Amongst the more promising non-viral DNA vehicles are liposomes, with those derived from cationic lipids showing significant potential, despite moderate transfection levels in vivo. This study has investigated the effect of liposome-anchored ionophore crown ethers on lipoplex-mediated gene transfer in vitro and in vivo. Several liposomes were constructed to include the cytofectin 3β[N(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the co-lipid dioleoylphosphatidylethanolamine (DOPE), and 5% (mole/mole) of the cholesteryl crown ethers RUI-128 (aza-18-crown-6) or RUI-129 (aza-15-crown-5). Liposome size and lamellarity were established by transmission electron microscopy. All liposome preparations were shown to bind, condense and protect DNA avidly in the respective band shift, ethidium displacement and nuclease protection assays. Lipoplex targeting to hepatocytes may be achieved via the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed on the human hepatoblastoma cell line HepG2. Therefore six additional liposomes were formulated to include 5% (mole/mole) of the cholesteryl galactosyl RUI-90 (Gal) and cholesteryl glucosyl RUI-92 (Glu) ligands. Their hepatotropic gene delivery was examined in the HepG2 cell line using the pCMV-luc plasmid. Transfection studies in the human embryonic kidney cell line HEK293 (ASGP-R-negative) revealed an increase in transgene activity in lipoplexes displaying the RUI-129 cholesteryl derivative. No ionophore-mediated enhancement of transfection activity was observed in HepG2 cells although Chol-T:DOPE, Chol-T:DOPE:RUI-128 and Chol-T:DOPE:RUI-129 liposomes achieved very high transfection levels, exceeding those of their hepatocyte targeted counterparts. Liposome-anchored crown ethers have been shown to potentiate in vitro transfection activity of lipoplexes in the HEK293 cell line. The novel cholesteryl glycosyl derivatives were, however, unable to enhance the targeted entry of lipoplexes into HepG2 cells. The three most effective preparations from in vitro studies were taken forward for in vivo assessment in NMRI mice at the University of the Witwatersrand Molecular Medicine and Haematology unit. Three groups of mice were employed for the evaluation of Chol-T:DOPE, Chol-T:DOPE:RUI-129 and Chol-T:DOPE:RUI-129-Gal lipoplexes with the Psi-CHECK plasmid. Mice treated with hydrodynamic injection and untreated animals made up two control groups. Luciferase activity was determined on examination of the harvested liver homogenates. All liposomes showed modest, but significant transfection activity (p<0.05) and were well tolerated. The assemblies examined therefore warrant further development. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2010.

RNA interference (RNAi), mediated by small interfering RNA (siRNA), has emerged as a powerful tool for elucidating gene function and also holds great potential for the treatment of acquired and inherited diseases. The delivery of siRNAs still remains a major obstacle for their therapeutic use. Cationic liposomes, a group of positively charged nanovesicles, represent a class of non-viral vectors that have shown the ability to efficiently bind and deliver siRNA. In this study, six cationic liposome formulations containing either cationic cholesterol derivative [N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or N,Ndimethylaminopropylaminylsuccinylcholesterylformyl- hydrazide (MSO9) were prepared with the neutral lipid dioleoylphosphatidylethanolamine (DOPE). Varying amounts of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), (0, 2 and 5 mole percent) were also included in the liposomal formulations as polyethylene glycol is known to improve the lipoplex bioavailability in vivo. We present evidence that siRNA may be delivered to mammalian cells, in vitro, using a novel cationic liposome carrier system and that siRNA binding and transfection efficiency of the cationic liposomes are affected by the degree of pegylation. Cryoelectron microscopy revealed that the liposome vesicles were unilamellar and were in the 30 - 130 nm size range, while band shift assays confirmed the formation of complexes between the siRNA and the liposome preparations. These siRNA lipoplexes were shown to afford protection to their siRNA cargoes against serum nuclease degradation and were also shown to be relatively non-toxic to the HeLa tat luc cell line which stably expresses the firefly luciferase gene. Cryoelectron microscopy revealed that an inverse relationship exists between the lipoplex size and the degree of pegylation. To determine the transfection efficiency of the cationic liposome preparations in the HeLa tat luc cell line, complexes were prepared with anti-luciferase siRNA, which is specific for the firefly luciferase gene, and knockdown of the luciferase gene was monitored in transfected cells. The results show that liposomes containing the cytofectin Chol-T were particularly effective, achieving up to 93.4% gene knockdown at the 30 nM siRNA concentration. The non- pegylated and pegylated cationic liposomes that have been formulated and examined in this study therefore warrant further development to facilitate in vivo studies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1978.

