Thesis (MSc (Biochemistry))--University of Stellenbosch, 2006. / This thesis is about mathematical modelling of sucrose accumulation in the storage perenchyma of Saccharum officinarum (sugarcane) In 2001, Rohwer & Botha (76) published a kinetic model that described the defining feature of this process ...

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Heartwater is a major threat to livestock in Africa due to its high mortality rate. The intracellular nature of the causative organism, Cowdria ruminantium, makes it difficult to study, hence an effective and user-friendly vaccine has been extremely difficult to obtain. Two C. ruminantium DNA libraries have recently been constructed, the lambda GEM11 bacteriophage DNA library and the lambda ZAPII bacteriophage DNA library, and this has lead to a renewed search for protective genes that could be used as a vaccine against heartwater. In this study, several molecular techniques including PCR, cloning and sequencing were used to identify genes in the lambda GEM11 bacteriophage DNA library that code for proteins, which could be used as vaccines to protect susceptible animals against heartwater. The lambda GEM11 library was screened with a rickettsial secretory protein gene sequence, known as seeD. One positive colony was selected from which the bacteriophage DNA was isolated. The C. ruminantium DNA was amplified from the bacteriophage DNA by using PCR and C. ruminantium-specific primers. The C. ruminantium DNA was screened with Mycoplasma, bovine and Cowdria DNA probes. The amplified DNA was subeloned into two vectors and the clones were screened by restriction analysis to identify clones containing inserts. The appropriate clones were sequenced and overlapping sequences matched, ordered and aligned. Two sequences were continuous with a short sequence of unidentified bases in between. Oligonucleotide primers were designed to amplify the DNA sequence between the two contiguous sequences. This led to the identification of the entire sequence of the C. ruminantium genome contained within the bacteriophage plaque. The single contiguous sequence was analysed and the putative protein-coding sequences were obtained and compared to DNA sequences of known organisms using the BLAST program. Five open reading frames were identified with homology to genes encoding specific proteins in bacteria. Two open reading frames showed homology to the genes encoding the transporter proteins, FtsY and the ABC transporter, and three open reading frames were found to be homologous to genes encoding the essential enzymes dethiobiotin synthetase, pro lipoprotein diacylglycerol transferase and the putative NADH-ubiquinone oxidoreductase subunit. The five open reading frames encode for genes, which are essential for the normal functioning of the C. ruminantium organism. However, these open reading frames might not be effective for use in a DNA vaccine since none of the open reading frames showed homology to obvious genes that could play a role in immunity and therefore confer protection. The open reading frames can be used in mutagenesis studies to produce attenuated strains of the organism that possess mutated versions of these proteins. These attentuated strains could be used for the vaccination of cattle, and thereby confer protection against viable pathogenic C. ruminantium isolates. / AFRIKAANSE OPSOMMING: Hartwater is 'n bedreiging vir vee in Afrika weens die hoë mortaliteitssyfer verbonde aan die siekte. Die intrasellulêre aard van die organisme wat hartwater veroorsaak, Cowdria ruminantium, bemoeilik navorsing aangaande die organisme. Dit het tot gevolg dat 'n effektiewe en gebruikersvriendelike entstof moeilik bekombaar is. Daar is onlangs sukses behaal met die konstruksie van twee C. ruminantium DNA genoteke, die lambda GEM11 bakteriofaag genoteek en die lambda ZAPII bakteriofaag genoteek. Dit het gelei tot 'n herlewing in die soektog na beskermende gene, wat in 'n entstof teen hartwater gebruik kan word. In hierdie studie is verskeie molekulêre tegnieke insluitende PKR, klonering en geenopeenvolging bepaling, gebruik om gene te identifiseer in die lambda GEM11 bakteriofaag genoteek wat kodeer vir proteïene wat in entstowwe gebruik kan