Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is an independent predictor of cardiovascular morbidity and allcause mortality. Significantly, it is considered a modifiable cardiovascular risk factor as its regression increases overall survival and reduces the frequency of adverse cardiac events. A clear understanding of LVH pathogenesis is thus imperative to facilitate improved risk stratification and therapeutic intervention. Hypertrophic cardiomyopathy (HCM), an inherited cardiac disorder, is a model disease for elucidating the molecular mechanisms underlying LVH development. LVH, in the absence of increased external loading conditions, is its quintessential clinical feature, resulting from mutations in genes encoding sarcomeric proteins. The LVH phenotype in HCM exhibits marked variability even amongst family members who carry the same disease-causing mutation. Phenotypic expression is thus determined by the causal mutation and additional determinants including the environment, epigenetics and modifier genes. Thus far, factors investigated as potential hypertrophy modifiers in HCM have been relatively removed from the primary stimulus for LVH; and the few studies that have been replicated yielded inconsistent results. We hypothesized that the factors that closely interact with the primary stimulus of faulty sarcomeric functioning, have a greater capacity to modulate it, and ultimately the LVH phenotype in HCM. Plausible candidate modifiers would include factors relating to the structure or function of the sarcomere, including known HCM-causal genes; and the enzymes that function in sarcomere-based energetics. Indeed, the literature highlights the relevance of sarcomeric proteins, Ca2+-handling and myocardial energetics in the development of LVH in HCM. This study, therefore, set out to evaluate the hypertrophy-modifying capacity of such factors by means of family-based genetic association testing in 27 South African HCM families in which one of three unique HCM-causing founder mutations segregates. Moreover, the single and combined effects of 76 variants within 26 candidate genes encoding sarcomeric or sarcomere-associated proteins were investigated. The study identified a modifying role in the development of hypertrophy in HCM for each of the candidate genes investigated with the exception of the metabolic protein-encoding gene, PRKAG1. More specifically, single variant association analyses identified a modifying role for variants within the genes MYH7, TPM1 and MYL2, which encode proteins of the sarcomere, as well as the genes CPT1B, CKM, ALDOA and PRKAB2, which encode metabolic proteins. Haplotype-based association analyses identified combined modifying effects for variants within the genes ACTC, TPM1, MYL2, MYL3 and MYBPC3, which encode proteins of the sarcomere, as well as the genes CD36, PDK4, CKM, PFKM, PPARA, PPARG, PGC1A, PRKAA2, PRKAG2 and PRKAG3, which encode metabolic proteins. Moreover, a number of variants and haplotypes showed statistically significant differences in effect amongst the three HCM founder mutation groups. The HCM-modifier genes identified were prioritised for future studies according to the number of significant results obtained for the four tests of association performed. The genes TPM1 and MYBPC3, which encode sarcomeric proteins, as well as the genes PFKM and PRKAG2, which encode metabolic proteins, were identified as stronger candidates for future studies as they delivered multiple significant results for various statistical tests. This study makes a novel contribution to the field of hypertrophy research as it tested the hypothesis that structural or energy-related factors located within the sarcomere may act as modifiers of cardiac hypertrophy in HCM, and succeeded in identifying a modifying role for many of the candidate genes selected. The significant results include substantial single and within-genecontext variant effects; and identified sizeable variation in the risk of developing LVH owing to the compound effect of the modifier and the individual founder mutations. Collectively, these findings enhance the current understanding of genotype/phenotype correlations and may, as consequence, improve patient risk stratification and choice of treatment. Moreover, these findings emphasize the potential for modulation of disease by further elucidation of some of the avenues identified. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is ‘n onafhanklike voorspeller van kardiovaskulêre morbiditeit en van mortaliteit weens alle oorsake. Van belang is dat dit ‘n wysigbare kardiovaskulêre risiko faktor is, aangesien die afname daarvan algehele oorlewing verhoog en die frekwensie van nadelige kardiale voorvalle verlaag. ‘n Duidelike begrip van LVH patogenese is dus noodsaaklik om verbeterde risiko stratifikasie en terapeutiese intervensie te fasiliteer. Hipertrofiese kardiomiopatie (HKM), ‘n oorerflike hart-siekte, is ‘n model-siekte vir die uitpluis van die molekulêre meganismes onderliggend aan die ontwikkeling van LVH. LVH, in die afwesigheid van verhoogde eksterne lading, is die kern kliniese simptoom van HKM en die gevolg van mutasies in die gene wat kodeer vir sarkomeriese proteïene. Die LVH fenotiepe in HKM toon merkbare veranderlikheid selfs in familie-lede wat dieselfde siekte-veroorsakende mutasie dra. Die fenotiepe word dus bepaal deur die siekte-veroorsakende mutasie asook addisionele determinante insluitend die omgewing, epigenetika en modifiserende gene. Potensiële hipertrofie-modifiseerders wat tot dusver bestudeer is, is betreklik verwyder van die primêre stimulus vir LVH en die paar studies wat gerepliseer is, het teenstrydige resultate gelewer. Ons hipoteseer dat die faktore wat in noue interaksie met die primêre stimulus van foutiewe sarkomeriese funksionering is, ‘n groter kapasitieit het om