The problems associated with the use of white clover in pastures in the eastern high potential areas of South Africa i.e. high P requirements, low tolerance to high Al levels and low pH in the soil as well as a limited survival time of approximately 30 months, were identified and found to be related to the inadequate root system of white clover cultivars. During the improvement programme cultivars available on the world market were introduced and evaluated under dryland conditions. Selections were made from these introductions on the basis of root conformation in high AI, low pH soils, their response to grazing and induced moisture stress. A laboratory technique for the improvement of Al tolerance was developed and the tolerance of white clover plants to high levels of Al was improved but due to the complexity of pasture plant improvement it was decided that the selection for tolerance to Al could be more effectively carried out in the field. The effectiveness of vesicular arbuscular mycorrhizas as phosphate gatherers indicated that local strains of mycorrhizas combined as effectively with white clover as the imported strains. As no seed production of white clover is undertaken in South Africa guidelines for local seed production were also established. As a result of the improvement programme, Trifolium repens cv. DUSI was developed as an open pollinated synthetic variety, based on thirty eight selected mother lines. DUSI has a greater tolerance to high AI, low pH, low P in the soil and due to an improved root system with a high percentage of secondary taproots produces better under dryland conditions and has a longer stand life than any of the cultivars of white clover available on the local market. Plant Breeders Rights were obtained for cv. DUSI and the cultivar was inscribed on the South African variety list. Limited amounts of Breeders seed have been made available to the South African Forage Seed Association for commercial seed production. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1988.

The high gene knockdown specificity and efficiency of RNA interference (RNAi) provides a potentially viable avenue for the development of a new class of nucleic acid therapeutics for gene-based disease conditions. However, serum instability, inefficient cellular trafficking and non-specific effects of small interfering RNAs (siRNAs), one of the functional mediators of RNAi, has necessitated the development of carriers to facilitate targeted cell delivery. The decline of viral vectors in human gene therapy as a consequence of safety issues has intensified the importance of non-viral vector development. Among the non-viral vectors available for siRNA delivery, cationic liposomes have emerged as an attractive option owing to their simplicity, versatility, relatively low toxicity and potential for cell-specific targeting. Although existing cationic lipids and liposomes traditionally used for DNA delivery have also been used for siRNAs, there still exists a need to develop cationic lipids tailored towards siRNA transfection for improved gene silencing efficiency. Among the cell specific targets available for RNAi therapeutics, hepatocytes expressing the asialoglycoprotein receptor (ASGP-R) are an ideal choice due to the large number of disease targets present for treatment. In this investigation four novel cationic liposome formulations were prepared from equi-molar quantities of either the cationic cholesterol derivative 3β [N-(N’,N’- dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3β [N-(N’, N’, N’- trimethylammoniumpropyl)-carbamoyl] cholesterol iodide (Chol-Q) and DOPE, with and without the hepatotropic ligand, cholesteryl-β-D-galactopyranoside. Electrophoretic gel analysis and SYBR®green displacement assays were employed to determine siRNA binding and condensation efficiencies for all cationic liposomes; while liposome and lipoplex size measurements were made by cryoTEM. SiRNAlipoplex stability in serum was determined by the nuclease protection assay. Cell studies performed on the ASGP-R+ human hepatoma cells, HepG2 and the ASGP-Rembryonic kidney cells, HEK293, to determine lipoplex toxicity and transfection efficiencies were also undertaken. We show that the cationic liposomes formulated for this investigation were able to bind synthetic siRNA optimally at a positive to negative charge ratio of ± 1 : 6. In addition, the cationic liposomes were able to afford siRNA duplexes substantial protection from ribonuclease digestion in serum. From the results obtained in this study, it appears that the cationic liposomes are well tolerated by both the HEK293 and HepG2 cells in vitro. More importantly, the results obtained demonstrated higher transfection efficiencies for the targeted lipoplexes compared with the untargeted controls, strongly supporting the notion that incorporation of the cholestryl-β-D-galactopyranoside into the liposome structure increases transfection efficiency to the targeted HepG2 cells in culture via ASGP receptor mediation. Comparative studies in the HEK293 cell line yielded low transfection effeciences in the order of 20%, with no significant difference being recorded between galactosylated and non-galactosylated lipoplexes. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2011.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Abruptio placentae is the premature separation of the normally implanted placenta from the uterine wall, resulting in haemorrhage before delivery of the fetus. This has serious maternal and neonatal implications, and is one of the leading causes of perinatal and maternal mortality and morbidity in South Africa. Placental vasculopathies, such as abruptio placentae, are believed to result from faults occurring in early placental development. Placental protein 13 (PP13) is a member of the pregnancy-related protein family, and is believed to function in a number of important physiological processes such as trophoblast invasion, placentation and implantation. The aim of this study was to investigate whether DNA sequence variants in the LGALS13 gene (encoding PP13), underlie and/or confer susceptibility to abruptio placentae. The gene was screened and genotyped in a cohort of patients whose pregnancies were complicated by abruptio placentae, as well as an ethnically matched control cohort. Statistical and in silico analyses were performed in order to identify potential susceptibility factors in this South African cohort and to predict whether the identified variants may impact on gene expression or the structure and function of PP13. In addition, the luciferase reporter gene assay was employed to investigate the functionality of the -98A/C variant identified in the 5’ untranslated region of the LGALS13 gene. Statistically significant differences were observed between patient and control groups at the following loci in the Coloured population: -98A/C, IVS2 -36G/A, IVS2 -22A/G and the hotspot variant in exon 3 (p<0.05). These variants could represent a susceptibility profile of this population or alternatively have implications in the pathogenesis of abruptio placentae. The deletion of a single thymine in exon 3 was shown to result in truncation of PP13 and subsequent disruption of a number of cysteine residues and putative phosphorylation sites, which could impact on dimerization and ultimately, the function of the protein. The reporter gene assay revealed a significant reduction (p=0.004) in luciferase activity by the -98 C allele. iii In silico analysis suggests that this reduction could be due the disruption of a NF1 or GR transcription factor binding site. This study provides evidence that variants in the LGALS13 gene may underlie and/or confer susceptibility to abruptio placentae by impacting on gene regulation or resulting in the expression of an aberrant form of the PP13 which could affect functionality and thereby result in the disruption of normal implantation and placentation.

Thesis (PhD (Genetics))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Currently, South African aquaculture is dominated by the cultivation of Haliotis midae, which is estimated as the most lucrative sector of the industry, with 934 t being export in 2008, totalling an income of ZAR 268 million (40 million USD) in 2008. This represents 81% of the total rand value of the aquaculture sector. Abalone was the highest aquaculture commodity exported during the last two years from South Africa, representing 24% of the total tonnage exported. Employment in the aquaculture sector increased by approximately 80% between 2005 and 2008, and was highest in the abalone sector where the number of people employed increased by 234%. Despite these high production rates, no hatchery procedures have been developed specifically for H. midae. Most procedures and protocols currently used in South African abalone hatcheries have been adopted from cultivation methods used for foreign species. Although certain aspects of reproduction are universally conserved between abalones, it is important to consider the physiology and the origin of the species studied. To date, no scientific research has been conducted on the reproduction of H. midae, except for a few studies in the early 1990s, which focused on the basic reproduction of this species. No further studies have been done on H. midae reproduction under intensive culture. Currently, hatch-out rates obtained by most abalone farms in South Africa averages 80%, with a 50% settlement rate, and a final hatchery output of only 30%. This study reports on various aspects of H. midae reproduction that can influence its commercial culture. A detailed histological characterisation of gametogenesis was developed. Findings indicated that cultured H. midae reaches 50% sexual maturity at a shell width of between 25 mm and 30 mm. During fertilisation trials, a sperm concentration of 50 000 sperm mL-1 and egg concentrations lower than 50 eggs mL-1 produced the highest hatch-out rates. Whilst fertilisation volume did not influence fertilisation success, fertilisation