word as beskerming teen hartwater. Die secD geen is gebruik om die lambda GEM11 bakteriofaag genoteek te sif. Een positiewe plaak is gevind waarna die DNA uit die bakteriofaag plaak geïsoleer en die C. ruminantium DNA vanuit die bakteriofaag plaak geamplifiseer is deur gebruik te maak van PKR en spesifieke C. ruminantium inleiers. Die C. ruminantium DNA is gesif met Mycoplasma, bees en Cowdria radioaktief gemerkte DNA peilers. Die C. ruminantium DNA is vervolgens in twee vektore gekloneer. Die klone is gesif deur middel van restriksie analise. Die DNA volgorde van die klone is bepaal en twee ononderbroke sekwense is geïdentifiseer met 'n gaping in die middel tussen die twee sekwense. Oligonukleotied inleiers is daarna ontwerp om die geenopeenvolging van die gaping tussen die twee sekwense te vul. Hierdeur kon die volledige geenopeenvolging van die genoom van C. ruminantium wat in die lambda GEM 11 bakteriofaag plaak voorkom, bepaal word. Hierdie volledige geenopeenvolging is vervolgens geanaliseer en die oop leesrame wat daarin voorkom geïdentifiseer. Vyf leesrame is gevind om homologie met gene wat kodeer vir proteïene wat in bakterieë voorkom, te toon. Twee leesrame het homologie met die gene wat kodeer vir transport proteïene, FtsYen die ABC transporter getoon, en drie leesrame het homologie met gene wat kodeer vir die essensiële ensieme detiobiotin sintetase, prolipoproteïen diasielgliserol transferase en die NADHubikinoon oksidoreduktase subeenheid getoon. Dié vyf leesrame het die potensiaal om as entstowwe gebruik te word aangesien al vyf leesrame kodeer vir gene wat 'n belangrike rol speel in die oorlewing van die C. ruminantium organisme. Alhoewel die leesrame moontlik nie so effektief sal wees in 'n DNA entstof nie, toon dit potensiaal om in mutasieeksperimente gebruik te word. Organismes wat die gemuteerde weergawe van die geen besit sal nie-funksionele proteïene produseer, wat 'n invloed kan hê op die normale fisiologiese funksies van die organisme en dus sal lei tot 'n minder virulente organisme. Die geattenueerde organisme kan moontlik gebruik word om diere te immuniseer en daardeur immuniteit aan diere lewer wat beskerming sal bied teen patogeniese C. ruminantium isolate.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The possible use of the bacterially produced antimicrobial peptides, and in particular class IIa bacteriocins as food preservatives is a motivating factor in studies on resistance to them by food-borne pathogens like Listeria monocytogenes. The high frequencies of resistance to class Ha bacteriocins have however sparked concern regarding their adequacy as potential biopreservatives. Activity of these cationic peptides was reported to occur by membrane permeabilisation due to pore formation, which results in the leakage of the intracellular contents followed by cell death. The cell envelope (cell wall and cell membrane) is therefore envisaged as a key site of modification in suscepti bility of bacteria to class Ha bacteriocins. Mutants of the L. monocytogenes 873 isolate, resistant to the class IIa bacteriocin, leucocin A, were generated at the start of the study to complement the existing array of L. monocytagenes wild-type and resistant isolates obtained from other sources. The fifty percent inhibitory concentrations using a highly sensitive and reproducible bioassay were determined. This allowed categorisation of the mutants into intermediate and highly resistant phenotypes. Analysis of the growth patterns of all these strains showed decreased growth rates and higher growth yields for all the resistant strains in general. This provided evidence for possible effects of membrane adaptation and metabolic changes in the resistant strains and prompted further investigation. The major focus of the study on the class Ha resistant mutants were: (1) analysis of membrane compositional changes and factors influencing cell surface charge; (2) assessment of physical changes in the membrane and bacteriocin itself using circular dichroism and fourier transform infrared spectroscopy; (3) and, determination of changes in glucose metabolism. Electrospray mass spectrometry analysis of the major listerial phospholipid, phosphatidylglycerol, revealed