Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sulfated galactans (agarans and carrageenans) are accumulated in the cell wall of various red algae (Rhodophyta) species. These polysaccharides are of commercial importance in the food, pharmaceutical and biotechnology industries due to their unique physicochemical properties. Although having received significant research attention over the last 20 years, events regarding their biosynthesis have not been elucidated. Aiming for the identification of galactosyltransferase (GalT) genes involved in sulfated galactan biosynthesis, cDNA expression libraries were constructed from the prolific agar-producing South African red seaweed Gelidium pristoides (Turner) Kützing and screened by functional complementation of UDP-galactose 4-epimerase deficient mutants (E. coli and S. cerevisiae). Regretfully, no GalTs were identified. The study however yielded the first UGE enzyme described for a red seaweed. Southern hybridization indicated the presence of two UGE copies and confirmed the gene originated from G. pristoides. Bioinformatic analysis of G. pristoides UGE shows amino acid sequence homology to known UGEs from various organisms. The enzyme was shown to be functional in E. coli crude extracts and showed affinity for UDP-D-galactose, similar to other UDP-galactose 4-epimerases. Further, the isolated G. pristoides UGE (GpUGE) was biochemically characterized and its kinetic parameters determined. We found that there was no kinetic difference between this enzyme and previously described UGE enzymes except enhanced activity in the presence of exogenously added NAD+. The UDP-galactose 4-epimerase (UDP-glucose 4-epimerase, UGE, EC 5.1.3.2) is an essential Leloir pathway enzyme facilitating the catalytic inter-conversion between UDP-D-glucose and UDP-D-galactose. UDP-D-galactose is the nucleotide sugar required by galactosyltransferases for the production of red algae sulfated galactans. UGE is suspected as being responsible for supplying UDP-D-galactose for the synthesis of sulfated galactans. In planta monitoring of GpUGE transcript levels with respect to dark and light cycling indicated high expression of the enzyme at night, while expression diminished during the day. The occurrence of increased nocturnal UGE expression correlates with floridean starch breakdown at night. Evidence for hydrolysis of floridean starch is also reflected in obtained G. pristoides transcriptome sequence data. In red algae, floridean starch degradation coincides with sulfated galactan production. The detection of starch hydrolysis enzyme transcripts alongside increased expression of GpUGE suggests the enzyme plays a role in supplying UDP-Dgalactose for sulfated galactan production. As far as we know, this the first report of sequencing and biochemical characterization of a UGE from red seaweed.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Increased demand of petroleum, declining fossil fuel reserves, geopolitical instability and the environmentally detrimental effects of fossil fuels have stimulated research to search for alternative sources of energy such as plant derived biofuels. The main feedstocks for production of first generation biofuels (bioethanol) are currently sucrose and starch, produced by crops such as sugarcane, sugarbeet, maize, and cassava. The use of food crop carbohydrates to produce biofuels is viewed as competing for limited agronomic resources and jeopardizing food security. Plants are also capable of storing sugars in their cell walls in the form of polysaccharides such as cellulose, hemicelluloses and pectin, however those are usually cross-linked with lignin, making their fermentation problematic, and are consequently referred to as lignocellulosics. Current technologies are not sufficient to degrade these cell wall sugars without large energy inputs, therefore making lignocellulosic biomass commercially unviable as a source of sugars for biofuel production. In the present study genes encoding for enzymes for cellulosic, hemicellulosic and starch-like polysaccharides biosynthesis were heterologously expressed to increase the amount of fermentable sugars in sugarcane. Transgenic lines heterologously expressing CsCesA, encoding a cellulose synthase from the marine invertebrate Ciona savignyi showed significant increases in their total cellulose synthase enzyme activity as well as the total cellulose content in internodal tissues. Elevation in cellulose contents was accompanied by a rise in hemicellulosic glucose content and uronic acid amounts, while total lignin was reduced in internodal tissues. Enzymatic saccharification of untreated lignocellulosic biomass of transgenic sugarcane lines had improved glucose release when exposed to cellulose hydrolyzing enzymes. Calli derived from transgenic sugarcane lines ectopically expressing galactomannan