potential of the eggs did decrease with time. Eggs older than 100 minutes showed a lower fertilisation potential than eggs fertilised earlier. A larval stress test was developed to evaluate larval resistance against chemical stress. It was determined that 50% of resultant larvae would exhibit morphological abnormalities after fertilised eggs were incubated in 0.7% dimethyl sulfoxide (Me2SO) for a 24 hour period. If larvae exhibited fewer abnormalities at this concentration, it could be deduced that the larvae had a high resistance to the negative effect of the toxicant, and could thus be seen as good quality larvae. When evaluating hybridisation potential between H. midae and H. spadicea, it was found that it was possible to fertilise spawned H. midae eggs with biopsied H. spadicea sperm. By incorporating the results obtained from the present study into current hatchery systems on South African abalone farms, higher hatchery yield could be achieved, which in turn would lead to an increase in commercial revenue. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse akwakultuur sektor word tans oorheers deur die produksie van Haliotis midae, en word gereken as die mees winsgewendste bedryf in die industrie, met 934 t uitgevoer in 2008, na-raming „n inkomste van ZAR 268 miljoen (40 miljoen Amerikaanse doller). Dit verteenwoordig sowat 81% van die totale rand waarde in die akwakultuur bedryf. Perlemoen was ook die grootste uitvoer kommoditeit gedurende die laaste twee jaar, en het tot sowat 24% van die totale uitvoer vanuit akwakultuur bygedra. Werksgeleenthede in die akwakultuur sektor het tussen 2005 en 2008 met ongeveer 80% gegroei, waarvan die hoogste groeisyfer in die perlemoen bedryf was, waar die aantal werknemers met 234% toegeneem het. Ten spyte van hierdie hoë produksie omset, is daar tans geen protokolle wat spesifiek vir die produksie van H. midae ontwerp is nie. Meeste van die tegnieke wat huidiglik gebruik word op Suid-Afrikaanse plase, is gebaseer op, en aangepas vanaf metodes daargestel in die internasionale bedryf vir uitheemse spesies. Alhoewel sekere aspekte van reproduksie tussen perlemoen spesies verband hou, is dit belangrik om die fisiologie en oorsprong van die spesie van belang, in ag te neem. Wetenskaplike navorsing gedoen op die reproduksie van H. midae is beperk tot studies in die vroeë 1990s, wat die basiese beginsels van die spesie se reproduksie ondersoek het. Daar is geen verdere studies op die reproduksie van H. midae, veral onder intensiewe teel toestande, gedoen nie. Tans toon die meeste perlemoen plase in Suid-Afrika „n produksie persentasie van ongeveer 80% larwes vanaf bevrugting, met „n afname na 50% met vestiging en „n gevolglike uitset van slegs 30%. Hierdie studie doen verslag oor verskeie reproduksie aspekte van H. midae wat die teel doeltreffendheid van perlemoen op kommersiële plase kan beïnvloed. „n Gedetaileerde histologiese karakterisering van gametogenese is ontwikkel. Daar is gevind dat geteelde perlemoen 50% geslagsrypheid bereik met „n skulp wydte van tussen 25 mm en 30 mm. Tydens bevrugting eksperimente is bepaal dat 50 000 sperm mL-1 en „n eier konsentrasie van laer as 50 eiers mL-1, die optimale gameet konsentrasies is vir effektiewe bevrugting. Alhoewel die volume water waarin bevrugting plaasvind nie „n invloed getoon het op bevrugtingsukses nie, is daar wel gevind dat die eiers se potensiaal om bevrug te word, afneem met verloop van tyd. Eiers ouer as 100 minute het „n verlaagde bevrugtingspotentiaal getoon teenoor eiers wat vroeër bevrug is. „n Larwale stres toets is ontwikkel om larwale weerstand teen chemiese stres te bepaal. Daar is gevind dat 50% van geproduseerde larwes morfologiese abnormaliteite sal toon indien bevrugte eiers vir „n periode van 24 uur in 0.7% dimetiel sulfoksied (Me2SO) geïnkubeer word. Indien larwes minder abnormaliteite toon by hierdie konsentrasie, beteken dit dat hierdie larwes meer weerstand kan bied teen die negatiewe effek van die toksiese middel, en dus beskou kan word as goeie kwaliteit larwes met hoë lewensvatbaarheid. Met die evaluering van hibridisasie potensiaal tussen H. midae en H. spadicea, is gevind dat dit moontlik is om vrygestelde H. midae eiers te bevrug met H. spadicea sperm wat verkry is deur „n biopsie. Die implementering van hierdie studie se bevindinge in kommersiële H. midae produksiesisteme sal daadwerklik bydra tot die optimisering van bestuurspraktyke en „n verhoging in die totale produksie doeltreffendheid van sulke sisteme.