitdrukking van die prostaat spesifieke antigeen (PSA) protei'en te onderdruk. Die resultate aangebied in hierdie tesis, in kombinasie met die beskikbare kennis oor AR funksie, dra by tot ‘n verbeterde kennis van AR funksionering. Verder word die belang van die definiering van die meganisme waardeur individuele verbindings hulle effekte veroorsaak, getoon. In hierdie verband is getoon dat twee verbindings (MPA en NET-A), wat gedeeltelike agonistiese aktiwiteit besit, hulle effekte via verskillende meganismes op die molekulere vlak veroorsaak. Deur hierdie verskille in die molekulere meganismes van aksie uit te wys, kan beter progestogene ontwikkel word, en verder sal dit vir dokters en hul pasiente help om die beste voorbehoedmiddel te kies. Laastens, die insig wat verkry is ten opsigte van die meganismes van anti-androgeniese aktiwiteit van VbgA mag nuttig wees in die ontwerp van terapeutiese middels vir die behandeling van siektetoestande met androgeen-afhanklikke etiologie (bv. prostaatkanker).

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, 2003. / ENGLISH ABSTRACT: Scientists and biochemical engineers alike are very interested in the control and regulation of free-energy metabolism in micro-organisms, whether the findings purely satisfy scientific curiosity or translate into the meeting of biotechnology company deadlines. We used a rather fundamental approach to investigate experimentally the control and regulation of yeast free-energy metabolism in anaerobic chemostat cultures using supply-demand analysis. This conceptually simple, quantitative framework, however, may lead to insight into the control properties of various metabolic pathways to be used in biotechnological applications. Supply-demand analysis is based on the theoretical framework of metabolic control analysis (MCA).Sections (of arbitrary size) of a metabolic pathway are grouped together around a linking metabolite. Those steps that produce the intermediate are combined into the supply block while the reactions that remove/consume the intermediate are grouped together as the demand. The elasticity coefficients of the supply and demand blocks (with regard to the linking metabolite concentration) can be used to determine the flux and concentration control coefficients by using the traditional MCAsummation and connectivity theorems. Supply and demand rate characteristics are a powerful visual approach to determine and display the control structure of the pathway under consideration and sets supply-demand analysis apart from traditional top-down analysis. Our first tool of analysis was a structured kinetic model of yeast growing in a chemos tat, constructed by using methods developed in our research group for modelling systems with variable volumes. Independent perturbations of the linking metabolite concentration resulted in a control profile where the control resided mainly in the demand (flux control coefficient of 0.92), as a result of a large negative supply elasticity. This elasticity, however, varied greatly under different conditions, leading to increased flux control by the supply in some cases. We extended our research to an experimental setup of Saccharomyces cerevisiae growing in a glucose-limited chemos tat supplemented with yeast extract as a source of carbon intermediates. This allowed glucose to act solely as the freeenergy source, as confirmed by balancing the glucose flux with the fluxes towards the fermentation products, ethanol and carbon dioxide. We obtained the supply rate characteristic by perturbing the ATPdemand through the addition of benzoate, which uncouples the proton gradient across the cell membrane. The demand rate characteristic was obtained by perturbing the ATP supply through changes in the dilution rate and thus the residual glucose concentration in the fermentor. The concentrations of ATPand ADPwere measured using a luciferase bioluminescence assay, while the fermentation products were measured with HPLCand C02 with an acoustic off-gas analyser. For our experimental conditions the flux-control of energy metabolism resided predominantly in the supply with respect to the linking metabolite [ATP]/[ADP](chosen as an indication of the free-energy state of the cell), i.e. a flux control coefficient of 0.90. Further, the [ATP]/[ADP]was under strong homeostatic control, as evidenced by the low [ATP]/[ADP]control coefficients of ± 0.12. We adjusted the structured kinetic model by varying strategic parameters, so that the results resembled the experimental observations more closely. However, the kinetics of our core model seem to be too simplistic to capture fully the extent of regulation displayed by the experimental system. The model did, however, reveal the regulatory importance of glucose transport into the cell. We conclude that the control and regulation of free energy metabolism in yeast strongly depend on the culturing conditions and on the steady state being analysed. / AFRIKAANSE OPSOMMING: Wetenskaplikes sowel as biochemiese ingenieurs is dikwels geïnteresseerd in die beheer en regulering van vry-energie metabolisme in mikro-organismes, hetsy die bevindinge suiwer wetenskaplike nuuskierigheid bevredig of die haalbaarheid van biotegnologie-maatskappy-mikpunte beteken. Ons het 'n redelik fundamentele