that membranes of resistant strains had increased levels of unsaturated and short-acyl-chain phosphatidylglycerol molecular species, indicating more fluid membranes. In addition, treatment with a desaturase inhibitor resulted in increased sensitivity of only the intermediate resistant strains to the class na bacteriocin, leucocin A. This indicated the influence of membrane adaptation in only lower levels of resistance. It is conceivable that more fluid membranes could also impact on decreased stability of pore formation by the bacteriocin. Complementary biophysical studies using fourier transform infrared spectroscopy indicated the possible occurrence of greater membrane fluidity of resistant cells, by the notable shift in the anti symmetric CH2 stretching vibration from 2921 cm-I to 2922 cm-I. Additionally, circular dichroism revealed a decreased a-helical and increased random structure of leucocin A in the presence of listerial liposomes derived from highly resistant cell membrane extracts. It is possible that this may result in reduced activity of the bacteriocin in resistant cell membranes as a-helical stucture is a critical feature for membrane insertion of cationic antimicrobial peptides. Cell surface charge was determined by quantification of alanine and lysine esterification of the anionic cell surface polymer, teichoic acid, and membrane phospholipids respectively. Increased D-alanine, which causes neutralisation of the cell surface, was observed in all resistant cells. A tendency for greater lysine content in membrane phospholipids, which also impacts on neutralisation of the anionic phospholipid of listerial membranes, was observed in highly resistant strains only. This neutralisation of the negative charge of the cell surface may interfere with initial electrostatic interaction of bacteriocin with the cell, and subsequent interactions required for permeabilisation of the cell membrane. These differences in alanine and lysine esterification were not the result of increased expression of certain associated genes (d/tA and /mo1695) and may be the result of post-transcriptional regulation. It was, however, found that all resistant L. monocytogenes strains, including the intermediate resistant strains, exhibited decreased expression of a putative docking molecule, the mannose-specific phosphotransferase system EIIAB subunit (EIlABMan).A clear correlation existed between the levels of resistance and EIIABMandown-regulation. Finally, analysis of the glucose metabolism in highly resistant and wild-type strains, indicated a more efficient metabolism with regards to higher growth yields and ATP yield, in contrast to a lower specific growth rate in a spontaneous and genetically defined (EIlABMan inactivated) highly resistant mutant. The switch in metabolic end-product observed, was attributed to the loss of the glucose transporter, EIlABMan,and may cast doubts on the feasibility of the use of class Ha bacteriocins as food preservatives in light of a stable and efficient resistant phenotype. / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Thesis (PhD)--Stellenbosch University, 1998. / ENGLISH ABSTRACT: Iturin A, an antifungal lipopeptide, is produced by Bacillus subtilis. This cyclic peptide consists of seven D- and L-amino acid residues (L-Asn2-D-Tyr3-D-Asn4-L-Gln5-L-Pro6-DAsn7- L-Ser8) and a beta-amino fatty acid residue. Eight analogues of iturin A2 were synthesised and purified by high performance chromatography (HPLC). Electrospray ionisation mass spectrometry (ESI-MS), amino acid analysis and HPLC confirmed high chemical purity of the synthetic products. The influence of primary structure on conformation, hydrophobicity, interaction with alkali metal ions and bioactivity was investigated using the purified peptides. Two low energy in vacuo structures of a linear iturin A2 analogue (8-Beta), one with a distorted W-backbone structure and one with a twisted S-backbone structure, were predicted with HyperChem®4.5. Nuclear magnetic resonance spectrometry confirmed the existence of two slow interconverting conformations, possibly a W<->S equilibrium. The predicted S-structure of 8-Beta includes two turns that approximate beta-turns. In natural iturin A, the same