Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / The aim of this study is the theoretical investigation of carbon fixation in sugarcane leaves. Sugarcane has a well known reputation for accumulating sucrose in the stalk to levels as high as 650 mM, almost a fifth of the plant’s fresh weight. Although this is an efficient accumulating mechanism, there is an even more efficient ‘carbon pump’ found in C4 plants. This is a well documented carbon concentrating mechanism and one of the first to be studied. However scientists are still trying to understand the carboxylating mechanism and the regulation thereof. It has been speculated that this mechanism is at its saturation level and elevating carbon dioxide will have little or no effect on further carbon fixation. Futher, studies suggest that the sucrose accumulating sink is able to regulate photosynthesis. Therefore a regulatory mechanism should exist from the sink to carbon fixation in order for such regulation to occur. Thework in this thesis therefore lays the foundation for investigating regulation of photosynthesis. The field of systems biology is the study of cellular networks by assemblingmodels. Pathways are considered as systems and notmerely collections of single components. This allows the interaction of pathway metabolites and the regulation that they have on one another to be studied. The questions asked pertaining to a pathway, will determine the types of model analysis. Structural analysis is useful for studying stoichiometric models, determining characteristics like energy consumption, futile cycles and valid pathways through a system at steady-state. Kinetic analysis on the other hand, gives insight into system dynamics and the control exerted by the system components, predicting time-course and steady states. In this thesis we begin to investigate photosynthesis in sugarcane leaves and the role it has in accumulating sucrose in the plant. Firstly, a structural model was developed incorporating carbon fixation, sucrose production in the leaf and subsequent transport of sucrose to the storage parenchyma and accumulation. The model was analysed using elementary mode analysis, showing that there are twelve routes for producing sucrose with no pathway beingmore energy efficient than any other. Further, it highlighted a futile cycle transporting triose phosphates and phosphoglycerate between the two photosynthetic compartments in the leaf. In the storage parenchyma, manymore futile cycleswere revealed,many of them energetically wasteful. Three other sets of elementary modes describe sucrose’s destination in either the vacuole or use in glycolysis or fibre formation, each with a different amount of required energy equivalents. The fourth set describes how sucrose cannot be converted to fibre precursors without also being used for glycolyis building blocks. Secondly, a kinetic model of carbon fixation in the leaf was assembled with the primary goal of characterising thismoiety-conserved cycle. This included the collation of kinetic data, incorporating volumes of the compartments and the areas of the location of the transporters into the model. This model was then analysed using metabolic control analysis. The model was able to predict metabolite concentration in the pathway at steady-state which were compared to those found experimentally. However, modifications need to be made to the model before further analysis is done so that the model predicted values match the experimental values more accurately. Time course analysis and response coefficients were also calculated for the carbon fixation cycle. Thework in this thesis therefore paves the way for understanding photosynthesis and its regulation in sugarcane leaves. Such work has the potential to pinpoint genetic engineering target points, allowing for better hybrid selection and propagation.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2005. / This study describes: 1. The cloning of baboon cytochrome P450 side-chain cleavage (CYP11A1) cDNA by in vitro site-directed mutagenesis. 2. The identification and sequencing of three baboon CYP11A1 mutants: CYP11A1a, CYP11A1b and CYP11A1c. 3. The expression and characterisation of baboon and human CYP11A1 cDNA, CYP11A1a, CYP11A1b and CYP11A1c in nonsteroidogenic COS-1 cells. The Km and V-values for the metabolism of 25-hydroxycholesterol were determined. 4. The construction of the first homology model of CYP11A1, using both mammalian (CYP2C5) and bacterial (CYP102) cytochromes P450 crystal structures as templates. 5. A structure/function study into the role of the amino acid residues Ile98, Lys103 and Thr291 in substrate binding and enzymatic activity. 6. The proposal of a topological model of the CYP11A1 active pocket as determined by substrate docking studies.