dit en uiteindelik die LVH fenotiepe in HKM, te moduleer. Aanneemlike kandidaat-modifiseerders sou insluit faktore wat betrekking het tot die struktuur en funksie van die sarkomeer insluitend HKM-oorsaaklike gene en die ensieme wat funksioneer in sarkomeer-gebaseerde energetika. Die literatuur beklemtoon inderdaad die relevansie van sarkomeriese proteïene, Ca2+-hantering en miokardiese energetika in die ontwikkeling van LVM in HKM. Hierdie studie het beoog om die hipertrofie-modifiserende kapasiteit van sulke faktore te evalueer deur middel van familie-gebaseerde genetiese assosiasie toetse in 27 Suid-Afrikaanse HKM families waarin een van drie unieke HKM-stigter mutasies segregeer. Verder was die enkel en gekombineerde effekte van 76 variante binne 26 kandidaat gene wat kodeer vir sarkomeer en sarkomeer-geassosieerde proteïene, ondersoek. Hierdie studie het ‘n modifiserende rol in die ontwikkeling van hipertrofie in HKM geïdentifiseer vir elk van die kandidaat gene wat ondersoek is, met uitsluiting van die PRKAG1, wat kodeer vir ‘n metaboliese proteïen. Meer spesifiek, enkel variant assosiasie analises het ‘n modifiserende rol geïdentifiseer vir variante in die gene MYH7, TPM1 en MYL2, wat kodeer vir sarkomeriese proteïene, asook die gene CPT1B, CKM, ALDOA en PRKAB2, wat kodeer vir metabolise proteïene. Haplotipe-gebaseerde assosiasie-analises het gekombineerde modifiserende effekte geïdentifiseer vir variante in die gene ACTC, TPM1, MYL2, MYL3 en MYBPC3, wat kodeer vir strukturele proteïene van die sarkomeer asook die gene CD36, PDK4, CKM, PFKM, PPARA, PPARG, PGC1A, PRKAA2, PRKAG2 en PRKAG3, wat kodeer vir metabolise proteïene. Verder het ‘n aantal variante en haplotipes statisties betekenisvolle verskille in effek tussen die drie HKM-stigter mutasie groepe getoon. Die HKM-modifiserende gene wat geïdentifiseer is, is verder geprioritiseer vir toekomstige studies volgens die aantal beduidende resultate wat vir die vier assosiasie toetse verkry is. Die gene TPM1 and MYBPC3, wat kodeer vir sarkomeriese proteïene, asook die gene PFKM and PRKAG2, wat kodeer vir metaboliese proteïene, is geïdentifiseer as sterker kandidate vir verdere studies omdat veelvuldige beduidende resultate vir die verskeie statistiese toetse deur hulle gelewer is. Hierdie studie maak ‘n nuwe bydrae tot die veld van hipertrofie navorsing omdat dit die hipotese dat strukturele en energie-verwante faktore, wat binne die sarkomeer geposisioneer is, potensieel as modifiseerders van kardiale hipertropfie in HKM kan optree, ondersoek het. Dit slaag ook daarin om ‘n modifiserende rol vir baie van die geselekteerde kandidaatgene te identifiseer. Die beduidende resultate sluit in aansienlike enkel en binne-geen-konteks variant-effekte en aansienlike variasie in die risiko vir LVH ontwikkeling verskuldig aan die gekombineerde effek van modifiseerder en individuele stigter mutasies. Gesamentlik verbeter hierdie bevindinge die huidige begrip van genotipe/fenotipe korrelasies en dit mag tot gevolg hê verbeterde pasiënt risiko stratifikasie en keuse van behandeling. Verder beklemtoon hierdie bevindinge die potensiaal vir siekte modulering deur verdere uitpluis van sekere van hierdie geïdentifiseerde navorsingsrigtings. / National Research Foundation / Dr. Paul van Helden / Stellenbosch University

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The multiplication of cells proceeds through consecutive phases of growth and division (G1, S, G2 and M phases), in a process known as the cell cycle. The transition between these phases is regulated by so-called checkpoints, which are important to ensure proper functioning of the cell cycle. For instance, mutations leading to faulty regulation of the G1/S transition point are seen as one of the main causes of cancer. Traditionally, models for biological systems that show rich dynamic behavior, such as the cell cycle, are studied using dynamical systems analysis. However, using this analysis method one cannot quantify the extent of control of an individual process in the system. To understand system properties at the process level, one needs to employ methods such as metabolic control analysis (MCA). MCA was, however, developed for steady-state systems, and is thus limited to the analysis of such systems, unless the necessary extensions would be made to the framework. The central question of this thesis focuses on quantifying the control in mathematical models of the G1/S transition by the individual cell cycle processes. Since MCA was never applied to the cell cycle, several new methods needed to be added to the framework. The most important extension made it possible to follow and quantify, during a single cell cycle, the control properties of the individual system processes. Subsequently, these newly developed methods were used to determine the control by the individual processes of an important checkpoint in mammalian cells, the restriction point. The positioning of the restriction point in the cell cycle was distributed over numerous system processes, but the following processes carried most of the control: reactions involved in the interplay between retinoblastoma protein (Rb) and E2F transcription factor, reactions responsible for the synthesis of Delayed Response Genes and Cyclin D/Cdk4 in response to growth signals, the E2F dependent Cyclin E/Cdk2 synthesis reaction, as well as the reactions involved in p27 formation. In addition it was shown that these reactions exhibited their control on the restriction point via the Cyclin E/Cdk2/p27 complex. Any perturbation of the system leading to a change in the restriction point could be explained via its e ect on the Cyclin E/Cdk2/p27 complex, showing a causal relation between restriction point positioning and the concentration of the Cyclin E/Cdk2/p27 complex. Finally, we applied the new methods, with