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.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This study describes: the evaluation of the current, and potential assay methods for the quantification of cholesterol, cholesteryl esters and free fatty acids in milk and the application thereof ; an account of the difficulties associated with the usage of FoodPro® Cleanline, an enzyme preparation used as processing aid, during ultra-high temperature processing of milk ; the development of activity assays which can be used for the kinetic characterization of glycerophospholipid cholesterol acyltransferase, the active enzyme in FoodPro® Cleanline ; the development of an accurate and facile activity assay, and the validation thereof, which can be used for the validation of enzyme activity prior to dosage of milk with FoodPro® Cleanline. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: die evaluering van die huidige, en potensiële, metodes vir die kwantifisering van cholesterol, cholesteriel esters en vryvetsure in melk, sowel as die toepassing van hieridie metodes ; 'n verduideliking van die moeilikhede wat ondervind word gedurende die gebruik van FoodPro® Cleanline, 'n ensiempreparaat vir gebruik as 'n verwerkingshulpmiddel, tydens ultrahoë-temperatuurprosessering van melk ; die ontwikkeling van aktiwiteitsbepalings metodes vir gebruik in kinetiese karakterisering van gliserofosfolipied cholesterol asieltransferase, die aktiewe ensiem in FoodPro® Cleanline ; die ontwikkeling van 'n akkurate, eenvoudige aktiwiteitsbepalings metode, en bevestiging van hierdie metode, wat gebruik kan word vir kwalitieitskontrole alvorens die dosering van melk met FoodPro® Cleanline.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Codes are ubiquitous in culture|and, by implication, in nature. Code biology is the study of these codes. However, the term `code' has assumed a variety of meanings, sowing confusion and cynicism. The rst aim of this study is therefore to de ne what an organic code is. Following from this, I establish a set of criteria that a putative code has to conform to in order to be recognised as a true code. I then o er an information theoretical perspective on how organic codes present a viable method of dealing with biological information, as a logical extension thereof. Once this framework has been established, I proceed to review several of the current organic codes in an attempt to demonstrate how the de nition of and criteria for identifying an organic code may be used to separate the wheat from the cha . I then introduce the `regulatory code' in an e ort to demonstrate how the code biological framework may be applied to novel codes to test their suitability as organic codes and whether they warrant further investigation. Despite the prevalence of codes in the biological world, only a few have been de nitely established as organic codes. I therefore turn to the main aim of this study which is to cement the status of the histone code as a true organic code in the sense of the genetic or signal transduction codes. I provide a full review and analysis of the major histone post-translational modi cations, their biological e ects, and which protein domains are responsible for the translation between these two phenomena. Subsequently I show how these elements can be reliably mapped onto the theoretical framework of code biology. Lastly I discuss the validity of an algorithm-based approach to identifying organic codes developed by G orlich and Dittrich. Unfortunately, the current state of this algorithm and the operationalised de nition of an organic code is such that the process of identifying codes, without the neccessary investigation by a scientist with a biochemical background, is currently not viable. This study therefore demonstrates the utility of code biology as a theoretical framework that provides a synthesis between molecular biology and information theory. It cements the status of the histone code as a true organic code, and criticises the G orlich and Dittrich's method for nding codes by an algorithm based on reaction networks and contingency criteria. / AFRIKAANSE OPSOMMING: Kodes is alomteenwoordig in kultuur|en by implikasie ook in die natuur. Kodebiologie is die studie van hierdie kodes. Tog het die term `kode' 'n verskeidenheid van betekenisse en interpretasies wat