Part of work presented in this thesis has been published: Regulation of gene expressions by GC-rich DNA cis-elements / J.P. Hapgood, J. Riedemann and S.D. Scherer in Cell biology international, vol. 25, 2001. / Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The sea urchin G-string binding factor 1 (suGF1) has previously been shown to bind with high affinity and selectivity to stretches of contiguous deoxyguanosine residues, a DNA motif found in the upstream regions of many unrelated genes from several organisms. It has been proposed that suGF1 plays a role in transcriptional regulation. Homopurine.homopyrimidine stretches have been shown to form unusual DNA structures, in vitro. To investigate the potential of the suGF1 binding site to form unusual structures under certain conditions, synthetic oligodeoxyribonucleotides containing the suGF1 poly(dG).(dC) binding site were subjected to circular dichroism (CD) analyses. The CD results indicate that the suGF1 binding site forms a mixture of unusual DNA structures, as deduced by comparison with the spectra obtained for B-DNA, triplex and quadruplex conformations. These results are consistent with the hypothesis that suGF1 specifically recognises G-strings that exhibit unusual structures. Exhaustive database searches showed that suGF1 has no significant homology with any previously identified proteins or cDNAs from any species. Given the relevance of mammalian models to medical science, and since no sea urchin cell lines are currently available, the identification of a mammalian functional homologue would facilitate determination of the in vivo function of such a potentially important, putative, novel DNAbinding protein in mammalian cell lines. In this study sequence analysis tools were used to identify hORFX, a putative human functional homologue of suGF1. Similarities in the domain organisation of the two proteins, prompted an investigation into the DNA-binding properties of hORFX, as well as a more detailed structure prediction analysis, with a view to determining whether hORFX is a functional homologue of suGF1. hORFX was successfully expressed in vitro, but lacked the ability to specifically bind G-strings. Theoretical predictions suggest that suGF1 has a DNA-binding domain belonging to a different family to that predicted for hORFX, consistent with differences in their respective DNA-binding specificities. suGF1 and hORFX were predicted to have helix-turn-helix and helix-loop-helix DNA-binding domains, respectively. Taken together the results do not support the hypothesis that hORFX is a suGF1 homologue. To date, no direct evidence for the in vivo function of suGF1 has been obtained. With a view to performing transactivation assays in the future, the expression of suGF1 in yeast was investigated in this project. An suGF1 expression construct was engineered and transformed into a protease-deficient yeast strain. Nuclear extracts were prepared and subjected to SOS-PAGE and electrophoretic mobility shift assays (EMSAs). suGF1 was shown to be successfully expressed in yeast cells and exhibited similar G-string-binding properties to that of native and in vitro transcribed and translated (IVT) suGF1. The suGF1 eDNA was also subjected to in si/ico expression, which together with the SDSPAGE results of yeast nuclear extracts and IVT suGF1, indicated that the protein might be expressed as multiple truncated products, due to the utilisation of multiple AUG translation start sites. These in vitro results are crucial for the ultimate outcome and correct interpretation of future transactivation experiments and lay the foundation for further investigation into the possible role of suGF1 in transcriptional regulation. / AFRIKAANSE OPSOMMING: In die verlede is bewys dat die seepampoentjie G-string-bindende faktor (suGF1) hoë affiniteit en spesifisiteit vir aaneenlopende volgordes van deoksiguanosien residue besit. Hierdie DNA motief kom algemeen voor in die stroom-op gebiede van verskeie gene in verskillende organismes. Daar is 'n veronderstelling dat suGF1 betrokke is by die regulering van geenuitdrukking. Vroeër is bewys dat homopurien.homopirimidien-ryke areas die vermoë besit om in vitro ongewone DNA-strukture te vorm. Die potentiaal van die suGF1-bindingsetel om ongewone DNA-strukture te vorm is gevolglik deur sirkulêre dikroïsme (SD) analise ondersoek. Vergelyking van die spektra vir B-DNA-, tripleks- en kwadrupleks-strukture met dié van die suGF1-bindingsetel, toon duidelik dat laasgenoemde 'n mengsel van ongewone DNA konformasies, onder die spesifieke eksperimentele omstandigehede, aanneem. Deeglike inspeksie van die beskikbare geen- en proteïendatabasisse vir alle spesies het aangetoon dat suGF1 geen merkbare kDNA- of proteïenhomoloë besit nie. As gevolg van die belang van soogdiermodelsisteme in die mediese wetenskappe, asook die onbeskikbaarheid van seepampoentjie-sellyne, is 'n soektog na 'n funktionele suGF1 homoloog in soogdiere geloods. Die ontdekking van só 'n homoloog sal dit moontlik maak om die rol van hierdie potensiaal belangrike en unieke DNA-bindingsproteïen te ondersoek. Tydens