biosynthetic sequences ManS and GMGT from the cluster bean (Cyamopsis tetragonoloba) were observed to be capable of producing a galactomannan polysaccharide. However, after regeneration, transgenic sugarcane plants derived from those calli were unable to produce the polymer although the inserted genes were transcribed at the mRNA level. While the ectopic expression of Deinococcus radiodurans amylosucrase protein in the cytosol had a detrimental effect on the growth of transgenic lines (plants showed stunted growth through the 18 months growth period in greenhouse), contrastingly targeting the amylosucrase protein into the vacuole resulted in 3 months old transgenic lines which were having high maltooligosaccharide and soluble sugar (sucrose, glucose and fructose) levels in leaves. After 18 months growing in the greenhouse, the mature transgenic lines were morphologically similar to the untransformed lines and also contained comparable maltooligosaccharide and soluble sugar and starch amounts. The non-biosynthesis of galactomannan and amylose polysaccharides in the matured transgenic plants may be due to post-transcriptional protein processing and or protein instability, possibly explainable by other epigenetic mechanisms taking place to regulate gene expression in the at least allo-octaploid species of sugarcane under investigation in this study.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-­‐virus interaction. This study made use of next-­‐generation sequencing to investigate the effect of virus-­‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐derived sRNAs and vsiRNAs. Broad and narrow size-­‐range datasets were generated using sRNA libraries prepared from total and size-­‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-­‐ and trans-­‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-­‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-­‐halves. Reads associated with tRNA-­‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-­‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple. / AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-­‐virus interaksie is beskikbaar. Hierdie studie het van volgende-­‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl. miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐afkomstige sRNAs en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-­‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte. Deur middel van bioinformatiese data-­‐ontleding is 130 genomiese loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-­‐beskikbare degradoom datastel. Cis-­‐ en trans-­‐natuurlike antisense transkripte (NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook geïdentifiseer. Vir 25 van hierdie loci is 'n in-­‐fase miRNA teiken geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs het ooreengestem met tRFs en tRNA-­‐helftes. Volgordes geassosieer met tRNA-­‐helftes was prominent in die breë grootte-­‐verspreiding datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek voorbereiding op die interpretasie van resultate. Ontleding van die differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies het geen verskil tussen gesonde en geïnfekteerde plante getoon nie, behalwe vir die tRNA-­‐afkomstige sRNAs, wat wel verandering die vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat in hierdie studie geïdentifiseer is, het die bestaande kennis van appel sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige funksionele studies in appel. Hierdie studie bied ook die eerste, en mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in appel.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: see item for full text / AFRIKAANSE OPSOMMING: sien item vir volteks

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines. / AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie. / The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work

Thesis (MSC)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: South Africa is shouldering the highest burden of HIV-infection. Inter-individual differences in response to antiretroviral treatment (ART) and the limited availability of second and third-line ART regimens call for optimising first-line ART in South African populations. Measuring antiretroviral drug levels in patients may be of clinical value as an intermediate indicator of treatment response and may moreover serve to assess the genetic variation underlying differential drug exposure. This study aimed to determine the effect of SNPs in the CYP2B6 gene and efavirenz (EFV) levels measured in hair on ART outcomes in females of two South African populations. Female Xhosa (XH) (n = 81) and Mixed Ancestry (MA) (n = 53) patients receiving the first-line regimen component EFV for at least three months donated saliva for genomic DNA extraction and 20 strands of hair for determination of EFV concentrations by high performance