heelwat verwarring veroorsaak. Die eerste doel van hierdie studie is dus om te bepaal wat 'n organiese kode is en 'n stel kriteria te formuleer wat 'n vermeende kode aan moet voldoen om as 'n ware kode erken te word. Ek ontwikkel dan 'n inligtings-teoretiese perspektief op hoe organiese kodes `n manier bied om biologiese inligting te hanteer as 'n logiese uitbreiding daarvan. Met hierdie raamwerk as agtergrond gee ek `n oorsig van 'n aantal van die huidige organiese kodes in 'n poging om aan te toon hoe die de nisie van en kriteria vir 'n organiese kode gebruik kan word om die koring van die kaf te skei. Ek stel die `regulering kode' voor in 'n poging om te wys hoe die kode-biologiese raamwerk op nuwe kodes toegepas kan word om hul geskiktheid as organiese kodes te toets en of dit die moeite werd is om hulle verder te ondersoek. Ten spyte daarvan dat kodes algemeen in die biologiese w^ereld voorkom, is relatief min van hulle onomwonde bevestig as organiese kodes. Die hoofdoel van hierdie studie is om vas te stel of die histoonkode 'n ware organiese kode is in die sin van die genetiese of seintransduksie kodes. Ek verskaf 'n volledige oorsig en ontleding van die belangrikste histoon post-translasionele modi kasies, hul biologiese e ekte, en watter prote endomeine verantwoordelik vir die vertaling tussen hierdie twee verskynsels. Ek wys dan hoe hierdie elemente perfek inpas in die teoretiese raamwerk van kodebiologie. Laastens bespreek ek die geldigheid van 'n algoritme-gebaseerde benadering tot die identi sering van organiese kodes wat deur G orlich en Dittrich ontwikkel is. Dit blyk dat hierdie algoritme en die geoperasionaliseerde de nisie van 'n organiese kode sodanig is dat die proses van die identi sering van kodes sonder die nodige ondersoek deur 'n wetenskaplike met 'n biochemiese agtergrond tans nie haalbaar is nie. Hierdie studie bevestig dus die nut van kodebiologie as 'n teoretiese raamwerk vir 'n sintese tussen molekul^ere biologie en inligtingsteorie, bevestig die status van die histoonkode as 'n ware organiese kode, en kritiseer G orlich en Dittrich se poging om organiese kodes te identi seer met 'n algoritme wat gebaseer is op reaksienetwerke en `n kontingensie kriterium.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: This study investigated the effect of several hormones on the rat corticosteroid binding globulin proximal promotor and for the first time showed that modulation occurs at the promotor level and can be correlated with changes in corticosteroid binding globulin mRNA and protein levels. The effect of various physical and psychological stressors on rat liver corticosteroid binding globulin mRNA levels was also tested and it was shown that voluntary running had no effect on rat corticosteroid binding globulin levels but that involuntary swimming and immobilization decreased rat corticosteroid binding globulin mRNA levels. Glucocorticoid responsiveness of the corticosteroid binding globulin promoter was investigated further by using truncated contructs of the corticosteroid binding globulin proximal promoter. Glucocorticoid responsiveness was delineated to between -296 and -145bp from the transcription start site an area that contains putative binding sites for D-site binding protein, hepatic nuclear factor-3 and CAAT/enhancer binding protein suggesting that these transcription factors may be involved in glucocorticoid responsiveness of the corticosteroid binding globulin proximal promoter. The dissociative glucocorticoid activity of medroxyprogesterone acetate and Compound A, both putative dissociated glucocorticoids, was compared to standard glucocorticoids by examining transactivation of glucocorticoid response element-containing reporter constructs and transrepression of corticosteroid binding globulin gene expression in hepatic cell lines. Results showed that medroxyprogesterone acetate, but not Compound A, trans activates only in the presence, but not in the absence, of co-transfected glucocorticoid receptor. Medroxyprogesterone acetate down modulated dexamethasone transactivation while the modulatory effect of Compound A depends on the order of addition of Compound A. If added together Compound A has no effect on dexamethasone transactivation, however, if Compound A was added before dexamethasone, Compound A significantly decreased dexamethasone transactivation. Both