benadering gevolg om die beheer en regulering van vry-energie metabolisme in gis eksperimenteel te bepaal in anaerobiese chemostaatkulture met behulp van aanbod- aanvraag analise. Dit is 'n konseptueel eenvoudige, kwantitatiewe raamwerk met die potensiaal om insig te gee in die beheereienskappe van verskeie metaboliese paaie wat nuttig kan wees in biotegnologiese toepassings. Aanbod-aanvraag analise is gebaseer op die teoretiese onderbou van metaboliese kontrole-analise (MKA).Dele (van arbitrêre grootte) van 'n metaboliese pad word gegroepeer rondom 'n verbindingsmetaboliet. Die stappe wat die intermediaat produseer word gekombineer as die aanbod terwyl die reaksies wat die intermediaat verbruik, saamgegroepeer word as die aanvraag. Die elastisiteitskoëffisiënte van die aanbod en aanvraag blokke (met betrekking tot die verbindingsmetabolietkonsentrasie) kan gebruik word om die fluksie en konsentrasie kontrolekoëffisiënte te bereken met behulp van die sommasie en konnektwiteit teoremas van MKA.Aanbod en aanvraag snelheidskenmerkgrafieke is 'n treffende visuele benadering om die kontroleprofiel van die betrokke metaboliese pad te bepaal en te vertoon. Hierdie kenmerk onderskei aanbod-aanvraag analise van bo-na-onder analise. Die eerste deel van ons ondersoek het behels 'n gestruktureerde kinetiese model (van gis wat groei in 'n chemostaat) met behulp van metodes wat in ons groep ontwikkel is om sisteme met variërende volumes te modelleer. Onafhanklike perturbasies van die verbindingsmetaboliet konsentrasie het gelei tot 'n kontroleprofiel waar die kontrole hoofsaaklik in die aanvraag gesetel was (fluksie kontrolekoëffisiënt van 0.92), as gevolg van 'n groot negatiewe aanbod-elastisiteit. Hierdie elastisiteit kan egter grootliks varieer tydens verskillende kondisies, wat lei tot 'n toenemende fluksle-beheer deur die aanbod in sommige gevalle. Ons het ons navorsing uitgebrei na 'n eksperimentele opstelling van Saccharomyces cerevisiae wat groei in 'n glukose-gelimiteerde chemostaat, aangevul met gisekstrak as 'n bron van koolstof-Intermediate. Dit bring mee dat glukose slegs as energiebron dien; dit is wel bevestig deur balanse op te stel van die koolstoffluksie vanaf glukose na koolstofdioksied en etanol as die fermentasieprodukte. Die aanbod snelheidskenmerkgrafiek is gegenereer deur die aanvraag van ATP te manipuleer deur middel van toevoeging van bensoaat, wat die protongradiënt oor die selmembraan ontkoppel. Die snelheidskenmerkgrafiek Vir die aanvraag is gegenereer deur die aanbod van ATP te manipuleer deur middel van 'n variasie in die verdunningstempo en sodoende die residuele glukose konsentrasie in die fermentor. Die konsentrasies van ATPen ADPis bepaal deur middel van 'n lusiferase bioluminessensie-essai, terwyl die fermentasieprodukte met 'n HPLCen CO2 met 'n akoestiese aflaatgasanaliseerder gemeet is. Vir die betrokke eksperimentele toestande was die flukste-kontrole van energiemetabolisme oorwegend in die aanbod met betrekking tot die verbindingsmetaboliet, [ATP]/[ADP](gekies as aanduiding van die vrye-energiestatus van die sel), naamlik 'n fluksie kontrolekoëffisiënt van 0.90. Verder was die [ATP]/[ADP]onder sterk homeostatiese beheer soos duidelik blyk uit die lae [ATP]j[ADP] kontrolekoëffisiënte van ± 0.12. Ons het die gestruktureerde kinetiese model aangepas deur strategiese parameters te verander om sodoende die eksperimentele gedrag te probeer naboots. Die kinetika van ons kernmodel blyk egter te simplisties te wees om die volle omvang van die regulering van die eksperimentele sisteem te vertoon. Die model het egter die belang van glukose transport oor die selmembraan aan die lig gebring. Ons kom tot die gevolgtrekking dat die beheer en regulering van vrye-energie metabolisme in gis sterk afhang van die groeitoestande sowel as die spesifieke bestendige toestand wat ondersoek word.

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.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) Characterization of traditional methodologies and testing methods used to purify and quantify trypsin and α-chymotrypsin b) Re-engineering / development of a new method for purifying trypsin and α-chymotrypsin that delivered higher product yields and improved control exercised over the process by investigating: i. Extraction methods ii. Centrifugation iii. Ultrafiltration iv. Chymotrypsinogen and trypsin crystallization v. Column chromatography vi. Investigation into different raw material sources for pancreatic enzyme production c) Development of kinetic and ELISA testing methodologies for in-process QC analysis. / AFRIKAANSE OPSOMMING: Hierdie Studie beskryf: a) Karakterisering van die ou prosessering metodes en toets metodes wat gebruik word om Tripsien en Alpha-chimotripsien te suiwer en te kwantifiseer. b) Herontwerp / ontwikkeling van 'n nuwe metode vir die suiwering Tripsien en Chimotripsien wat „n hoër opbrengs lewer en meer kontrole oor die proses uit oefen deur ondersoek in te stel na: i. Ekstraksie- metodes ii. Sentrifugering iii. Ultrafiltrasie iv. Chymotripsienogeen - en tripsien kristallisasie v. Kolom chromatografie vi. Ondersoek na verskillende rou materiaal bronne vir die produksie van pankreas ensieme. c) Die ontwikkeling van kinetiese- en ELISA toets metodes vir die in-proses kwaliteitkontrole.