two peptide moieties, beta-aminotetradecanoyl-L-Asn2-D-Tyr3-D-Asn4 and L-Gln5-L-Pro6-DAsn7- L-Ser8, each adopt a type II beta-turn conformation. ESI-MS fragmentation patterns of sodiated 8-Beta indicated that the sodium interacts with the majority of the amide bond oxygens in the predicted turns. The linear peptides associated with either one or two alkali metal ions, while the cyclic analogues associated only with one ion. The alkali metal ion selectivity sequence of all the lipopeptides was Na+>K+>Rb+, indicating a size limitation in interaction cavities. Iturin A possibly has a direct interaction with alkali metal ions and it is proposed that these ions are chelated by the carbonyl oxygens in either one of the two beta-turns of natural iturin A. It was found that the more hydrophobic the iturin A2 analogue, the better it interacted with lipid membranes and octadecanoylsilane matrices (HPLC retention), except if it had a high tendency to aggregate in solution. Aggregation in the membrane is part of iturin A’s mechanism of action. It is proposed that solution-phase aggregates are not the active form of iturin A as the lipopeptide preparations, which self-aggregated in solution, lost their antibacterial activity. Circular dichroism (CD) spectra of the peptides in liposomes revealed the possibility of type II b-turns in all the octalipopeptides. There is, however, a marked difference between the overall cyclic and linear structures in membranes, although diastereomers, differing in configuration of b-aminotetradecanoic acid (b-NC14) residue, had similar structures. The possibility of self-assembly of synthetic iturin A2 in antiparallel beta-sheets was also indicated by CD. Haemolytic activity of the iturin A2 analogues depended on cyclisation, inclusion of L-Asn2 and b-NC14 configuration. This activity is possibly stereoselective as synthetic iturin A2 and its linear analogue were the most haemolytic. Growth inhibition of Micrococcus luteus mainly depended on hydrophobic interaction and not on cyclisation or configuration of the beta-NC14 residue, therefore this activity differs in mechanism of action from that of haemolysis. Lysis of M. luteus protoplasts, however, decreased with decrease in peptide length: 8-Beta>7-Beta>6- Beta. The activity against Botrytis cinerea depended mainly on cyclisation. The hydrophobic hub, formed by the invariant Tyr residue and the beta-NC14 residue, is a possible key to antifungal activity. This hub is absent in the predicted S-structure of 8-Beta and may be influenced in cyclic 8-Beta and shorter analogues by the configuration of the beta-NC14 residue, resulting in good overall bioactivity of only the synthetic iturin A2. / AFRIKAANSE OPSOMMING: Iturin A, ’n antifungiese lipopeptied, word deur Bacillus subtilis geproduseer. Hierdie sikliese peptied bestaan uit sewe D- en L-aminosuurresidue (L-Asn2-D-Tyr3-D-Asn4-L-Gln5-L-Pro6- D-Asn7-L-Ser8) en ’n beta-aminovetsuurresidu. Agt analoë van iturin A2 is gesintetiseer en m.b.v. hoë doeltreffendheid chromatografie (HPLC) gesuiwer. Die hoë chemiese suiwerheid van die sintetiese produkte is deur elektrosproei-ionisasie massaspektrometrie (ESI-MS), aminosuuranalise en HPLC bevestig. Die gesuiwerde peptiede is gebruik om die invloed van primêre struktuur op konformasie, hidrofobisiteit, interaksie met alkalimetaal-ione en bioaktiwiteit te ondersoek. Twee lae-energie in vacuo strukture van die lineêre iturin A2 analoog (8-Beta), een met ‘n verwronge W-ruggraatstruktuur en een met ’n gedraaide S-ruggraatstruktuur, is deur HyperChem®4.5 voorspel. Kernmagnetiese resonansspektrometrie het die bestaan van twee interomskakelende konformasies bevestig, moontlik ’n W<->S ewewig. Die voorspelde S-struktuur van 8-Beta bevat twee draaie wat neig na beta-draaie. Dieselfde twee peptiedeenhede, beta-aminotetradekanoiël-L-Asn2-D-Tyr3-D-Asn4 en L-Gln5-L-Pro6-D-Asn7-L-Ser8, neem elk ’n tipe II beta-draai konformasie in die natuurlike iturin A aan. ESI-MS fragmentasiepatrone van die 8-Beta natruimaddukte het aangedui dat die natriumione met die meeste van die amiedbinding-suurstowwe in die voorspelde draaie interaksie het. Die lineêre peptiede het met een of twee alkalimetaal-ione geassosieer, terwyl die sikliese