medroxyprogesterone acetate and Compound A, like glucocorticoids, transrepressed the rat corticosteroid binding globulin proximal promoter. The potency of repression was similar but Compound A repressed with a higher efficacy than medroxyprogesterone acetate. We conclude that Compound A is a completely dissociated glucocorticoid in contrast to medroxyprogesterone acetate that displays only partial dissociation, which is dependent on glucocorticoid receptor levels. / AFRIKAANSE OPSOMMING: Tydens hierdie ondersoek is die effek van verskeie hormone op die rot kortikosteroied bindings globulien proksimale promoter ondersoek en vir die eerste keer is getoon dat modulering plaasvind op promoter-vlak en dat repressie korrileer met die verandering in kortikosteroied bindings globulien mRNA-en proteinvlakke. Die effek van verskeie fisiese en fisiologiese stressors op rotlewer kortikosteroied bindings globulien-mRNAvlakke is ook getoets en daar is getoon dat willekeurige hardloop geen effek op rot kortikosteroied bindings globulien-mRNA-vlakke het nie maar dat gedwonge swem en immobilisering rot kortikosteroied bindings globulien-mRNA-vlakke verlaag. Glukokortikoied responsiewiteit van die kortikosteroied bindings globulien proksimale promoter is verder ondersoek deur verkorte konstrukte van die kortikosteroied bindings globulien te toets. Glukokotikoied responsiewiteit is afgebaken tot tussen -296 en - 145bp vanaf die transkripsie beginplek 'n area wat beweerde bindings setels vir D-setel bindings protein, hepatosiet faktoor-3 en CCAAT-bindings protein-2 bevat en dus suggereer dat hierdie transkripsie faktore betrokke mag wees met glukokortikoied effekte op die kortikosteroied bindings globulien-proksimale promoter. Die dissosiatiewe glukokortikoied aktiwiteit van medroksiprogesteroon asetaat en Verbinding A, beide beweerde dissosiatiewe glukokortikoiede, relatief tot standaard glukokortikoiede is vergelyk deur transaktivering van glukokortikoied reseptor e1elment-bevattende konstrukte en onderdrukking van kortikosteroied bindings globulien geen ekspressie in lewersellyne te bestudeer. Medroksiprogesteroon asetaat, maar nie Verbinding A nie, transaktiveer slegs in die teenwoordigheid, maar nie in die afwesigheid, van ko-getransfekteerde glukokortikoied reseptore. Medroksiprogesteroon asetaat moduleer deksametasoon transaktivering afwaarts terwyl die modulerende effek van Verbinding A afhanklik van die orde van Verbinding A byvoeging is. Indien saam bygevoeg het Verbinding A geen effek op deksametasoon transaktivering nie, maar indien Verbinding A voor deksametasoon bygevoeg word verlaag Verbinding A deksametasoon transaktivering. Beide medroksiprogesteroon asetaat and Verbinding A, soos glukokortikoiede, onderdruk die rot kortikosteroied bindings globulien-proksimale promoter. Die sterkte van onderdrukking is dieselfde maar Verbinding A onderdruk met 'n hoër effektiwiteit as medroksiprogesteroon asetaat. Ons toon dat Verbinding A 'n totale dissosiatiewe glukokortikoied is in teenstelling met medroksiprogesteroon asetaat, wat slegs gedeeltelik dissosiatief is afhangende van glukokortikoied reseptor-vlakke.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new antimicrobial drugs especially with the rise in resistance to almost all available drugs by public health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat to public health. One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research found that the six major Trcs were active against Listeria monocytogenes and Plasmodium falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide unit has an important role in activity. We set out to extend the qualitative structure to activity relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either naturally or by chemical synthesis produced and characterised in terms of chemical character and biological activity. The Trcs were significantly more active than RW peptides, although much more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an aromatic amino acid residue for selective activity towards the leucocin A resistant L. monocytogenes B73-MR1. However, structural properties favouring a tighter membrane interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There was also need for an amphipathic balance between hydrophobicity and size/steric parameters for optimal activity. From our