Thesis (MSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Membrane bound cytochrome b5 is a ubiquitous protein with an average molecular weight of 16 kDa. The protein is involved in a number of reactions providing electrons directly to cytochrome P450 enzymes or to other enzymes involved in lipid biosynthesis. It is also known that the protein influences the activities of certain enzymes via an allosteric effect. It has been accepted in the literature that the cytochrome b5 exists primarily in the monomeric form, however, recently it has been shown that it forms homomeric complexes in vivo. In this study, we investigate the cytochrome b5 complex formation using a variety of analytical tools. Cytochrome b5 was isolated from ovine liver microsomes and the purity verified using sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrospray ionisation mass spectrometry. The latter analysis confirmed the presence of a single heme containing protein with Mr=15865 Da, while separation on the polyacrylamide gel revealed oligomeric complex formation with the tetrameric form the most prominent oligomer. Using different and particularly harsh denaturing conditions we found that the observed oligomeric aggregates persisted, indicating highly stable complexes. The most prominent tetrameric aggregate was identified to be cytochrome b5 by mass spectrometric sequencing. Further complex formation studies, using a fluorescent dye (1-anilinonaphthalene-8-sulfonic acid) that interact with hydrophobic cavities formed during oligomerisation, provided evidence of protein assembly in oligomeric complexes or aggregation. The formation of the cytochrome b5 complexes was dependent on ionic strength and protein concentration. Previously it was shown that the hydrophobic membrane anchoring domain plays a pivotal role in the cytochrome b5’s homomeric complexes. Using a peptide (IITTIDSNSS), resembling a portion of this domain, together with circular dichroism we showed more organized structure present for the wildtype peptide vs. a mutated control peptide (LLSSLKAVAV). A modified ELISA interaction assay also revealed that the wild-type peptide had a specific interaction with cytochrome b5, providing further evidence that the membrane anchoring domain plays a role in complex formation. These studies also indicated that a hydrogen bond network in this domain may be important for the formation of the homomeric complexes of cytochrome b5. / AFRIKAANSE OPSOMMING: Membraan-gebonde sitochroom b5 is ’n alomteenwoordige proteïen met ’n gemiddelde molekulêre massa van 16 kDa. Die proteïen is betrokke in reaksies waar dit elektrone direk aan sitochroom P450 ensieme verskaf, sowel as ensieme betrokke in lipiedbiosintese. Dit is ook bekend dat die proteïen die aktiwiteite van sekere ensieme via ’n allosteriese effek beïnvloed. Dit is geredelik in die literatuur aanvaar dat sitochroom b5 as ’n monomeer voorkom, maar daar is kort gelede gerapporteer dat homomeriese komplekse in vivo vorm. In hierdie studie is die sitochroom b5-kompleksvorming ondersoek deur gebruik te maak van verskeie analietiese metodes. Sitochroom b5 is vanuit skaaplewer mikrosome geïsoleer en die suiwerheid met behulp van natrium-dodesiel-sulfaat-poliakrielamied-gel-elektroforese en elektrosproei-ionisasie massa-spektrometrie geverifieer. Met die laasgenoemde bevestig dat ’n enkele heem-bevattende proteïen met Mr =15865 teenwoordig was, terwyl poliakrielamied gel-skeiding kompleksvorming getoon het, met tetrameer as die mees prominente oligomeer. Deur verskeie denaturerings kondisies, intsluitend besondere kondisies, is gevind dat hierdie aggregate behoue bly, wat baie stabiele oligomere aandui. Die mees prominente tetrameriese aggregaat is as sitochroom b5 geïdentifiseer met behulp van massa spektrometriese volgordebepaling. Kompleksvorming is verder bewys deur ’n verdere ondersoek met behulp van ’n fluoresserende kleurstof (1-anilinonaftaleen-8-sulfoonsuur) wat met die hirdofobiese holtes, wat vorm tydens oligomermerisasie, interaksie het. Die kompleksvorming was afhanklik van ioniese sterkte, sowel as proteïenkonsentrasie. Voorheen was dit bewys dat die deurslaggewende faktor in die vorming sitochroom b5 se homomeriese komplekse die hidrofobiese membraan-anker-domein is. Deur gebruik te maak van ’n peptied (IITTIDSNSS) wat lyk soos ’n gedeelte van hierdie domein, tesame met sirkulêre dichroisme, is gewys dat meer georganiseerde struktuur teenwoordig was vir die wilde tipe peptied vs. ’n gemuteerde kontrole peptied (LLSSLKAVAV). ’n Gemodifiseerde ELISAinteraksie- essai het ook getoon dat die wilde-tipe peptied spesifieke interaksie met sitochroom b5 het, ’n verdere bewys dat hierdie membraan-anker-domein ’n rol speel in kompleksvorming. Hierdie studies het ook aangedui dat ’n waterstofbinding netwerk in die domein belangrik kan wees vir die vorming van die homomeriese komplekse van sitochroom b5.