hierdie soektog is spesiale analise-programme gebruik en 'n potensiële menshomoloog van suGF1, hORFX, is geïdentifiseer. Die mees prominente ooreenkoms tussen die twee proteïene is die soortgelyke rangskikking van funksionele motiewe. Gevolglik is die DNA-bindings eienskappe van die hORFX-proteïen ondersoek, insluitende 'n detaileerde struktuur-funksie-voorspelling ten einde vas te stel of dit wél 'n homoloog van suGF1 is. hORFX is suksesvol uitgedruk in vitro, maar besit nie die vermoë om dieselfde G-string waaraan suGF1 spesifiek bind te herken nie. Teoretiese analise het voorspel dat suGF1 en hORFX aan verskillende DNA-bindings proteïen-families behoort, aangesien suGF1 'n heliks-draai-heliks en hORFX 'n heliks-lus-heliks motief bevat. Hierdie inligting, tesame met die eksperimentele resultate, dui aan dat hORFX nie 'n homoloog van suGF1 is nie. Tot op hede is daar niks bekend aangaande suGF1 se funksie in vivo nie. Met die oog op transaktiveringseksperimente in die toekoms, is die ekspressie van suGF1 in gisselle tydens hierdie navorsingsprojek ondersoek. 'n suGF1 ekspressievektor is berei en gebruik om 'n protease-negatiewe gissellyn te transformeer. Kernekstrakte is ondersoek deur SDS-PAGE en elektroforetiese mobiliteitsessais. Daar is gevind dat suGF1 suksesvol uitgedruk is in die gisselle. Die rekombinante suGF1 besit G-volgorde bindingsaktiwiteite soortgelyk aan dié van suGF1 in kernekstrakte van seepampoentjies, asook in vitro getranskribeerde-en getransleerde suGF1. Die kDNA vir suGF1 is ook in silico uitgedruk. Tesame met die SDS-PAGE-resultate het laasgenoemde aangetoon dat die suGF1-kDNA veelvuldige AUG-kodons bevat vir die inisiasie van proteïentranslasie. Dit lei moontlik tot die translasie van 'n reeks proteïenprodukte wat verkort is aan die N-terminale kant, afgesien van die volledige suGF1-proteïen. Die in vitro resultate in geheel is essensieel vir die toekomstige uitvoering en interpretasie van transaktiveringseksperimente. Hierdie projek lê gevolglik die fondasie vir 'n verdere ondersoek na die rol van suGF1 in die regulering van geenuitdrukking.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: At the Mondi Kraft paper mill in Piet Retief, paper machine effluent is pre-treated by means of dissolved air flotation (DAF) and a mierostrainer prior to ultrafiltration (UF). Despite the rigorous pre-treatment of the effluent, severe fouling of the UF membranes still persisted, resulting in a sharp decrease in operational flux. In an attempt to improve the flux performance of the UF membranes an investigation was launched into the possible causes of membrane fouling. The study yielded the following results: Ultraviolet-visible (UV-Vis) spectrophotometric analyses of varIOUS effluent samples collected from different locations at the mill indicated the presence of aromatic compounds. Lignosulphonate appeared to be the main constituent in all the samples analysed. UV-Vis spectrophotometry was also performed on fouling extracted from membranes In order to evaluate the different approaches attempted to reduce membrane fouling. Most of the UV-Vis spectra obtained did not show the absorbance maxima below 210 nm that were seen for the paper machine effluent, DAF product, lignosulphonate and mierostrainer product. This indicated that the compounds with absorbance at lower wavelengths did not foul the membranes to the same extent as the aromatic substances with absorbance maxima between 230 and 400 nm. The influence of pH on the absorption of the various effluent samples was also investigated. An increase in pH resulted in (1) a "shift" in the wavelength scans from a lower to a higher wavelength, suggesting ionisation (deprotonation) with a subsequent delocalization of electrons and (2) an increase in the turbidity. The increase in turbidity which accompanied the Increase in pH could be explained by complex formation between the carboxylate ions, phenolic groups and divalent metal ions present in the effluent. Inductively coupled plasma analyses of several effluent samples with pH values 7 and 13 indicated the presence of significant amounts of Ca2+ ions in the effluent. There was a significant decrease in the Ca2+ levels with an increase in pH, which supported the hypothesis that Ca2+ might contribute to complex formation. This resulted in a decrease in solubility and an increase in turbidity. The addition of a chelating agent (ethylenediaminetetra-acetic acid disodium salt) to an effluent solution at pH 13 redissolved the precipitate and considerably reduced the turbidity. The subsequent addition of CaCh again induced precipitation and increased turbidity, confirming the role of Ca2+ in complex formation. Gel permeation chromatographic analyses of mierostrainer product at pH 13 showed the formation of high molecular mass organo-calcium complexes. The exact molecular mass of the complexes present in the mierostrainer product could not be determined by electro spray mass spectrometry because of their poor ionisation ability. Atomic force microscopy and scanning electron microscopy (SEM) showed distinct differences in the membrane