liquid chromatography. Regulatory and exonic regions in the CYP2B6 gene, which codes for the major metabolising enzyme of EFV, were subjected to bi-directional sequence analysis in 15 XH and 15 MA individuals to assess common genetic variation in these populations. Out of 45 single nucleotide polymorphisms (SNPs) identified, 17 SNPs of known or predicted functional importance in EFV metabolism, including four novel SNPs, were genotyped in the entire patient cohort by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. All SNPs were tested for Hardy-Weinberg equilibrium (HWE) and maximum likelihood haplotypes and assessed for an association with EFV levels measured in hair, likelihood of developing adverse drug reactions (ADRs) and virological response to EFV-based treatment. After correcting for age and ethnicity, homozygous carriers of c.516G>T (CYP2B6*6) had significantly increased EFV levels (p = 0.0021; mean: 12.0 ng/mg; IQR: 3.95 – 6.99 ng/mg; n = 12), as did heterozygotes of c.983T>C (CYP2B6*18) (p = 0.0005; mean: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). No CYP2B6*18 homozygotes were detected. No association between EFV levels and virological response was evident (p = 0.8467), but CYP2B6*6 predicted increased odds of virological failure (VL > 80 copies/ml) after correcting for adherence, race, age, weight, time on treatment, baseline CD4, smoking, alcohol and WHO disease stage (p = 0.0328). Carriers of the CYP2B6*1 allele had increased odds (OR = 5) of favourable treatment outcome (VL < 80 copies/ml). In accordance with other studies, this study provides evidence that genetically predisposed poor metabolisers of EFV may be at increased risk of virological failure, possibly following non-adherence. Concurrently, these patients may be more vulnerable to adverse drug reactions and are more frequent in the XH (13%) than MA (4%). These results should be verified in larger patient cohorts, but contribute to a better understanding of the effect of genetic factors on EFV exposure and ART outcome in two South African populations. The outcomes of this study may thus provide recommendations for prospective studies and impact future clinical decisions. / AFRIKAANSE OPSOMMING: Suid-Afrika dra die grootste las van MIV-infeksies. Inter-individuele verskille in reaksie op anti-retrovirale terapie (ART) en die beperkte beskikbaarheid van tweede- en derde-linie ART-reekse regverdig die optimisering van eerste-linie ART in Suid-Afrikaanse bevolkings. Meting van antiretrovirale middel-vlakke in pasiënte, as ‘n intermediêre aanduiding van reaksie op behandeling, kan van kliniese belang wees en kan ook die waarde van die bepaling van genetiese variasie, onderliggend aan differensiële blootstelling aan middels, bepaal. Die doel van hierdie studie is om die effek van enkel-nukleotied polimorfismes (SNPs) in die CYP2B6-geen en efavirenz (EFV)-vlakke in hare op ART-uitkoms te bepaal in vroue van twee Suid-Afrikaanse bevolkingsgroepe. Vroulike Xhosa (XH) (n = 81) en Gemengde Herkoms (GH) (n = 53) pasiënte wat EFV as deel van eerste-linie ART vir ten minste drie maande ontvang het, het speekselmonsters vir genomiese DNA-ekstraksie en 20 hare vir die bepaling van EFV-konsentrasies deur hoë werkverrigting vloeistofchromatografie (“HPLC”) geskenk. Regulatoriese en eksoniese areas in die CYP2B6-geen, wat vir die vernaamste metaboliserende ensiem van EFV kodeer, is deur middel van tweerigting-volgordebepalings-analise in 15 XH en 15 GH individue ondersoek om gemeenskaplike genetiese variasie in hierdie bevolkings te bepaal. Uit ‘n totaal van 45 SNPs wat geïdentifiseer is, is 17 SNPs wat bekende of voorspelde belangrike rolle in EFV-metabolisme speel, insluitend vier nuwe SNPs, ondersoek. Hierdie SNPs is in die volledige pasiënt-kohort gegenotipeer deur polimerase-ketting reaksie gebaseerde restriksie fragment lengte-polimorfisme (PKR-RFLP) analise. Alle SNPs is getoets vir Hardy-Weinberg-ewewig (HWE) en maksimum waarskynlikheidshaplotipes en is geassesseer vir assosiasie met EFV-vlakke gemeet in hare, die waarskynlikheid om ongunstige reaksies tot die middel te ontwikkel en virologiese reaksie op EFV-gebaseerde behandeling. Nadat vir ouderdom en herkoms gekorrigeer is, het homosigotiese draers van c.516G>T (CYP2B6*6) beduidend verhoogde EFV-vlakke (p = 0.0021; gemiddeld: 12.0 ng/mg; IQR: 3.95 – 6.99; n=12) getoon, so ook heterosigote vir c.983T>C (CYP2B6*18) (p = 0.0005; gemiddeld: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). Geen CYP2B6*18 homosigote is gevind nie. Daarbenewens is geen duidelike assosiasie tussen EFV-vlakke en virologiese reaksie gevind nie (p = 0.8467), maar CYP2B6*6 het verhoogde waarskynlikheid op virologiese mislukking (VL > 80 kopieë/ml) getoon nadat daar vir mddel-getrouheid, ras, ouderdom, gewig, tydsduur van behandeling, basis-CD4, rook, alkohol en Wêreld Gesondheids Organisasie