a modular approach, to compare a number of cell cycle models for Saccharomyces cerevisiae (budding yeast) and mammalian cells with respect to the existence of a mass checkpoint. Such a checkpoint ensures that cells would have a critical mass at the G1/S transition point. Indeed, in budding yeast, a correction mechanism was observed in the G1 phase, which stabilizes the size of cells at the G1/S transition point, irrespective of changes in the specific growth rate. This in contrast to the mammalian cell cycle models in which no such mass checkpoint could be observed in the G1 phase. In this thesis it is shown that by casting specific questions on the regulation and control of cell cycle transition points in the here extended framework of MCA, it is possible to derive consensus answers for subsets of mathematical models. / AFRIKAANSE OPSOMMING: Die selsiklus bestaan uit agtereenvolgende groei- en delingsiklusse wat tot selvermeerdering lei. Die siklus word gekenmerk deur onderskeie fases (G1, S, G2 en M) wat deur sogenaamde beheerpunte gereguleer word. Hierdie beheerpunte verseker dat selvermeerdering nie ongekontroleerd kan plaasvind nie en mutasies wat lei tot foutiewe regulering van die G1/S transisiepunt word as een van die hoofoorsake van kanker beskou. Die hoofdoel van hierdie studie was om die beheer wat selsiklusprosesse op die G1/S transisie uitoefen met behulp van wiskundige modelle te kwantifiseer. Omdat biologiese sisteme soos die selsiklus ryk dinamiese gedrag vertoon, word hulle tradisioneeldeur middel van dinamiese sisteemanalise bestudeer. Die analisemetode beskik egter nie oor die vermoë om die hoeveelheid beheer wat afsonderlike sisteemprosesse op 0n sisteemeienskap uitoefen te kwantifiseer nie. Om sisteemeienskappe op prosesvlak te verstaan moet metodes soos metaboliese kontrole analise (MKA) ingespan word. MKA was egter ontwikkel om sisteme in 0n bestendige toestand te analiseer en aangesien MKA nog nooit vantevore vir selsiklus analises gebruik was nie, moes nuwe MKA tegnieke gedurende die studie ontwikkel word. Die belangrikste van die metodes maak dit moontlik om beheer (soos uitgeoefen deur die onderskeie sisteemprosesse) oor 0n enkele selsiklus na te volg en te kwantifiseer. Die nuut-ontwikkelde metodes was vervolgens gebruik om te bepaal hoe een so 0n beheerpunt in soogdierselle - die restriksiepunt - deur die onderskeie sisteemprosesse beheer word. Die studie het aangedui dat die posisie van die restriksiepunt tydens die selsiklus deur ’n verskeidenheid sisteemprosesse beheer word. Die bevinding was dat vier prosesse beduidend meer beheer op die posisie van die restriksiepunt uitoefen: Reaksies wat betrekking het op die wisselwerking tussen retinoblastoma proteïen (Rb) en E2F transkripsiefaktor; reaksies verantwoordelik vir die sintese van vertraagde responsgene en Siklien D/Cdk4 in respons tot groeiseine; die E2F afhanklike Siklien E/Cdk2 sintesereaksie; sowel as die reaksies betrokke in p27 vorming. Daar was ook aangetoon dat hierdie reaksies hul beheer op die posisie van die restriksiepunt deur die Siklien E/Cdk2/p27 kompleks uitoefen, siende enige sisteemversteuringe (wat tot veranderinge in die restriksiepuntposisie aanleiding gee) deur veranderinge in die kompleks verklaar kon word - 0n observasie wat aandui dat daar 0n kousale verhouding is tussen die posisie van die restriksiepunt en die Siklien E/Cdk2/p27 kompleks. Die nuut-ontwikkelde metodes was verder gebruik om 0n verskeidenheid selsiklusmodelle van Saccharomyces cerevisiae (bakkersgis) en soogdierselle met 0n modulêre aanpak te vergelyk om te bepaal of daar 0n massa beheerpunt in beide soogdier- en bakkersgisselle bestaan. Daar word gepostuleer dat hierdie beheerpunt verseker dat selle 0n kritiese massa by die G1/S transisiepunt bereik. Die resultate van die studie dui daarop dat bakkersgis, anders as soogdierselle, oor so 0n korreksiemeganisme beskik. Die meganisme stabiliseer die grootte van selle in die G1 fase ondanks veranderinge in die groeitempo van die selle, sodat massa homeostaties by die G1/S transisiepunt gehandhaaf word. Die studie het getoon dat moeilike vrae met betrekking tot die selsiklus beantwoord kan word deur van wiskundige modelle gebruik te maak en die probleme in die nuut-ontwikkelde metaboliese kontrole analise raamwerk te giet.

Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Skeletal muscle adapts to stimuli by modifying structural and metabolic protein expression. Furthermore, a muscle group may vary within itself to accommodate specialisation in regions. Structural and metabolic characteristics of an individual are regulated partly by genotype, but contraction duration and intensity may play a greater role in muscle phenotype. The aims of this dissertation were to investigate: structural and metabolic regionalisation in a muscle group, possible relationships between training volume and intensity and hybrid fibres, muscle characteristics of athletes from two different ethnic groups, and muscle adaptation in already well-trained athletes subjected to high intensity interval training. Myosin heavy chain (MHC) isoform content and citrate synthase (CS) activities were measured in the Quadriceps femoris (QF) muscle of 18 female rats. Muscle was divided into superficial, middle and deep, distal, central and proximal parts. MHC IIb and IIx were more abundant in superficial regions (P < 0.05) with low CS activities compared to deeper parts. Isoform content varied along the length of deep regions. This study showed that the QF has regional specialisation. Therefore, standardisation of sampling site is important. Hybrid fibre proportions in muscle biopsies of 12 middle distance runners and 12 non-runners were investigated. MHC IIa/IIx correlated with training volume/week in runners (r = -0.66, P < 0.05) and MHC IIa/IIx correlated with