surface texture before and after fouling. Furthermore, SEM images of the UF membranes exposed the limited ability of the 30 urn microstrainer, installed downstream from the DAF unit, to remove residual fibres from the DAF product. Static fouling experiments performed on all the flocculants and coagulants used In the paper-making process at the mill showed that none of these substances fouled the UF membranes. Cleaning of the UF membranes with Triton XIOO®, a nOn-IOnIC surfactant, caused a temporary increase in the operating flux to values higher than that of the initial flux. Mechanical cleaning of the UF membrane surface with spongebalIs proved to be one of the most effective and successful methods to prevent flux loss caused by fouling. Pre-coating of the UF membranes with Plutonic" FI08, another non-ionic surfactant, did not promote membrane productivity. Evaluation of various types of membranes indicated that hydrophilic or negatively charged membranes withstood membrane fouling more effectively than hydrophobic UF membranes under the same operating conditions. / AFRIKAANSE OPSOMMING: By Mondi Kraft se papier meule in Piet Retief word afloopwater vanaf die papiermasjiene vir hergebruik met behulp van ultrafiltrasie (UF) behandel. Opgeloste lugflotasie (OLF) en mikrosiwwing word as voorbehandeling vir die UF membraanproses ingespan. Ondanks die intensiewe voorafbehandeling wat toegepas word, vind daar geweldige aanvuiling van die UF membrane plaas wat tot die vinnige verlaging in bedryfsfluks aanleiding gee. 'n Ondersoek na die moontlike oorsake van membraan-aanvuiling het die volgende bevindinge opgelewer: Ultraviolet-sigbare (UV-Vis) spektroskopie van water monsters wat by die meule versamel is, het die teenwoordigheid van aromatiese komponente aangetoon, met lignosulfonaat die hoofkomponent in al die monsters wat ontleed is. Ekstrakte afkomstig van aangevuilde membrane is ook met behulp van UV-Vis-spektroskopie geanaliseer om verskeie benaderings te evalueer om 'n afname in membraan-aanvuiling te bewerkstellig. Die oorgrootte meerderheid spektra het nie die absorpsie maksima onder 210 nm aangetoon wat teenwoordig was in monsters van die papier masjien afloopwater, OLF uitvloeisel, lignosulfonaat en mikrosif produkwater nie. Dit het aangedui dat die komponente wat by laer golflengte absorbeer nie die UF membrane in dieselfde mate aanvuil as daardie komponente wat by hoër golflengtes (tussen 230 en 400 nm) absorbeer nie. Die invloed wat pH op die absorpsie van komponente teenwoordig in die onderskeie afloopwatermonsters het, is ook ondersoek. 'n Toename in pH het bygedra tot (1) 'n verskuiwing in die spektra vanaf 'n lae na 'n hoër golflengte vanweë ionisasie (deprotonering) met gevolglike delokalisasie van elektrone en (2) 'n toename in turbiditeit. Die toename in turbiditeit wat verband hou met die toename in pH was verduidelik aan die hand van kompleksvorming tussen die karboksilaat ione, fenoliese groepe en divalente metaal ione in die afloopwater. Induktief gekoppelde plasma analise van verskeie water monsters by pH 7 en 13 het die teenwoordigheid van 'n groot hoeveelheid Ca2+ aangetoon. 'n Verlaging in die vlakke van opgeloste Ca2+ het met die toename in pH verband gehou. Dit het die moontlike verbintenis tussen Ca2+ en kompleksvorming ondersteun wat bygedra het tot die afname in oplosbaarheid en toename in turbiditeit. Die byvoeging van etileendiamientetra-asynsuur-dinatriumsout, 'n kelerings reagens by afloopwater (pH 13) het die presipitaat weer in oplossing gebring en die turbiditeit merkwaardig verlaag. Die byvoeging van CaCh het weer presipitasie geïnduseer, met 'n gevolglike toename in turbiditeit. Hiermee is Ca2+ se rol in kompleksvorming bevestig. Gelpermeasie-chromatografiese analise van die mikrosif produk (pH 13) het die vorming van hoë molekulêre massa organo-kalsium komplekse bevestig. Dit was egter nie moontlik om met behulp van massaspektrometrie die korrekte molekulêre massa van die komplekse te bepaal nie vanweë hul onvermoë om te ioniseer. Atomiese krag mikroskopie en skandeer elektron mikroskopie (SEM) het duidelik die voor en na verskil getoon wat aanvuiling op die membraantekstuur gehad het. 'n SEM foto van die aangevuilde UF membraan het die onvermoë van die mikrosif blootgelê om oorblywende vesels vanuit die OLF produkwater te verwyder. Resultate bekom gedurende passiewe aanvuilingseksperimente het aangetoon dat al die in-proses flokkulante en koagulante wat gebruik word by die papier meule geen bydrae tot die aanvuiling van die UF membrane maak nie. Skoonmaak van die UF membrane met Triton XIOO® bring 'n verhoging in bedryfsvloed teweeg, maar die verhoging, wat hoër as die oorspronklike vloed is, is kortstondig. Meganiese skoonmaak van die buismembrane met behulp van sponsballe blyk die mees effektiewe skoonmaakmetode te wees. Voorafbehandeling van die UF membrane met Plutonic" F 108 het nie die membraanproduktiwiteit verhoog nie. Daar is ook bevind dat hidrofiliese of negatief gelaaide membrane groter weerstand bied teen aanvuiling in vergelyking met hidrofobiese UF membrane onder dieselfde bedryfstoestande.