siekte-fase gekorrigeer is (p = 0.0328). Draers van die CYP2B6*1-alleel het verhoogde waarskynlikheid (OR = 5) op gunstige behandelingsuitkomste getoon (VL < 80 kopieë/ml). In ooreenstemming met ander studies verskaf hierdie studie bewyse dat pasiënte wat geneties geneig is tot stadige metabolisme van EFV ‘n hoër risiko kan hê vir virologiese mislukking, wat moontlik ‘n gevolg is van middel-ontrouheid. Hierdie pasiënte kan ook meer geneig wees tot vatbaarheid vir ongunstige middel-reaksie en kom meer voor in die XH (13%) as in die MA (4%). Hierdie resultate moet in groter pasiënt-kohorte gestaaf word, maar dra by tot ‘n beter begrip van die effek van genetiese faktore op blootstelling aan EFV en ART-uitkoms in twee Suid-Afrikaanse bevolkings. Die uitkomste van hierdie studie kan dus as aanbevelings gebruik word vir voornemende studies en ook toekomstige kliniese besluite beïnvloed. / The Medical Research Fund (MRC) for funding this project. The University Centre for Studies in Namibia (TUCSIN) and Deutscher Akademischer Austausch-Dienst (DAAD) for financial support

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / Breast cancer is a major cause of morbidity and mortality as it is the most common invasive cancer in women worldwide. The lifetime risk for South African women to develop breast cancer is one in 31. A family history of the disease is a well-established risk factor and germline mutations in the BRCA1 (breast cancer one) and BRCA2 (breast cancer two) tumour suppressor genes markedly increase the risk of developing breast cancer. A few hundred mutations spanning the entire coding sequences of both genes have already been reported. Numerous other breast cancer susceptibility loci have been identified, but results from association studies are discrepant. The checkpoint kinase gene, CHEK2, and specifically the CHEK2*1100delC variant has, however, consistently been implicated as a candidate low-penetrance breast cancer allele. To date, few comprehensive molecular-genetic studies have been completed for the various South African breast cancer populations. The aim of this study was to determine the BRCA1 and BRCA2 mutation spectrum and prevalence in two South African populations, namely Mixed Ancestry and Caucasian. The frequency of the CHEK2*1100delC mutation was also investigated. The patient group comprised 101 unrelated patients (98 women and 3 men), presenting with invasive breast cancer. Patients with a moderate family history of breast cancer (n=48) were screened for the CHEK2*1100delC allele and the coding sequences of the BRCA1 (partly completed in a previous study) and BRCA2 genes. Patients without a family history of the disease (n=53) were only screened for the CHEK2*1100delC allele. Mutation detection was done using combined single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA), followed by DNA sequencing of the identified variants. Due to its size (~5kb), exon 11 of BRCA2 was sequenced directly after amplification, in seven overlapping fragments. Three deleterious BRCA1 mutations, 1623_1627delTTAAA, E881X and 5313delC have previously been identified in three patients from the study population. No additional pathogenic mutations have been detected in this gene during this study. Two deleterious BRCA2 mutations, 6677_6678insTA and 8162delG, were identified in two and three patients respectively. Overall, BRCA1 and BRCA2 mutations have been identified in 17% of the Mixed Ancestry patients and in 15.8% of the Caucasian patients. Together BRCA1 and BRCA2 mutations account for 16.7% of breast cancer in the study population. In addition, a number of silent polymorphisms as well as variants of unknown functional significance, both known and novel, were identified. The E881X variant, which has been reported as an Afrikaner founder mutation (Reeves et al. 2004), was identified in one patient of Mixed Ancestry, but none of the published European founder mutations have been detected in our patient group. This suggests a unique mutation spectrum for South African breast cancer patients. The prevalence of the BRCA2 mutations, 8162delG and 6677_6678insTA, has to be elucidated within a larger study group. Haplotype analysis will reveal whether these patients have a common ancestor. Our findings do not suggest the presence of the CHEK2 variant in South African breast cancer patients, but a larger study population has to be analysed to confirm this. The results of this study are in agreement with those from other populations, indicating that less than 20% of breast cancers that occur in individuals with a moderate-risk for developing breast cancer are due to BRCA1 and BRCA2 mutations. By determining the contribution of BRCA1 and BRCA2 mutations to breast cancer in this group of patients, one can assess the appropriateness of predictive or diagnostic DNA testing in the clinical setting.