exercise hours/week in non-runners (r = -0.72, P < 0.01). Average preferred racing distance (PRDA) correlated better with MHC IIa/IIx in runners (r = -0.85, P < 0.001). MHC IIa/IIx may therefore be more closely related to exercise intensity than previously thought. Fibre type characteristics and performance markers were investigated in 13 Xhosa and 13 Caucasian distance runners, matched for performance, training volume and PRDA. Xhosa runners had less MHC I and more MHC IIa fibres in muscle biopsies than Caucasian runners (P < 0.05). Xhosa runners had lower plasma lactate at 80% peak treadmill speed (PTS) (P < 0.05), but higher lactate dehydrogenase (LDH) (P < 0.01) and phosphofructokinase (P = 0.07) activities in homogenate muscle samples. LDH activities in MHC I (P = 0.05) and IIa (P < 0.05) fibre pools were higher in Xhosa runners. Xhosa athletes may thus have a genetic advantage or they may have adapted to running at a higher intensity. Six weeks of individually standardised high intensity interval treadmill training (HIIT) were investigated in 15 well-trained runners. PTS increased after HIIT (P < 0.01), while maximum oxygen consumption (VO2max) only showed a tendency to have increased as a result of HIIT (P = 0.06). Sub-maximal tests showed lower plasma lactate at 64% PTS (P = 0.06), with lower heart rates at workloads from 64% to 80% PTS (P < 0.01) after HIIT. No changes were observed for cross-sectional area, capillary supply and enzyme activities in homogenates muscle samples. LDH activity showed a trend (P = 0.06) to have increased in MHC IIa pools after HIIT. Higher HIIT speed was related to decreases in MHC I fibres, but increases in MHC IIa/IIx fibres (r = -0.70 and r = 0.68, respectively, P < 0.05). Therefore, HIIT may alter muscle fibre composition in well-trained runners, with a concomitant improvement in performance markers. / AFRIKAANSE OPSOMMING: Skeletspier kan adapteer deur strukturele en metaboliese protein ekspressie te verander as gevolg van stimulante. ‘n Spiergroep kan ook intern verskil om spesialisering in spierdele toe te laat. Strukturele en metaboliese karaktereienskappe van ‘n individu word deels gereguleer deur gene, maar kontraksie tydperk en intensiteit mag ‘n groter rol speel in spierfenotipe. Die doelwitte van hierdie tesis was om ondersoek in te stel in: strukturele en metaboliese eienskappe in spiergroepstreke, moontlike verhoudings tussen oefeningsvolume of intensiteit en baster vesels, spier eienskappe in atlete van twee etniese groepe, en spier adaptasie in goed geoefende atlete blootgestel aan hoë intensiteit interval oefening. Miosien swaar ketting (MSK) isovorm inhoud en sitraat sintase (SS) aktiwiteite is gemeet in die Quadriceps femoris (QF) spier van 18 wyfie rotte. Spiere was opgedeel in oppervlakkig, middel en diep, asook distaal, sentraal en proksimale dele. MSK IIb en IIx was meer oorvloedig in oppervlakkige dele (P < 0.05) met lae SS aktiwiteite in vergelyking met dieper dele. Isovorm inhoud het ook verskil oor die lengte van diep dele. Dus bevat die QF gespesialiseerde streke en is die area van monsterneming belangrik. Baster vesel proporsies is ondersoek in spiermonsters van 12 middel afstand hardlopers en 12 niehardlopers. MSK IIa/IIx van hardlopers het met oefeningsvolume/week gekorreleer (r = -0.66, P < 0.05), asook MSK IIa/IIx van nie-hardlopers met oefeningsure/week (r = -0.72, P < 0.01). Gemiddelde voorkeur wedloop afstand (VWAG) het beter met MSK IIa/IIx gekorreleer in hardlopers (r = -0.85, P < 0.001). MSK IIa/IIx mag dus meer verwant wees aan oefeningsintensiteit. Veseltipe eienskappe en prestasie merkers was ondersoek in 13 Xhosa en 13 Caucasian langafstand atlete, geëweknie vir prestasie, oefeningsvolume en VMAG. Xhosa hardlopers het minder tipe I en meer tipe IIA vesels in hul spiermonsters gehad as die Caucasian hardlopers (P < 0.05). Xhosa hardlopers het laer plasma laktaat by 80% van hul maksimale trapmeul spoed (MTS) (P < 0.05), maar hoër laktaat dihidrogenase (LDH) (P < 0.01) en fosfofruktokinase (P = 0.07) aktiwiteite in homogene spiermonsters gehad. LDH aktiwiteite in MSK I (P = 0.05) en IIa (P < 0.05) veselbondels was hoër in Xhosa hardlopers. Xhosa atlete mag dus ‘n genetiese voorsprong geniet, of hulle het geadapteer om by hoër intensiteite te hardloop. Ses weke van geïndividualiseerde gestandardiseerde hoë intensiteit interval trapmeul oefening (HIIT) was ondersoek in 15 goed geoefende hardlopers. MTS het verhoog na HIIT (P < 0.01), en maksimale surrstof verbruik (VO2max) het ‘n neiging getoon om te verhoog het na HIIT (P = 0.07). Submaksimale toetse het laer plasma laktaat by 64% MTS getoon (P = 0.06), met laer harttempos by werkladings 64% tot 80% MTS (P < 0.01). Geen veranderings was gemerk vir deursnit area, kapillêre toevoer en ensiem aktiwiteite in homogene spiermonsters nie. LDH aktiwiteit het ‘n neiging getoon om te verhoog het (P = 0.06) in MSK IIa veselbondels na HIIT. Hoër HIIT snelhede was verwant aan ‘n daling in MSK I vesels, maar ‘n verhoging in MSK IIa/IIx vesels (r = -0.70 en r = 0.68, respektiwelik, P < 0.05). HIIT mag dus spier veseltipe verander in goed geoefende hardlopers, met gevolglike verbetering in prestasie merkers.