QSAR studies we predict as lead peptide for a future library of antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport system or bacterial dehydrogenases. We confirmed, as previously found, that the Trcs have potent antimalarial activity that is sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in activity. From these results a proposed lead peptide for future research is cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages. This indicated that membrane structures containing neutral lipids, as well as chromatin may be targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic towards each other’s activity. This indicated a shared target and we propose the food vacuole as another of the Trc targets in P. falciparum. / AFRIKAANSE OPSOMMING: Antimikrobiese peptiede (AMPe) is tans die mees nagevorsde groep verbindings in die soeke na nuwe antimikrobiese middels, veral weens 'n toenemende weerstandigheid van patogene in die openbare gesondheidsektor teen alle beskikbare middels. Die doel van hierdie studie was om klein, sikliese AMPe in terme van hul aktiwiteit teenoor twee menslike patogene wat 'n bedreiging vir openbare gesondheid is, Listeria monocytogenes, 'n voedsel-oordraagbare patogeen wat listeriose veroorsaak, asook Plasmodium falciparum, die parasiet verantwoordelik vir malaria, te karakteriseer. Een van die klein, sikliese peptiedbiblioteke wat ondersoek is, is die tyrocidines (Trcs) en analoë (sikliese dekapeptiede [siklo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu]). Hierdie peptiede deur die Gram-positiewe bakterie Bacillus aneurinolyticus word wat nie-ribosomaal gesintetiseer as deel van die tirotrisien kompleks word tydens sporulasie. Vorige navorsing het gewys dat die ses hoof Trcs teen Listeria monocytogenes en Plasmodium falciparum aktief is en dat die identiteit van die aromatiese residue in die aromatiese dipeptiedeenheid 'n belangrike rol speel in die Trc-aktiwiteit. Ons het gepoog om die kwalitatiewe struktuur-aktiwiteit-verwantskap (QSAR) studies uit te brei deur meer Trc analoë en klein sintetiese Arg- en Trp-ryke sikliese peptiede (RW-peptiede) te gebruik en sodoende essensiële struktuur-motiewe en voorvereistes vir aktiwiteit vas te stel. Agt natuurlike en drie sintetiese Trc analoë, asook vyftien RW-peptiede is of deur natuurlike of chemiese sintese geproduseer en gekarakteriseer in terme van chemiese karakter en biologiese aktiwiteit. Die Trcs het beduidend meer aktiwiteit as RW-peptiede getoon, maar is ook meer hemolities en dus meer toksies. Die resultate dui op die belang van waterstofbinding met 'n aromatiese aminosuurresidu vir die selektiewe aktiwiteit teenoor die leucocin A weerstandige L. monocytogenes B73-MR1. Strukturele eienskappe wat tot 'n sterker membraan-interaksie lei, verhinder egter die werkingsmeganisme. Ons het vasgestel dat Gln en die hidroksielgroep van Tyr betrokke kan wees in die interaksie met die vermeende teenmiddelteiken in L. monocytogenes. 'n Balans tussen amfipatiese/hidrofobiese en grootte/steriese parameters is ook noodsaaklik vir optimale aktiwiteit. Vanuit ons QSAR studies word die peptied siklo-(VOMe3LfPWfNQY) as die voorloper vir 'n toekomstige peptiedbiblioteek van antilisteriale Trcs voorgestel. Verder is daar gevind dat die antilisteriese aktiwiteit van die Trcs oorwegend lities en sout-verdraagsaam is, terwyl die RW-peptiede nie-lities en Ca2+ sensitief is. Ons het bevestig dat Ca2+ die Trc antilisteriese aktiwiteit verbeter, deur die Trc se antimetaboliese aktiwiteit verhoog, maar terselfdertyd 'n nie-litiese werkingsmeganisme induseer. Vanuit model-membraan studies word voorgestel dat Trc se nie-litiese werkingsmeganisme, soos teweeggebring deur Ca2+, die gevolg kan wees van nadelige lipied vermenging, die teenwoordigheid van 'n kalsium geïnduseerde Trcsensitiewe nie-membraan teiken in 'n vervroegde kalsium geïnduseerde intrasellulêre anaerobiese vorm van Listeria monocytogenes, en/of dat die Trc-Ca2+ komplekse belangrike komponente soos ’n membraan-gebonde elektron transport sisteem of bakteriële dehidrogenases inhibeer. Daar is ook bevestig, soos voorheen gevind, dat die Trcs kragtige, antimalaria aktiwiteit besit wat volgorde-spesifiek en nie-lities is. Die RW-peptiede het swak aktiwiteit getoon, maar ons resultate het weereens bewys dat peptiede wat meer hidrofobies en hemolities is, meer aktief is, veral die RW-peptiede wat die Trp analoog β-(bensoteïen-3-iel)-alanien (Bal) bevat. 