analoë slegs met een ioon geassosieer het. Al die lipopeptiede se alkalimetaal-ioon selektiwiteitsvolgorde was Na+>K+>Rb+, wat aandui dat daar ’n grootte limiet is in die interaksieholtes. Iturin A het moontlik ’n direkte interaksie met alkalimetaal-ione en dit word voorgestel dat hierdie ione deur die karbonielsuurstowwe in enige een van die twee beta-draaie van natuurlike iturin A gechelateer word. Daar is gevind dat hoe meer hidrofobies die iturin A2 analoog is, hoe beter is die interaksie daarvan met lipiedmembrane en oktadekanoïelsilaanmatrikse (HPLC retensie), behalwe as dit ’n groot tendens het om in oplossing te aggregeer. Aggregasie in die membraan is deel van iturin A se meganisme van aksie. Daar word voorgestel dat die aggregate in oplossing nie die aktiewe vorm van iturin A is nie, omdat die lipopeptiedpreparate wat in oplossing selfaggregeer antibakteriële aktiwiteit verloor. Sirkulêre dichroïsme (CD) spektra van die peptiede in liposome het die moontlikheid van tipe II beta-draaie in al die oktalipopeptiede uitgewys. Alhoewel daar merkbare verskille tussen die totale sikliese en lineêre strukture in die membrane voorgekom het, was die diastereomere, wat verskil t.o.v. konfigurasie van beta-aminotetradekanoësuurresidu (beta-NC14), se strukture baie dieselfde. Die moontlikheid van selfverpakking van sintetiese iturin A2 in antiparallele beta-plate is ook deur CD aangedui. Hemolitiese aktiwiteit van die iturin A2 analoë het afgehang van siklisering, die insluiting van L-Asn2 en die beta-NC14-konfigurasie. Hemolise is moontlik stereoselektief want sintetiese iturin A2 en sy lineêre analoog was die aktiefste. Groei-inhibisie van Micrococcus luteus het hoofsaaklik afgehang van hidrofobiese interaksie en nie van siklisering of die konfigurasie van die beta-NC14-residu nie, dus verskil hierdie aktiwiteit in meganisme van aksie van diè van hemolise. Die lise van M. luteus protoplaste daarenteen neem af met verkorting van peptiedketting: 8-Beta>7-Beta>6-Beta. Die aktiwiteit teen Botrytis cinerea het hoofsaaklik afgehang van siklisering. Die hidrofobiese eenheid, gevorm deur die invariante Tyr-residu en die beta-NC14-residu, is ’n moontlike sleutel tot die antifungiese aktiwiteit. Hierdie eenheid is afwesig in die voorspelde S-struktuur van 8-Beta en mag ook in die sikliese 8-Beta en korter analoë beïnvloed word deur die konfigurasie van die beta-NC14-residu, wat lei tot goeie algemene bioaktiwiteit van slegs die sintetiese iturin A2.

Dissertation (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study: 1. Compares various heterologous expression systems for high-level expression of cytochromes P450. Limitations of the existing cytochromes P450 expression systems are discussed and possibilities to improve the expression yields of human steroidogenic enzymes, are suggested. In addition the potential applications of human steroidogenic cytochromes P450 expressed in Pichia pastoris are illustrated. 2. Describes the cloning and extracellular expression of a recombinant full-length human cytochrome P450 17a-hydroxylase (p45017a) enzyme in Saccharomyces cerevisiae. After the optimisation of expression conditions, it was shown that this system is not suitable for the expression of full-length human P45017a. 3. Describes the cloning and extracellular expression of the full-length human cytochromes P45017a, aromatase, bs and truncated human cytochrome P45017a in P. pastoris. The limitations using P. pastoris as an export system for expressed P450 enzymes were pointed out. 4. Describes the cloning and intracellular expression of the full-length human cytochrome P45017a in P. pastoris as well as the functional expression of human P45017a in P. pastoris, showing progesterone conversion to 17ahydroxyprogesterone and 16a-hydroxyprogesterone in vivo, for the first time. 5. Evaluates developed methods for the preparation of mierosomes from P. pastoris expressing human P45017a and the spectral characterisation of detergent solubilised human P45017a. 6. Describes the development of protocols for the purification of human P45017a from P. pastoris microsomes. / AFRIKAANSE OPSOMMING: Hierdie ondersoek: 1. Vergelyk verskillende heterologiese proteïen uitdrukkings-sisteme vir die preperatiewe produksie van sitochrome P450. Die tekortkomings van bestaande sitochroom P450-uitdrukkings-sisteme word bespreek en moontlikhede om die opbrengs van menslike steroïedogeniese ensieme te verbeter word voorgestel. Die potensiële aanwendings van menslike steroïedogeniese sitochrome P450, wat in Pichia pas/oris uitgedruk word, word ook geïllustreer. 