Dissertation (PhD)--University of Stellenbosch, 2004 / ENGLISH ABSTRACT: Newcastle disease (NO) and mycoplasma infections in ostriches have considerable economic implications for the South African ostrich industry in that NO is a limiting factor in the export of ostrich products to the European Union and mycoplasma infections cause stock losses, reduced production, reduced hatchability and downgrading of carcasses. In the first section of this dissertation, the role of passively acquired and mucosal immunity in protection of ostrich chicks against Newcastle disease virus (NOV) was investigated. Ostrich hen serum IgG and yolk IgY were isolated and characterized, and the transfer of maternal anti-NOV antibodies to the egg yolk was determined using an enzyme-linked immunosorbent assay (ELISA). Results indicated that anti-NOV antibodies were successfully transferred from the ostrich hen to the egg yolk. In addition, ostrich IgA was isolated, characterized and rabbit anti-ostrich IgA antibodies produced and used for measuring mucosal anti- NOV IgA antibodies produced in response to mucosal vaccination. Results indicated that the live La Sota vaccine stimulates IgA production and thus mucosal immunity in ostrich chicks. In the second section of this dissertation, ostrich mycoplasmas were isolated and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches carry three unique mycoplasmas, which are phylogenetically quite divergent. The 16S rRNA gene sequences of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches by PCR. The last section of this dissertation focuses on avian malaria in African penguins and the management of this disease during rehabilitation. The Foundation for the Conservation of Coastal Birds (SANCCOB) is a seabird rescue and rehabilitation centre, which is largely dedicated to the rehabilitation of diseased, injured and oiled penguins. Significant mortalities due to avian malaria occur at this facility. The aim of this study was the development of an ELISA for the purpose of assessing the natural levels of anti-Plasmodium antibodies in African penguins on entry into the SANCCOB facility and during rehabilitation. Results indicated significant increases in anti- Plasmodium antibody levels after entry, which was not influenced by oiling. Infection with malaria and not parasite recrudescence was viewed to be the cause of this increase, indicating a possible role of the SANCCOB facility in exposing penguins to avian malaria. / AFRIKAANSE OPSOMMING: Newcastlesiekte (NS) en mikoplasmainfeksies in voltruise het geweldige ekonomiese implikasies vir die Suid-Afrikaanse volstruisbedryf. Die rede hiervoor is dat NS 'n beperkende faktor in die uitvoer van volstruisprodukte na die Europese Unie is, en mikoplasmainfeksies tot kudde verliese, verlaagde produksie en uitbroei asook lae gradering van karkasse lei. In die eerste gedeelte van hierdie proefskrif is die rol van passiewe- en mukosale-immuniteit in die beskerming van volstruiskuikens teen NS virus (NSV) ondersoek. Volstruishenserum IgG en eier IgY is geïsoleer en gekarakteriseer en die oordrag van maternale anti-NSV antiliggame na die eier ondersoek met behulp van 'n 'enzyme-linked immunosorbent assay' (ELISA). Resultate het getoon dat anti-NSV antiliggame suksesvol van die hen na die eier oorgedra is. Volstruis IgA is ook geïsoleer, gekarateriseer en konyn anti-volstruis IgA antiliggame geproduseer wat gebruik is vir die bepaling van mukosale anti-NSV IgA antiliggame in reaksie op mukosale immunisering. Resultate het getoon dat lewendige La Sota entstof IgA produksie stimuleer en dus tot mukosale-immuniteit in volstruiskuikens lei. In die tweede gedeelte van hierdie proefskrif is volstruismikoplasmas geïsoleer en geïdentifiseer met behulp van 16S rRNA geenopeenvolgingsbepalings. Hierdie volgordes het getoon dat drie unieke mikoplasmas in volstruise voorkom wat filogeneties verskillend blyk te wees. Die 16S rRNA geenopeenvolgings van die volstruismikoplasmas is gebruik vir die ontwikkeling van spesifieke inleiers vir die PKR identifisering en diagnose van mikoplasmainfeksies in volstruise. Die laaste gedeelte van hierdie proefskrif fokus op voëlmalaria in die Afrika pikkewyn en die bestuur van hierdie siekte gedurende rehabilitasie. Die 'South African Foundation for the Conservation of Coastal Birds' (SANCCOB) is 'n seevoëlreddingsen rehabilitasie-sentrum vir siek, beseerde en ge-oliede pikkewyne. Hierdie sentrum het egter aansienlike vrektes as gevolg van voëlmalaria. In hierdie studie is 'n ELISA ontwikkel vir die bepaling van natuurlike anti-Plasmodium antiliggaamvlakke van pikkewyne by aankoms en tydens rehabilitasie by SANCCOB. Resultate het 'n toename in anti-Plasmodium antiliggaamvlakke getoon na toelating wat nie beïnvloed is deur olie nie. Hierdie toename kan toegeskryf word aan nuwe malariainfeksies en nie 'n heruitbraak van bestaande infeksies nie wat daarop dui dat pikkewyne aan voëlmalaria blootgestel word by die SANCCOB-sentrum.