'n Nuwe bevinding is dat een van die meer polêre Trc C analoë, genaamd triptosidien C (Tpc C), in teenstelling met Trc C, sterk antimalaria aktiwiteit het, wat 'n aanduiding is van die spesifieke volgorde en die rol van die Trp7 in aktiwiteit. Vanuit hierdie bevindinge word die peptied siklo- (VOLfP(Bal)fNQ(Bal)) as 'n voorloper vir toekomstige navorsing aangedui. Vir ons soeke na die Trc en Tpc C teiken(s), het ons hoë resolusie fluoressensie mikroskopie aangewend. Resultate toon dat Trc tot die ontwrigting van 'n neutrale lipied strukture en chromatien lei en sodoende groei beperk in die laat trofosoïet/vroeë skisont fases. Dit het aangedui dat die membraanstrukture wat neutrale lipiede bevat, sowel as chromatien, deur die Trcs geteiken word. 'n Verdere nuwe bevinding in hierdie studie was dat chloroquine (CQ) weerstandigheid nie net korreleer met weerstandigheid teen Trcs nie, maar dat die Trcs en CQ antagonisties optree teenoor mekaar se aktiwiteite. Dit dui op 'n gemeenskaplike teiken en die kosvakuool as 'n addisionele Trc teiken in P. falciparum word voorgestel.

Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: A new membrane based affinity separation system that is bio-specific, biocompatible, well characterised and capable of being regenerated or re-used is described. The amphiphilic non-ionic surfactant Pluronic® F108, was covalently derivatised to form two novel bioligands (Pluronic-Biotin and Pluronic-DMDDO) for the bio-specific immobilisation of avidin conjugated proteins and histidine tagged proteins respectively. Pluronic was also used to non-covalently functionalise nonporous membranes for ligand attachment and to simultaneously shield the surfaces from non-specific protein adsorption. Each component of this bioaffinity system (from the membrane matrix to the elution/desorption of the ligate/ligand system) was studied with the aim of producing a well characterised system and key quantitative data for the development of a robust, reliable, re-usable and scalable technology. Specifically, this study describes: 1. The fabrication and partial characterisation of nonporous planar and capillary membranes as model affinity matrices. 2. The development and evaluation of a robust protocol for solvent desorption and accurate colorimetric quantification of Pluronic® F108 and its derivatives. 3. Interfacial analysis of Pluronic adsorption onto nonporous affinity membranes, including the direct solid-state analysis of model, halogenated Pluronic derivatives using nuclear microprobe analysis. 4. Development of a surfactant based protocol for affinity membrane regeneration and re-use. 5. Specific bioaffinity immobilisation of avidin conjugated peroxidase onto biotinylated membranes in the presence of model protein foulants. 6. Cloning and expression of C-terminal hex-histidine tagged human cytochrome b5 into the bacterial expression system E. coli BL-21 DE3. 7. Development and characterisation of an immobilised metal affinity membrane system for metal chelation (Ni2+, Cu2+ and Zn2+) using a new chelator Pluronic- N,N-dicarboxymethyl-3,6-diazaoctanedioate and the bio-specific immobilisation of N-terminal hex-histidine tagged pantothenate kinase. / AFRIKAANSE OPSOMMING: 'n Nuwe membraan-gebaseerde affiniteitskeidingsisteem word beskryf wat biospesifiek, bioversoenbaar en goed gekarakteriseer is, en geregenereer of hergebruik kan word. Die amfifiliese nie-ioniese surfaktant Pluronic is kovalent gederivatiseer om twee nuwe bioligande (Pluronic-Biotien en Pluronic-DMDDO) te vorm vir biospesifieke immobilisering van proteïnligate. Pluronic is ook gebruik om nie-poreuse membrane niekovalent te funksionaliseer vir ligandaanhegting en om hulle oppervlaktes teen niespesifieke proteïen-adsorbsie af te skerm. Elke komponent van hierdie bioaffiniteitsisteem (van die membraanmatriks tot die uitwas/desorpsie van die ligaat/ligand sisteem) is ondersoek met die doel om 'n goed-gekarakteriseerde sisteem te produseer en om kwantitatiewe data te genereer vir die ontwikkeling van 'n robuuste, betroubare, herbruikbare en opskaleerbare tegnologie. Hierdie studie beskryf spesifiek: 1. Die fabrisering en gedeeltelike karakterisering van nie-poreuse planêre en kapillêre membrane as model affiniteitsmatrikse. 