2. Beskryf die klonering en ekstrasellulêre uitdrukking van die rekombinante vollengte menslike sitochroom P45017a-hidroksilase (P45017a) ensiem in Saccharomyces cerevisiae. Na optimisering van die kondisies vir die uitdrukking kon aangetoon word dat hierdie sisteem nie geskik is vir die uitdruk en sekresie van vollengte menslike P45017a nie. 3. Beskryf die klonering en ekstrasellulêre uitdrukking van die vollengte menslike sitochrome P45017a, aromatase, b5 en verkorte menslike sitochroom P45017a in P. pas/oris. Die beperkinge van P. pas/oris as 'n uitvoersisteem vir die uitdrukking van P450 ensieme word bespreek. 4. Besryf die klonering en intrasellulêre ekspressie van die vollengte menslike sitochroom P450 17a. Die funksionele ekspressie van menslike sitochroom P450 17a in P. pas/oris is vir die eerste keer gekarakteriseer. 5. Evalueer die ontwikkelde metodes vir die voorbereiding van mikrosome van P. pas/oris wat menslike P45017a uitdruk en karakteriseer die detergent opgelosbare menslike P45017a t.o.v. spektroskopiese eienskappe. 6. Beskryf die ontwikkelling van protokolle vir die suiwering van die uitgedrukte menslike P45017a vanuit P. pas/oris mikrosome.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: The implication of biologically active peptides from different organisms on one another in complex ecological communities is largely unknown at this stage. The elucidation of the nature of this influence may have practical implications in terms of organism resistance and the conservation of an optimal agricultural environment. This study was aimed to elucidate the effect of antimicrobial peptides from different co-habitational organisms, on each other, both in terms of bioactivity and interaction. The two peptides investigated were gramicidin S, a decapeptide from Bacillus brevis, and surfactin, a heptalipopeptide from Bacillus subtilis. Preliminary studies were also done on iturin A and synthetic analogues of iturin A and iturin C, both octalipopeptides from Bacillus subtilis. Analytical antimicrobial assay systems were used to study the effect of surfactin on the antibiotic action of gramicidin S towards three different target cells namely, a Gram-positive bacterium (Micrococcus luteus), a Gram-negative bacterium, (Escherichia coli) and a fungus (Penicillium corylophilium). The investigation of the antifungal activity was hampered by the insensitivity and subjectivity of the majority of antifungal assays and necessitated the development of two new testing methodologies. The investigation showed that surf actin had an antagonistic effect on the antimicrobial activity of gramicidin S against all three of the target cells. This antagonism is dose-dependent at concentrations lower than required for surfactin to exert biological activity. Electrospray mass spectrometry (ESMS) showed the formation of surfactin-gramicidin S complexes in 1:1 and 2: 1 ratios with enhanced complex formation in an apolar environment. Dissociation experiments indicated that the peptide complexes were slightly less stable than the peptides alone. The presence of NaCI up to 80 mM had little effect on the stability of preformed complexes. Incubating surfactin with NaCI and CaCh before titration with gramicidin S also did not affect complex formation. Furthermore, results from the pre-incubation studies with CaCh indicated that surfactin-gramicidin S complexes might be formed through the displacement of the metal ion. The mechanism of this displacement is unlikely to be direct competition but rather the result of conformational' changes induced by peptide-peptide interaction/interactions. A likely point of interaction the p-tums in the peptide ring. Linear iturin A2 and iturin C analogues were synthesised (8-Beta and 8-Betac) with solid phase peptide synthesis and purified using self-assembly and high performance liquid chromatography. The