Thesis (MPhil)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Tilapia is presumed to have a well-defined genetic mechanism of sex determination, but not all sex ratios are compatible with a monofactorial sex determination model. A theory of autosomal gene influence, as well as temperature sex determination (TSD) has been proposed in order to explain large variations in sex ratios. This study assessed the variation in progeny sex ratio in O. mossambicus as a basis for the application of YY male technology in the production of all-male progeny groups. Three populations of O. mossambicus were sampled that are representative of the geographical distribution of the species in Southern Africa. Progeny groups were produced from randomly selected parents and maintained at constant temperature during the labile period of sex differentiation. Variation in sex ratios between different families of the same strain as well as between different strains was calculated. No significant differences were observed in sex ratio between strains, though a significant intra-group variation was identified. This study identified both male and female-biased sex ratios. The data in general conform to a monofactorial sex determination model. Male-biased sex ratio observed in one strain can possibly be ascribed to temperature sex determination (TSD). This strain of O. mossambicus originated from an area with different annual temperature patterns and the possibility of TSD having an adaptive advantage is discussed. This thesis also presents the results of a program to produce monosex male tilapia through the application of the YY male technology in O. mossambicus. Viable XY female and YY male genotypes were produced. XY females sired progenies ranging from 68-94% male ofspring, while YY males sired a mean progeny of 94% male phenotypes. From these results it is concluded that YY male technology provide a viable method for the production of all-male progeny in O. mossambicus. Once available on a commercial scale, the technology can be made more reliable through the application of the appropriate selection methods. / AFRIKAANSE OPSOMMING: Tilapia spesies is oorwegend onderworpe aan ‘n goed gedefinieerde enkelfaktor model van genetiese geslagsbepaling, hoewel afwykings van sodanige geslagsbepalingsmodel soms waargeneem word. Die invloed van outosomale gene, sowel as omgewings temperatuur word voorgehou as verklaring van die waargenome variasies in geslagsverhoudings. Hierdie studie behels die evaluasie van variasie in geslagsdifferensiasie van O. mossambicus as basis vir die implementering van die YY manlike tegnologie vir die produksie van slegs-manlike nageslag groepe. Monsters is bekom van drie populasies van O. mossambicus verteenwoordigend van die geografiese verspreiding van die spesie in Suider Afrika. Nageslag groepe is geproduseer vanaf ewekansig gekose ouerpare en by ‘n konstante temperatuur gehuisves tydens die sensitiewe tydperk van geslagsdifferentiasie. Variasie in geslagsverhouding tussen verskillende families binne dieselfde subpopulasie sowel as binne verskillende populasies is bepaal. Geen betekenisvolle verskille is waargeneem in die geslagsverhouding tussen die verskillende populasies nie terwyl betekenisvolle verskille tussen families binne ‘n populasie waargeneem is. Die data voldoen aan ‘n enkelfaktor genetiese geslagsbepalingsmodel, met die uitsondering van een populasies waar ‘n moontlike omgewingsverwante geslagsbepalingseffek waargeneem is. Die tesis sluit ook in die resultate in van ’n program om YY-tegnologie in O. mossambicus te vestig ten einde manlike nageslag groepe te produseer. Funksionele vroulike XY- en manlike YY genotipes is ontwikkel. Die vroulike XY-genotipes het manlike nageslag groepe opgelewer wat wissel van 68%-94% manlik, terwyl manlike YY-genotipes nageslag groepe gelewer het wat gemiddeld 94% manlik was. Die resultate bevestig die lewensvatbaarheid van YY-tegnologie vir die kommersiële produksie van manlike nageslaggroepe in O. mossambicus.