2. Die ontwikkeling en evaluering van 'n robuuste protokol vir oplosmiddel desorpsie en akkurate kolorimetriese kwantifikasie van Pluronic® F108 en afgeleides daarvan. 3. Intervlakanalises van Pluronic adsorpsie op nie-poreuse affiniteitsmembrane, insluitend die direkte vastetoestand analise van model ligand-gemodifiseerde Pluronic deur die gebruik van kern-mikrosonde analise. 4. Ontwikkeling van 'n surfaktant-gebaseerde protokol vir affiniteitsmembraan regenerering en hergebruik. 5. Spesifieke bioaffiniteitsimmobilisering van avidien-gekonjugeerde peroksidase op gebiotinileerde membrane in die teenwoordigheid van model bevuilende proteïne. 6. Klonering en uitdrukking van C-terminaal hex-histidien geëtiketeerde menslike sitochroom b5 in die bakteriële uitdrukkingsisteem E. coli BL-21 DE3. 7. Ontwikkeling en karakterisering van 'n geïmmobiliseerde metaalaffiniteitsmembraansisteem vir metaalchelering (Ni2+, Cu2+ en Zn2+) met behulp van die nuwe cheleerder Pluronic-N,N-dikarboksimetiel-3,6- diasaoktaandioaat en die bio-spesifieke immobilisering van N-terminaal hexhistidiengeëtiketerde pantotenaatkinase.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Twenty five years after the last major morphological revision, phylogenetic relationships were inferred on the basis of a new DNA dataset for the African orchid subtribe Disinae, which includes the large genus Disa and the small genus Schizodium. One nuclear gene region (ITS) and two plastid gene regions (trnLF and matK) were sequenced for 136 ingroup, representing 70% of all known Disinae species, as well as for 7 outgroup taxa. The combined data matrix contained 4094 characters and was analysed using parsimony and Bayesian inference. The generic status of Schizodium can no longer be supported, as it is deeply embedded within the genus Disa. Furthermore, the currently recognised subgenera do not reflect the phylogenetic relationships. Several of the currently recognised sections are monophyletic, others contain misplaced elements, while some are polyphyletic. These results necessitate a re-classification of the Disinae. A monotypic subtribe Disinae and a subdvision of Disa into eighteen sections is formally proposed. These sections are monophyletic, well-supported, morphologically distinguishable and are delimited to maximize the congruence with the previous classification. All currently known species are enumerated and assigned to sections. Likelihood optimisation onto a dated molecular phylogeny is subsequently used to explore the historical biogeography of Disa, as well as of three other Cape lineages (Irideae p.p., the Pentaschistis clade and Restionaceae), to find out where these lineages originated and how they spread through the Afrotemperate region. Three hypotheses have been proposed: (i) a tropical origin with a southward migration towards the Cape; (ii) a Cape origin with a northward migration into tropical Africa and (iii) vicariance. None of these hypotheses, however, has been thoroughly tested. In all cases, tropical taxa are nested within a predominantly Cape clade and there is unidirectional migration from the Cape into the Drakensberg and from there northwards into tropical Africa. Dating estimates show that the migration into tropical East Africa has occurred in the last 17 million years, consistent with the Mio-Pliocene formation of the mountains in this area. The same technique is then utilised to reconstruct the temporal occurrence of ancestral ecological attributes of the genus Disa. The first appearance of species in the grassland and savanna biomes, as well as in the subalpine habitat, are in agreement with the existing, reliable geological and paleontological information. This suggests that phylogenies can be used to date events for which other information is lacking or inconclusive, such as the age of the fynbos biome and the start of the winter rainfall regime in southern Africa. The results indicate that these are much older than what is currently accepted and date back to at least the Oligocene. / AFRIKAANSE OPSOMMING: Vyf-en-twintig jaar na die laaste groot morfologiese hersiening, is die filogenetiese verwantskappe van die Afrika orgideë subtribus Disinae, wat die groot genus Disa en die klein genus Schizodium insluit, in hierdie studie op grond van ‘n nuwe DNA datastel afgelei. Daar is van 136 binnegroep, wat 70% van alle bekende Disinae