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: With the recent advances in the field of molecular biology, there is an increased need to integrate data on the various constituents of the cell in kinetic models that can predict and describe cellular behavior. When working towards a description of the entire cell using such kinetic models, the question arises: How do we compare different models for a given biological network? This is the central question addressed in my thesis and I developed and applied mathematical and computational methods for comparing dozens of existing models of erythrocyte and yeast glycolysis. To compare the steady-state behavior in models of erythrocyte glycolysis, I focussed on the function of the pathway, which is to supply the cell with Gibbs-free energy (γ- phosphate of ATP). I used supply-demand analysis in the framework of metabolic control analysis to make this comparison, which revealed that the ATP concentrations were homeostatically buffered at varying supply rates. I also applied this approach to compare steady-state behavior in models of yeast glycolysis, finding that they were not necessarily optimized for homeostatic maintenance of the ATP concentration and that in models for this organism the rate of ATP production is often determined by the supply reactions of glycolysis. In addition, I tested whether a kinetic model can describe novel behavior if it is adjusted to conditions different from those for which the model was originally constructed. More specifically, using a model of steady-state yeast glycolysis, I showed that small adjustments to the original enzyme concentrations are enough to obtain an oscillating model, which shows a remarkable resemblance to the experimentally observed oscillations. Importantly, some of these enzyme concentrations changes are known to occur during the pre-treatment of the cells which is necessary to obtain oscillatory behavior. To the best of my knowledge, the resulting model is the first detailed kinetic model that describes the experimentally observed strong synchronization of glycolytic oscillations in yeast populations. To analyze the dynamic behavior of yeast glycolytic models and to compare different models in terms of dynamics, I introduced a framework used in physics and engineering to create a vector based, two dimensional graphical representation of the oscillating metabolites and reactions of glycolysis. Not only was it possible to make a concise comparison of the set of models, but with the method I could also quantify the contribution of the interactions in the network to the transduction of the oscillations. Furthermore I could distinguish between different mechanisms of oscillation for each of the models, and demonstrated how the framework can be used to create such representations for experimental data sets. / AFRIKAANSE OPSOMMING: Met die onlangse vooruitgang in die veld van molekulere biologie, is daar ?n toenemende behoefte om data rakende die verskeie komponente van die sel in kinetiese modelle te integreer, om sodanig selgedrag te voorspel en te beskryf. As daar gepoog word om ’n beskrywing van die sel as geheel te verkry d.m.v. sulke kinetiese modelle, onstaan die vraag: Hoe vergelyk ons verskillende modelle van ’n gegewe biologiese netwerk? Dit is die sentrale vraag wat my tesis aanspreek en ek het wiskundige en numeriese metodes ontwikkel en toegepas om talle bestaande modelle van gis- en eritrosietglikolise te vergelyk. Om die bestendige-toestand gedrag in modelle van eritrosietglikolise te vergelyk, het ek gefokus op die funksie van die padweg, naamlik om die sel met Gibbs-vrye energie (γ-fosfaat van ATP) te voorsien. Ek het vraag-aanbod analiese in die raamwerk van metaboliese kontrole analiese gebruik om hierdie vergelyking te maak, wat getoon het dat die ATP konsentrasies homeostaties gebuffer was by verskillende aanbod tempos. Ek het ook hierdie aanpak gebruik om die bestendige-toestand gedrag in modelle van gisglikolise te vergelyk, en het bevind dat hulle nie noodwendig geoptimiseer is om ?n homeostatiese balans in die ATP konsentrasie te handhaaf nie, en dat in modelle vir hierdie organisme, die tempo van ATP produksie dikwels bepaal word deur die aanbod reaksies van glikoliese. Ek het verder ook bepaal of so ?n kinetiese model nuwe soorte gedrag kan beskryf, as dit aangepas word aan omstandighede wat verskil van dié waarvoor die model oorspronklik gekonstrueer was. Meer spesifiek, deur ?n model van bestendige-toestand gisglikolise te gebruik, kon ek wys dat klein veranderinge aan die oorspronkline ensiem konsentrasies genoeg was om ?n ossilerende model te verkry, wat opmerklik ooreenstem met die eksperimenteel waargenome ossilasies. Let ook daarop dat sommige van hierdie ensiem konsentrasie veranderinge plaasvind tydens die voorafbehandeling van die selle, wat essensieel is om die ossilasies waar te neem. Tot die beste van my kennis is die model wat ek met hierdie prosedures verkry het, die eerste gedetaileerde kinetiese model wat die eksperimenteel waargenome sterk sinkronisasie in ossilerende gis populasies voorspel. Om gis glikolitiese modelle te vergelyk in terme van hul dinamiese gedrag, het ek ?n raamwerk wat in fisika en ingeneurswese gebruik word, ingespan om ?n vektor-gebasseerde, twee dimensionele grafiese voorstelling van die ossilerende metaboliete en reaksies te maak. Hierdie raamwerk het dit nie net moontlik gemaak om ?n kompakte vergelyking van ?n stel modelle te maak nie, maar ek kon ook die bydrae van interaksies in die netwerk tot transduksie van die ossilasies kwantifiseer. Ek kon verder onderskeid tref tussen die verskillende ossilasiemeganismes vir elk van die modelle, en het ook gedemonstreer hoe die raamwerk gebruik kan word om sulke voorstellings vir eksperimentele datastelle te skep.