products of the syntheses wete analysed by ESMS and found to be correct. The products, together with commercially obtained iturin A, were used in biological assays and it was found that iturin A antagonises the antibiotic activity of gramicidin S but the linear analogues had no effect. Complex formation between iturin A and gramicidin S was observed using ESMS but no complexes were detected for the analogues, which reinforces the hypothesis that antagonism is related to the formation of inactive complexes. In general, the formation of peptide-gramicidin S complexes may indicate that a defence mechanism may be present in which toxic peptides of the competitor organism are inactivated by peptides from co-habiting organisms. / AFRIKAANSE OPSOMMING: Die invloed wat biologiese aktiewe verbindings van verskillende mikro-organismes in komplekse ekologiese omgewings op mekaar het, is onbekend. Die ontrafeling van die rol mag verskeie vrae ten opsigte van weerstandbiedenheid en ontwrigting van ekologiese landbou-omgewings beantwoord. Die doel van hierdie studie was om die invloed wat antimikrobiese peptiede, afkomstig van verskillende ko-habiterende organismes, op mekaar het te ondersoek- beide in terme van biologiese aktiwiteit en interaksie. Die twee peptiede wat ondersoek is, was gramisidien S, 'n dekapeptied geproduseer deur Bacillus brevis en surfaktien, 'n heptalipopeptied, geproduseer deur Bacillus subtilis. Voorlopige ondersoeke is ook uitgevoer op iturin A, en sintetiese iturin A en iturin C analoë, beide oktalipopeptiede van B. subtilis. Analitiese antimikrobiese toetsstelsels is gebruik om die effek van surfaktien op die biologiese aktiwiteit van gramisidien S te bepaal. Drie teikenselle is gebruik nl. 'n Grampositiewe bakterium (Micrococcus luteus), 'n Gram-negatiewe bakterium (Escherichia coli) en 'n fungus (Penicillium corylophilium) Die gebrek aan sensitiwiteit van bestaande antifungiese toetsstelsels het die ontwikkelling van twee nuwe toetstelsels genoodsaak. Die ondersoek het aangetoon dat surfaktien 'n antagonistiese effek op gramisidien S se antimikrobiese werking teen al drie teikenselle het. Die antagonisme is waarneembaar by surfaktien konsentrasies veel laer as wat nodig is vir biologiese aktiwiteit. Elektrosproeimassaspektrometrie (ESMS) van surfaktien en gramisidien S mengsels het aangedui dat komplekse in 'n 1:1 en 2: 1 stoichiometrie voorkom. Die vorming van peptiedkomplekse word ook deur 'n nie-polêre omgewing bevorder. Die stabiliteit van die peptiedkomplekse is ook geëvalueer met dissosiasie eksperimente en daar is gevind dat die komplekse minder stabiel is as die peptiede alleen - dit is 'n aanduiding van kompleksdissosiasie. Die teenwoordigheid van NaCI tot en met 80 mM het 'n minimale invloed op die stabiliteit van voorafgevormde peptiedkomplekse gehad. Inkubasie van surfaktien met NaCl en CaCh voor titrasie met gramisidien S, het ook nie die vorming van peptiedkomplekse beïnvloed nie en die studies het aangetoon dat die komplekse moontlik gevorm word deur die verplasing van die alkaliemetaalioon. Dit is onwaarskynlik dat die meganisme van ioon-verplasing direkte kompetisie is, maar eerder as gevolg van interaksie in een van die p-draaie. Liniêre iturin A2 en liniêre iturin Canaloë (8-Beta en 8-Betac) is gesintetiseer met behulp van soliede fase peptiedsintese en gesuiwer deur middel van "self-assembly" en "high performance liquid chromatography (HPLC)". Volgens die ESMS analise is die korrekte produkte verkry. Die analoë en kommersieel beskikbare iturin A is aan biologiese toetsing onderwerp en daar is gevind dat iturin A, maar nie die analoë nie, die antibiotiese effek van gramisidien S ophef. Die vorming van iturin en gramisidien S komplekse, wat met ESMS waargeneem is, versterk die teorie dat opheffing van aktiwiteit verband hou met die vorming van inaktiewe komplekse. Verder, die analoë het nie komplekse met gramisidien S gevorm me. Dit blyk vanuit hierdie studies dat die vorming van peptied komplekse moontlik deel kan uitmaak van 'n tipe verdedigingsmeganisme waar toksiese peptiede van kompeterende organismes, deur peptiede van ko-habiterende organismes, geïnaktiveer word.

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.