spesies verteenwoordig, sowel as sewe buitegroep taksa geenopeenvolgings van een nukleêre geen streek (ITS) en twee plastiedgeen streke (trnLF en matK) bepaal. Die gekombineerde data matriks het 4094 karakters bevat en is met die parsimonie en Bayesian metodes ontleed. Die generiese status van Schizodium kan nie hieruit ondersteun word nie, en is diep ingebed binne die genus Disa. Die huidiglik aanvaarde subgenera word ook nie deur hierdie filogenie ondersteun nie. Verskeie van die huidiglik herkende seksies is bevind om monofileties te wees, ander bevat verkeerd geplaasde spesies, terwyl ander polifileties blyk te wees. ’n Monotipiese subtribus Disinae en ’n onderverdeling van Disa in agtien seksies word formeel voorgestel. Dié seksies is monofilities, goed ondersteun, morfologies onderskeibaar en omskryf om maksimaal ooreen te stem met die vorige klassifikasie. Alle huidiglik bekende spesies word gelys en toegewys aan seksies. Waarskynlikheidsoptimalisering op ’n gedateerde molekulêre filogenie is dan gebruik om die historiese biogeografie van Disa te ondersoek, tesame met drie ander Kaapse groepe (Irideae p.p., die Pentaschistis klade en Restionaceae), om te bepaal waar hierdie groepe hulle oorsprong gevind het en hoe hulle na die “Afrotemperate“ streek versprei het. Drie hipoteses word voorgestel: (i) ’n tropiese oorsprong met ’n suidwaartse migrasie na die Kaap; (ii) ’n Kaapse oorsprong met ’n noordwaartse migrasie na tropiese Afrika, en (iii) vikariansie. Geen van hierdie hipoteses is egter vantevore deeglik getoets nie. In alle gevalle is bevind dat die tropiese taksa oorwegend binne ’n Kaapse klade gesetel is, en dat daar ’n eenrigting migrasie is van die Kaap na die Drakensberge en van daar noordwaarts na tropiese Afrika. Dateringsskattings toon dat die migrasie na tropiese Oos-Afrika in die laaste 17 miljoen jaar plaasgevind het, ooreenstemmend met die Mio-Plioseen vorming van die berge in die area. Dieselfde tegniek is daarna aangewend om die temporale voorkoms van voorvaderlike ekologiese eienskappe van die genus Disa te rekonstrueer. Die eerste voorkoms van die spesies in die grasveld en savanna biome, sowel as die subalpiene habitat, is in ooreenstemming met bestaande, betroubare geologiese en paleontologiese informasie. Dit suggereer dat filogenieë gebruik kan word om gebeurtenisse te dateer waarvoor daar informasie ontbreek of nie beslissend is nie, soos die ouderdom van die Fynbos bioom en die begin van die winterreënval stelsel in suider-Afrika. Die resultate dui daarop dat dit heelwat ouer is as wat tans aanvaar word en terugdateer na ten minste die Oligoseen.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This dissertation describes how the isolation and characterisation of ovine liver cytochrome b5 was accomplished by referring to the following goals achieved in this study: - The optimisation of the isolation and purification procedure for ovine liver microsomal cytochrome b5 in order to obtain sufficient material for aggregation and immunological studies. - The removal of the membrane binding domain of cytochrome b5 by means of tryptic digestion to establish the role of the carboxyl terminal in ovine cytochrome b5 aggregation. - The raising of antibodies against both the trypsin truncated and intact forms of cytochrome b5 to study the aggregation of the protein. - The investigation into the influence of purified cytochrome b5 on steroidogenesis in ovine adrenal microsomes. / AFRIKAANSE OPSOMMING: Die isolering en karakterisering van skaaplewersitochroom b5, soos beskryf in hierdie proefskrif, is uitgevoer deur die volgende doelwitte suksesvol af te handel: - Die optimalisering van die prosedure vir die suksesvolle isolering en suiwering van skaaplewersitochroom b5 ten einde genoegsame hoeveelhede van die suiwer proteïen te hê vir die bestudering van die aggregasie van die proteïen sowel as ‘n immunologiese studie. - Die verwydering van die membraanbindingsdomein van sitochroom b5 om die invloed van die karboksielterminaal op die aggregering van die proteïen te bestudeer. - Die gebruik van sowel die tripties gesnyde as die intakte vorms van sitochroom b5 om ‘n immuunrespons in hase op te wek vir die verkryging van sitochroom b5 spesifieke anti-liggame. - Die gebruik van die gesuiwerde proteïene om die invloed van sitochroom b5 op adrenale steroïdogenese te bestudeer.