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The literature contains conflicting ideas about the effectiveness of diagrams, and their constituent symbolism as teaching and learning tools. In addition, only limited research has been specifically conducted on the presentation and interpretation of arrow symbolism used in biology diagrams, let alone on the nature, source and remediation of student difficulties caused by arrows. On the basis of this limited research and 30 years of experience of teaching biology at secondary-level, the author suspected that students might have difficulties interpreting arrow symbolism in diagrams used as explanatory tools and decided to thoroughly investigate this issue. The hypothesis, 'Secondary-level students have difficulty with the use of arrow symbolism in biology diagrams' was formulated and the following broad research questions defined to address the hypothesis: 1. How much of a problem is arrow symbolism in diagrams? 2. How effectively is arrow symbolism used in diagrams to promote the communication of intended ideas? 3. To what extent does the design of arrow symbolism in diagrams influence students ' interpretation and difficulties? 4. How can the emerging empirical data and ideas from literature be combined to illustrate the process of interpretation of arrow symbolism? 5. What measures can be suggested for improving the presentation and interpretation of arrow symbolism in biology diagrams at secondary-level? To address Research question 1, a content analysis of all arrow symbolism in seven popular secondary-level biology textbooks was undertaken. This revealed a wide diversity of arrow styles, spatial organisations, purposes and meanings that could be confusing to students. These results suggested the need for an evaluation of the effectiveness of arrow symbolism (Research question 2). As there was no definitive set of guidelines available for specifically evaluating arrows, general guidelines from the literature on diagrams were used to develop a set of 10 criteria, to evaluate the syntactic, semantic and pragmatic dimensions of arrow symbolism, which were validated by selected educators, students and a graphic design expert. Application of the criteria (which constituted expert opinion) to the arrow symbolism used in 614 realistic, stylised and abstract diagram types, revealed a relatively high incidence (30%) of inappropriately presented arrow designs that could mislead students. To establish whether this problem could be the cause of student difficulties, and to thereby address Research question 3, a stylised and an abstract diagram were selected and evaluated according to the criteria. The results of the evaluation were compared to the responses given by 174 students to a range of written and interview probes and student modified diagrams. In this way, student performance was correlated with expert opinion. The results confirmed that students experience a wide range of difficulties (26 categories) when interpreting arrow symbolism, with some (12 categories) being attributable to inappropriately presented arrow symbolism and others (14 categories) to student-related processing skills and strategies at both surface- and deeper-levels of reasoning. To address question 4, the emerging empirical data from the evaluation and student studies was combined with a wide range of literature, to inform the development of a 3-level, non-tiered model of the process of interpretation of arrow symbolism in diagrams. As this model emphasised the importance of both arrow presentation in diagrams and arrow interpretation by students, it could be used as an effective explanatory tool as well as a predictive tool to identify sources of difficulty with the use of arrow symbolism. This model was, in turn, used to inform the compilation of a range of guidelines for improving the presentation and interpretation of arrow symbolism, and so target Research question 5. These, and other guidelines grounded in the data and relevant literature, were suggested for all role players, including students, educators, textbook writers, graphic artists and researchers, to use as remedial tools. Future research should focus on the implementation of these guidelines and studying their effectiveness for improving the presentation and interpretation of diagrams with arrow and other types of symbolism. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

This study forms part of an investigation into the possible relevance of the lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this study, the main technique adopted was the Tokuyasu "cryo" method, in which the tissues were fixed, frozen and sectioned and labelled using the relevant antibodies, which were detected with protein A gold probes. In order to implement the Tokuyasu technique, it was necessary to rebuild a knife maker, for the production of adequately sharp glass knives, and to modify a sputter-coater into a glow-discharger, for rendering carbon-coated grids hydrophilic, to promote adhesion of hydrated sections. This study was directed towards human tissues and peptide antibodies were investigated as a means of avoiding isolation of proteins from scarce human tissue, and as a means of obtaining