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This thesis provides the first scientific evidence on the cancer modulating properties of two unique South African herbal teas, rooibos (Aspalathus Iinearis) and honeybush (Cyclopia intermedia) utilizing in vitro as well as in vivo carcinogenesis assays by: • Demonstrating the in vitro antimutagenic activity of aqueous extracts of the herbal teas against the metabolic activated mutagens, 2-acetylaminofluorene (2- AAF) and the mycotoxin, aflatoxin B1 (AFB,) as well as, to a certain extent, against the direct acting mutagen, hydrogen peroxide, utilizing the Salmonella typhimurium mutagenicity assay. • Increasing the activity of hepatic drug metabolizing enzymes, glutathione Stransferase alpha and UPD-glucuronosyl transferase, and reduced the oxidative stress by stabilizing the level of reduced glutathione (GSH) resulting in an increased hepatic reduced to oxidized glutathione ratio (GSG:GSSG). No toxic effects were noticed in rats consuming the herbal teas for 10 weeks as their sole source of drinking fluid. • Demonstrating the ex vivo modulation of 2-AAF- and AFB1-induced mutagenesis by sub- cellular hepatic fractions of rats consuming the herbal teas in the Salmonella mutagenicity assay. Hepatic cytosolic fractions protected against mutagenesis of both mutagens, while the microsomal fractions exhibited a reduced capacity to metabolize AFB1 to its active mutagenic metabolite. • Providing evidence for the in vivo modulation of tumour promotion using the liver as well as the two-stage skin carcinogenesis animal models. The unprocessed herbal teas arrested proliferation of the placental form of glutathione-Stransferase (GSTP+) altered cells as well as reduced the total number of enzyme altered foci in the liver of rats. Topical application of polyphenolic fractions of the various herbal teas prior to 12-0-tetra-decanoylphorbol-13-acetate (TPA) tumour promotion, reduced tumour formation in mouse skin initiated with 7,12-dimethylbenz[ ajanthracene (DMBA). The protective effect was illustrated by a decreased tumour incidence, a reduction in tumour volume as well as a delayed onset of tumour development. The f1avanol/proanthocyanidin content of the fractions could playa major role in the protection against skin tumour promotion. • Proposing possible mechanisms whereby rooibos and honeybush herbal teas could exert their cancer modulating properties with respect to in vitro and ex vivo antimutagenicity, in vivo oxidative status and reduced tumour promotion. • Providing evidence that the herbal teas mimic the cancer modulating properties of green and black teas although differences exist, presumably due to differences in the polyphenolic constituents. • Suggesting that rooibos and honeybush herbal teas may play an important role as chemopreventive agents in the modulation of cancer. / AFRIKAANSE OPSOMMING: Hierdie tesis bevat die eerste ondersoek na die effek van waterige en polifenoliese ekstrakte van rooibos (Aspalathus Iinearis) en heuningbos (Cyclopia intermedia) op verskeie aspekte van kankerontwikkeling. Die twee kruietees is uniek aan Suid-Afrika en kan 'n belangrike rol speel in die voorkoming van kanker. Verskillende in vitro so wei as in vivo studies het die volgende getoon: • Antimutageniese aktiwiteite teen die metabolies-geaktiveerde mutagene, 2- asetielaminofluoreen (2-AAF) en die mikotoksien, aflatoksien B1 (AFB1) in die Salmonella fyphimurium mutagenisiteitstoets. 'n Beperkte mate van beskerming is ook verleen teen die oksidatiewe mutageen, waterstofperoksied, sonder metaboliese aktivering. • Verhoogde aktiwiteite van die fase II ensieme, glutatioon S-tranferase alfa en UDP-glukuronidase, wat liggaamsvreemde verbindings metaboliseer. Die kruietees verlaag die oksidasietoestand soos weerspieel word deur 'n toename van gereduseerde glutatioon tot die geoksideerde vorm in die lewer van rotte wat 10 weke hierdie kruietees gedrink he!. Die kruietees het geen toksiese uitwerking op die rotte gehad nie. • Antimutageniese aktiwiteite van subselluiE~re fraksies van die lewer teenoor 2- AAF en AFB1 in die Salmonella toets. Die sitosolfraksie van die rotlewer bied beskerming teen die ge"induseerde mutagenese van beide mutagene, terwyl die mikrosomale fraksie ook die metaboliese aktivering van AFB1 na die aktiewe mutageniese metaboliet verminder. • In vivo modulering van kankerpromosie met behulp van bekende rotlewer en muisvel kankerontwikkelingsmodelle. In die lewermodel het die ongeprosesseerde kruietees beide die ontwikkeling en getal van GSTP+ fokusse onderskeidelik vertraag en verminder. In die geval van die velkankermodel het aanwending van polifenoliese fraksies van die kruietees beskerming gebied teen die ontwikkeling van velkankers by muise. Die aantal en grootte van die tumors het afgeneem terwyl die verskyning daarvan ook vertraag is. • Verskeie meganismes waardeur rooibos- en heuningboslee moonllik kanker kan moduleer word voorgeslel. Verskille in die polifenoliese sameslelling asook hul onderskeie konsenlrasies kan 'n belangrike rol speel in die kankerveranderende effekle van die lees. • Oal gereelde inname van rooibos- en/of heuningboslee moonllik 'n belangrike rol kan speel in die voorkoming van dieel- en omgewings-geYnduseerde kankers.

This study reports on recurring difficulties experienced by undergraduate students with respect to understanding and interpretation of certain symbolism, nomenclature, terminology, shorthand notation, models and other visual representations employed in the field of Molecular Biology to communicate information. Based on teaching experience and guidelines set out by a four-level methodological framework, data on various topic-related difficulties was obtained by inductive analyses of students’ written responses to specifically designed, free-response and focused probes. In addition, interviews, think-aloud exercises and student-generated diagrams were also used to collect information. Both unanticipated and recurring difficulties were compared with scientifically correct propositional knowledge, categorized and subsequently classified. Students were adept at providing the meaning of the symbol “Δ” in various scientific contexts; however, some failed to recognize its use to depict the deletion of a leucine biosynthesis gene in the form, Δ leu. “Hazard to leucine”, “change to leucine” and “abbreviation for isoleucine” were some of the erroneous interpretations of this polysemic