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / In South Africa, pre-eclampsia is the second highest cause of maternal deaths. The incidence of this disease in the Western Cape alone is 6.8% and places a large burden of health care facilities. The placenta and implantation thereof is thought to play the most significant role in the onset of this disease. Among the many theories for its aetiology, is the acknowledged two - stage theory. This is based on evidence that pre-eclamptic placentas demonstrate altered remodelling and invasion into the uterine endometrium and myometrium. The sub-optimal endometrium invasion leads to less oxygenation of the placental environment causing transient hypoxia. Consequently, the placenta is thought to release unknown factors into the maternal circulation which then culminates in clinical features associated with pre-eclampsia. Neurokinin B is thought to be one of these placental factors and subsequently binds to the NKB receptor in the maternal system. Endothelium-derived nitric oxide synthase has recently been shown to activate this receptor. The aim of this study was to investigate the role of neurokinin B (TAC3) and the neurokinin B receptor (TACR3) genes in the predisposition of pre-eclampsia and their interaction with eNOS in the South African coloured population together with a matched control cohort.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The abalone Haliotis midae is the most important aquaculture species in South Africa. The industry is dependent on export to Far Eastern markets in a variety of forms, including live, frozen, canned or dried. The species is considered undomesticated in the sense that the current commercial broodstock has been obtained from natural populations through a process of random collection. Global competition has necessitated the South African industry to introduce a genetic improvement program to increase biological productivity and financial profitability. The objective of this study was to assess the genetic variation and to estimate key parameters in terms of growth and yield related traits, between family groups that form part of the breeding program. The study reports on heritability estimates of growth rate (0.14 ± 0.05), canning yield (0.08 ± 0.03), and drip loss during live export (0.03 ± 0.02). The high genetic correlation (0.94 ± 0.34) between shell length and live weight enables industry to utilise either weight or shell length as a criteria during operational practices such as sorting, grading and harvesting. The correlation of 0.85 ± 0.01 between live weight and canning loss indicates that animals that weigh more have a lower dressing percentage. Based on these low heritability values obtained for yield related traits it is recommended not to include these traits in the selection program at this stage. The findings of the study were however, compromised by the availability of a limited number of family groups, the age differences between families and the effect of different locations on the variance in phenotypes. Further investigation is needed to confirm the credibility of the results. / AFRIKAANSE OPSOMMING: Die perlemoen Haliotis midae is die belangrikste akwakultuurspesie in Suid Afrika. Die industrie is afhanklik van uitvoere na markte in die Verre Ooste. ‘n Verskeidenheid van produkte word uitgevoer, insluitend lewendige, gevriesde, gedroogte en verblikte perlemoen. Die spesie word as ongedomestikeerd beskou aangesien die huidige teeldiere op ‘n lukrake wyse uit natuurlike populasies versamel is. Globale kompetisie het die Suid Afrikaanse industrie genoodsaak om ‘n genetiese verbeteringsprogram in werking te stel om sodoende die biologiese produktiwiteit en finansiële winsgewendheid te verbeter. Die studie poog om genetiese variasie in groei en opbrengsverwante kenmerke tussen familiegroepe wat deel uitmaak van die teelprogram te ondersoek. Oorerflikheid van groeitempo (0.14 ± 0.05), opbrengs na verblikking (0.08 ± 0.03), en vogverlies na lewendige uitvoer (0.03 ± 0.02) is beraam. Die hoë korrelasie (0.94 ± 0.34) tussen gewig en skulplengte stel die industrie in staat om beide massa en skulplengte as kriteria te geruik tydens operasionele praktyke van sortering, groepering en oes. Die korrelasie van 0.85 ± 0.01 tussen gewig en verlies na verblikking dui aan dat swaarder diere ‘n laer uitslagpersentasie het. As gevolg van die lae oorerflikheidswaardes vir opbrengsverwante kenmerke word daar aanbeveel dat hierdie kenmerke nie op hierdie stadium ingesluit word in die seleksieprogram nie. Resultate is egter beïnvloed deur ‘n beperkte aantal familie groepe, ouderdomsverskille tussen families en die effek van verskillende lokaliteite op die variansie in die fenotipes. Verdere ondersoeke is nodig om die geloofwaardigheid van die bevindinge te bevestig.