antibodies that will target specific regions of proteins of interest. Peptide antibodies were also considered promising for studies of proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer therapy. Various prediction programmes were investigated for their effectiveness in predicting whether a given peptide sequence will elicit antibodies that will react with the native protein. Successful prediction would increase the success rate of peptide antibody production and thus lower the cost. Leucocytes were studied as a model of an invasive cell, since they are more readily available than tumour cells and serve the purpose during the development of methods. In the course of these studies, an optimal protocol for the fixation of PMNs was developed, involving lateral fixation of cut sections, that should be useful for future studies on these cells. Elastase and cathepsins D and G were found on the surface of activated PMNs and could thus play a role in the invasive properties of these cells. Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed Neo-T counterparts revealed that upon transformation, lysosomes shift from a perinuclear position, to a more peripheral position. None of the cathepsins studied was found on the cell surface of either the normal or ras-transfected cells, suggesting that surface distribution of these enzymes may not be a requirement for invasiveness. These studies suggest that immunocytochemical investigation of cells, in the process of invading through a barrier membrane, might be profitable in elucidating the role of proteinases in invasive cancer. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.

Trypanosoma brucei and T. congolense are protozoan parasites that infect humans, domestic livestock and wildlife in Africa. These parasites undergo complex morphological and biochemical changes, during the various stages of their life cycle. These changes correlate with alterations in the levels of trypanosomal lysosomal cysteine proteases, suggesting a role for transcriptional regulation of the cysteine protease in these parasites. The mechanism of this regulation is not yet understood nor have the promoter regions of the cloned trypanosome cysteine protease genes been investigated. This study involved an attempt to clone the T. brucei and T. congolense DNA fragments containing the promoter regions as the initial step in the investigation of the control elements of the cysteine protease gene. Trypanosomes were isolated from infected rat blood employing a combination of the methods of isopicnic isolation on Percoll gradients and DEAE-cellulose anion exchange resin chromatography. Approximately 5 x 10⁹ viable trypanosome cells were isolated from the infected rat blood and chromosomal DNA (approximately 500 μg) was extracted by alkaline-lysis method. Trypanosome genomic libraries were initially constructed in Eschericia coli HB101 employing the positive selection vector pEcoR251. The Trypanosoma brucei pEcoR251 library contained 6 000 recombinants and the Trypanosoma congolense library contained 15 000 recombinants. Plasmid DNA was then extracted from pools of recombinants, employing the alkaline-lysis method, digested with EcoRl restriction endonuclease and resolved by agarose gel electrophoresis. After Southern hybridisation, the pEcoR251 libraries did not reveal any putative clones containing the fragment of interest when probed with both an oligonucleotide probe and the PCR generated dsDNA probe. Genomic libraries were then constructed in the phagemid pUC119. The T. brucei and T. congolense genomic libraries contained 33 000 and 27 000 recombinants respectively. Recombinants from the T. brucei and T. congolense libraries were pooled in lots of 400 and 300 respectively. Of the 80 T. brucei plasmid pools that were screened 30 pools contained fragments that hybridised with the probe whilst 12 pools from the 90 T. congolense library pools that were screened contained fragments that hybridised with the probe. Putative clones identified appeared to contain inserts, ranging between two and seven kb in size. A partial T. congolense library consisting of approximately 12 pools was screened by colony hybridisation for identification of individual clones and 76 putative clones were identified. After confirmation of these putative clones on a dot blot using a DIG-labelled dsDNA probe, a selection of 30 putative clones were subjected to Southern hybridisation using a DIG-labelled DNA probe. Following Southern hybridisation 23 putative clones were identified to contain DNA inserts of interest in the range of two to seven kb. Five clones, designated pCPC1, pCPC2, pCPC3, pCPC4 and pCPC5 were then selected for further restriction mapping. Clone pCPC4 contains a seven kb fragment of T. congolense genomic DNA. A partial T. brucei library consisting of approximately 30 pools was screened by colony hybridisation for the identification of individual putative clones. Although plasmid pools containing putative clones were identified repeatedly by Southern blotting and DNA/DNA hybridisation, it was not possible to identify individual putative clones following transformation into E. coli MV1184 and colony hybridisation. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.