symbol. Investigations on these definitions suggest a constructivist approach to knowledge construction and the inappropriate transfer of knowledge from prior mental schemata. The symbol, “::”, was poorly differentiated by students in its use to indicate gene integration or transposition and in tandem gene fusion. Idiosyncratic perceptions emerged suggesting that it is, for example, a proteinaceous component linking genes in a chromosome or the centromere itself associated with the mitotic spindle or “electrons” between genes in the same way that it is symbolically shown in Lewis dot diagrams which illustrate covalent bonding between atoms. In an oligonucleotide shorthand notation, some students used valency to differentiate the phosphite trivalent form of the phosphorus atom from the pentavalent phosphodiester group, yet the concept of valency was poorly understood. By virtue of the visual form of a shorthand notation of the 3,5 phosphodiester link in DNA, the valency was incorrectly read. VSEPR theory and the Octet Rule were misunderstood or forgotten when trying to explain the valency of the phosphorus atom in synthetic oligonucleotide intermediates. Plasmid functional domains were generally well-understood although restriction mapping appeared to be a cognitively demanding task. Rote learning and substitution of definitions were evident in the explanation of promoter and operator functions. The concept of gene expression posed difficulties to many students who believed that genes contain the entity they encode. Transcription and translation of in tandem gene fusions were poorly explained by some students as was the effect of plasmid conformation on transformation and gene expression. With regard to the selection of transformants or the hybridoma, some students could not engage in reasoning or lateral thinking as protoconcepts and domain-specific information were poorly understood. A failure to integrate and reason with factual information on phenotypic traits, media components and biochemical pathways were evident in written and oral presentations. DNA-strand nomenclature and associated function were problematic to some students as they failed to differentiate coding strand from template strand and were prone to interchange the labelling of these. A substitution of labels with those characterizing DNA replication intermediates demonstrated erroneous information transfer. DNA replication models posed difficulties integrating molecular mechanisms and detail with line drawings, coupled with inaccurate illustrations of sequential replication features. Finally, a remediation model is presented, demonstrating a shift in assessment score dispersion from a range of 0 - 4.5 to 4 - 9 when learners are guided metacognitively to work with domain-specific or critical knowledge from an information bank. The present work shows that varied forms of symbolism can present students with complex learning difficulties as the underlying information depicted by these is understood in a superficial way. It is imperative that future studies be focused on the standardization of symbol use, perhaps governed by convention that determines the manner in which threshold information is disseminated on symbol use, coupled by innovative teaching strategies which facilitate an improved understanding of the use of symbolic representations in Molecular Biology. As Molecular Biology advances, it is likely that experts will continue to use new and diverse forms of symbolic representations to explain their findings. The explanation of futuristic Science is likely to develop a symbolic language that will impose great teaching challenges and unimaginable learning difficulties to new generation teachers and learners, respectively. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Brewing is defined as “the combined processes of preparing beverages from the infusion of sound grains that have undergone sprouting, and the subsequent fermentation of the sugary solution produced, by yeast-whereby a proportion of the carbohydrate is converted to ethanol and carbon-dioxide.” It is a complex process that requires knowledge of concepts from disciplines such as biochemistry, chemistry, engineering, microbiology and physics. The micro-brewery apparatus at the University of KwaZulu-Natal is used by the discipline of microbiology as part of a brewing exercise to introduce students to industrial microbiology with the aim of developing their conceptual understanding of the process. So far, though, no research has been conducted in order to fully establish the effectiveness of this exercise in developing such understanding of the brewing process. The aim, therefore, of this study was to investigate the effectiveness of a micro-brewing Teaching-Learning Sequence (TLS) that incorporates the micro-brewery, for promoting students‟ understanding of the scientific concepts of relevance to the brewing process. The following research questions were addressed: 1) What concepts are essential for understanding the process of beer brewing? 2) Did those students with sound conceptions develop deeper understanding during the TLS? 3) Did students show any conceptual difficulties with the brewing concepts? 4) Did any remediation of such difficulties occur during the TLS? 5) Did students show retention of (mis)understanding two months after the brewing practical? 6) What were students‟ attitudes and motivational levels like during the brewing practical? 7) How well did students rate their experiences of the whole TLS? 8) How well did students‟ motivational levels and their rating of the TLS correlate with any changes in understanding? The study involved ten microbiology honours students subjected to a TLS which consisted of: i) three brewing lectures aimed at introducing students to the brewing process; ii) pre- & post tests including concept mapping tasks aimed at addressing research questions 2, 3 & 4; iii) a brewing practical aimed at facilitating students‟ development of mental models and conceptual understanding of the brewing process and their motivation and attitude to this exercise (addressing question 6 & 8); iv) a group discussion which involved a group tasting session and the evaluation and discussion of each group‟s final beer product; v) semi-structured interviews to establish the source (s) of students‟ difficulties and their retention of knowledge or difficulties (questions 2, 4, & 5 addressed); and vi) an evaluation questionnaire aimed at obtaining student opinion of the TLS (addressing question 7). The data obtained was analyzed via inductive analysis. The results revealed the following brewing difficulties: i) belief that glycolysis reactions are non-consecutively linked chemical reactions which are independent of one another; ii) confusion that whirl-pooling cools the wort; and iii) belief that the final specific gravity value is a measure of the amount of sugars converted to ethanol. Comparison between the pre- & post test responses indicated that some students‟ (B, D & K) conceptual understanding including integrated knowledge of the brewing process improved during the TLS and their brewing difficulties were remediated. In contrast, other students‟ (A, C, E, G, H, J & I) conceptual understanding did not improve during the TLS and their brewing difficulties were not remediated. There was also a positive correlation between student attitudes and motivation towards the brewing practical and the quality of their learning outcomes. Students (B, D & K) who showed high motivational levels and cognitively and physically took part in the TLS showed improved conceptual understanding of the brewing process and retention of knowledge, while those showing low motivational levels did not improve. Furthermore, there are students (G, H & J) who showed high motivational levels during the TLS but their conceptual understanding of the brewing process did not improve. The results obtained suggest that the TLS, based on the micro-brewery apparatus, was at least partially effective in facilitating the development of students‟ conceptual understanding and visualization of the brewing process and the remediation of some of their difficulties, which in some case correlated well with their motivational levels and attitudes towards the brewing exercise. More research is however required to fully confirm the usefulness of such TLSs in brewing education. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Scientific external representations (ERs), such as diagrams, images, pictures, graphs and animations are considered to be powerful teaching and learning tools, because they assist learners in constructing mental models of phenomena, which allows for the comprehension and integration of scientific concepts. Sometimes, however, students experience difficulties with the interpretation of ERs, which· has a negative effect on their learning of science, . . including biochemistry. Unfortunately, many educators are not aware of such student difficulties and make the wrong assumption that what they, as experts, consider to be an educationally sound ER will necessarily promote sound. learning and understanding among novices. On the contrary, research has shown that learners who engage in the molecular biosciences can experience considerable problems interpreting, visualising, reasoning and learning with ERs of biochemical structures and processes, which are both abstract and often represented by confusing computer-generated symbols and man-made markings. The aim of this study was three-fold. Firstly, to identify and classify students' conceptual and reasoning difficulties with a selection of textbook ERs representing· IgG structure and function. Secondly, to use these difficulties to identify sources of the difficulties and, therefore, factors influencing students' ability to interpret the ERs. Thirdly, to develop a model of these factors and investigate the practical applications of the model, including guidelines fOf improving ER design and the teaching and learning with ERs. The study was conducted at the University of KwaZulu-Natal, South Africa and involved a total of 166 second and third-year biochemistry students. The research aims were addressed using a p,ostpositivistic approach consisting of inductive and qualitative research methods. Data was collected from students by means of written probes, audio- and video-taped clinical interviews, and student-generated diagrams. Analysis of the data revealed three general categories of student difficulties, with the interpretation of three textbook ERs depicting antibody structure and interaction with antigen, termed the process-type (P), the. structural-type (S) and DNA-related (D) difficulties. Included in the three general categories of difficulty were seventeen sub-categories that were each classified on the four-level research framework of Grayson et al. (2001) according to v how much information we had about the nature ofeach difficulty and, therefore, whether they required further research. The incidences of the classified difficulties ranged from 3 to 70%, across the student populations and across all three ERs. Based on the evidence of the difficulties, potential sources of the classified difficulties were isolated. Consideration of the nature of the sources of the exposed difficulties indicated that at least three factors play a major role in students' ability to interpret ERs in biochemistry. The three factors are: students' ability to reason with an ER and with their own conceptual knowledge (R), students' understanding (or lack thereof) of the concepts of relevance to the ER (C), and the mode in which the desired phenomenon is represented by the ER (M). A novel three-phase single interview technique (3P-SIT) was designed to explicitly investigate the nature of the above three factors. Application of3P-SIT to a range of abstract to realistic ERs of antibody structure and interaction with antigen revealed that the. instrument was extremely useful for generating data corresponding to the three factors.. In addition; analysis of the 3P-SIT data showed evidence for the influence ofone factor on another during students' ER interpretation, leading to the identification of a further four interactive factors, namely the reasoning-mode (R-M), reasoning conceptual (R-C), conceptual-mode (C-M) and conceptual-reasoning-mode (C-R-M) factors. The Justi and Gilbert (2002) modelling process was employed to develop a model of the seven identified factors. Empirical data generated using 3P-SIT allowed the formulation and validation of operational definitions for the seven factors and the expression of the model as a Venn diagram, Consideration ofthe implications of the model, yielded at least seven practical applications of the model, including its use for: establishing whether sound or unsound interpretation, learning and visualisation of an ER has occurred; identifying the nature and source of any difficulties; determining which of the factors of the model are positively or negatively influencing interpretation; establishing what approaches to ER design and teaching and learning with ERs will optimise the interpretation and learning process; and, generally framing and guiding researchers', educators' and authors' thinking about the nature of students' difficulties with the interpretation of both static and animated ERs in any scientific context. In addition, the study demonstrated how each factor of the expressed model can be used to inform the design of strategies for remediating or preventing students' difficulties with the interpretation of scientific ERs, a target for future research. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The use of external representations (ERs) such as diagrams and animations in science education, particularly in the Molecular Life Sciences (MLS), has rapidly increased over the past decades. Research shows that ERs have a superior advantage over text alone for teaching and learning. Research has also indicated a number of concerns coupled with the use of ERs for education purposes. Such problems emanate from the mode of presentation and/or inability to use ERs. Regarding the later, a number of factors have been identified as major causes of student difficulties and they include visual literacy as one of the major factors. Given that little has been done to understand the nature of VL in the MLS the current study was conducted with the general aim of investigating this area and devising a way to measure the visual literacy levels of our students. More specifically, this study addressed the following research questions: i) What is the nature of visual literacy in MLS?; ii) Can specific levels of visual literacy be defined in the MLS?; and iii) Is a taxonomy a useful way of representing the levels of visual literacy for MLS? To respond to these questions, the current literature was used to define the nature of visual literacy and the visualization skills (VSs). These were then used to develop a Visual Literacy Test made up on probes in the context of Biochemistry. In these probes, the VSs were incorporated. The test was administered to 3rd year Biochemistry students who were also interviewed. Results were analysed qualitatively and quantitatively. The later analysis utilized the Rasch model to generate an item difficulty map. The results of the current study show that visual literacy is multifaceted in nature and is context based in that it requires specific propositional knowledge. In line with this, it was found that visual literacy is expressed through a cognitive process of visualization which requires VSs. Based on the performance of these skills, learners’ optimal visual literacy in the context of the MLS can be defined. Such performance can be assessed through the development of probes in the Biochemistry context. Furthermore, the current research has shown that using probes, the difficulty degree of each VS can be determined. In this instance, the Rasch model is a preferred method of ranking VSs in the context of Biochemistry in order of difficulty. From this, it was shown that given the uniqueness of each skill’s degree of difficulty, each skill can thus be regarded as a level of visual literacy. Such levels were defined in terms of the norm difficulty obtained in the current study. Given the multifaceted nature of visual literacy, the current study adopted the view that there are infinite number of VSs and hence the number of levels of visual literacy. From the variation in the degree of difficulty, the study showed that there are nonvisualization and visualization type difficulties which contribute to the differences in visual literacy levels between Biochemistry students. In addition to this, the current study showed that visual literacy in the MLS can be presented through a taxonomy. Such a taxonomy can be used to determine the level of each VS, its name and definition, typical difficulties found in the MLS as well as the visualization stage at which each skill is performed. Furthermore, this taxonomy can be used to design models, assess students’ visual literacy, identify and inform the remediation of students’ visualization difficulties. While the study has successfully defined the nature of visual literacy for the MLS and presented visual literacy in a taxonomy, more work is required to further understand visual literacy for the MLS, a field where visual literacy is very prevalent. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2007.