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Iron is an essential micronutrient that is an absolute requirement for correct cellular function in all eukaryotic organisms. However, ferrous iron has the ability to catalyze the formation of potentially toxic reactive oxygen species and regulation of iron metabolism is therefore of critical importance. Currently, there is little known about the co-ordinated regulation of the plethora of genes coding for proteins involved in this biochemical pathway, with the exception of the well characterized post-transcriptional IRE/IRP system. Regulation of gene expression in eukaryotic organisms is a highly intricate process. Transcriptional regulation is the first step and is controlled by the presence of specific cis-regulatory regions (cis-motifs), residing within the promoter region of genes, and the functional interactions between the products of specific regulatory genes (transcription factors) and these cismotifs. A combinatorial bioinformatic and functional approach was designed and utilized in this study for the analysis of the promoter architecture of genes of the iron metabolic pathway. The upstream non-coding region (~2 kb) of 18 genes (ACO1, CP, CYBRD1, FTH1, FTL, HAMP, HEPH, HFE, HFE2, HMOX1, IREB2, LTF, SLC11A2, SLC40A1, STEAP3, TF, TFRC, TFR2), known to be involved in the iron metabolism pathway, was subjected to computational analyses to identify regions of conserved nucleotide identity utilizing specific software tools. A subset of nine (CYBRD1, FTH1, HAMP, HFE, HFE2, HMOX1, IREB2, LTF, TFRC) of the genes were found to contain a genomic region that demonstrated over 75% sequence identity between the genes of interest. This conserved region (CR) is approximately 140 bp in size and was identified in each of the promoters of the nine genes. The CR was subjected to further detailed examination with comparative algorithms from different software for motif detection. Four specific cis-motifs were discovered within the CR, which were found to be in the same genomic position and orientation in each of the CR-containing genes. In silico prediction of putative transcription factor binding sites revealed the presence of numerous binding motifs of interest that could credibly be associated with a biological function in this pathway, including a novel MTF-1 binding site in five of the genes of interest. Validation of the bioinformatic predictions was performed in order to fully assess the relevance of the results in an in vitro setting. Luciferase reporter constructs for the nine CRcontaining genes were designed containing: 1) the 2 kb promoter, 2) a 1.86 kb promoter with the CR removed and 3) the 140 bp CR element. The expression levels of these three reporter gene constructs were monitored with a dual-luciferase reporter assay under standard culture conditions and simulated iron overload conditions in two different mammalian cell lines. Results of the luciferase assays indicate that the CR promoter constructs displayed statistically significant variation in expression values when compared to the untreated control constructs. Further, the CR appears to mediate transcriptional regulatory effects via an iron-independent mechanism. It is therefore apparent that the bioinformatic predictions were shown to be functionally relevant in this study and warrant further investigation. Results of these experiments represent a unique and comprehensive overview of novel transcriptional control elements of the iron metabolic pathway. The findings of this study strengthen the hypothesis that genes with similar promoter architecture, and involved in a common pathway, may be co-regulated. In addition, the combinatorial strategy employed in this study has applications in alternate pathways, and could serve as a refined approach for the prediction and study of regulatory targets in non-coding genomic DNA. / AFRIKAANSE OPSOMMING: Yster is ‘n noodsaaklike mikrovoedingstof wat ‘n vereiste is vir korrekte sellulêre funksie in alle eukariotiese organismes. Yster (II) of Fe2+ het egter die vermoë om die vorming van potensiële toksies reaktiewe suurstof spesies te kataliseer en dus is die regulasie van die yster metaboliese padweg van kardinale belang. Tans is daar beperkte inligting oor koördineerde regulasie van die gene, en dus proteïene waarvoor dit kodeer, in hierdie padweg. ‘n Uitsondering is die goed gekarakteriseerde na-transkripsionele “IRE/IRP” sisteem. Regulasie van geenuitdrukking in eukariotiese organismes is ‘n ingewikkelde proses. Transkripsionele regulasie is die eerste stap en word beheer deur die teenwoordigheid van spesefieke cis-regulatoriese elemente (cis-motiewe), geleë in die promotor area van gene, en die funksionele interaksies wat plaasvind tussen die produkte van spesifieke regulatoriese faktore (of transkripsie faktore) en hierdie cis-motiewe. ‘n Gekombineerde bioinformatiese en funksionele benadering was ontwerp en daarna gebruik in dié studie vir die analise van die promotor argitektuur van gene wat ‘n rol speel in die yster metaboliese padweg. Die stroomop nie-koderende streek (~2 kb) van 18 gene (ACO1, CP, CYBRD1, FTH1, FTL, HAMP, HEPH, HFE, HFE2, HMOX1, IREB2, LTF, SCL11A2, SLC40A1, STEAP3, TF, TFRC, TFR2), bekend vir hul betrokkenheid in die yster metabolisme padweg, was bloodgestel aan bioinformatiese analises om die streke van konservering te identifiseer met die hulp van spesifieke sagteware. Slegs nege (CYBRD1, FTH1, HAMP, HFE, HFE2, HMOX1, IREB2, LTF, TFRC) van die geanaliseerde gene het ‘n genomiese area bevat wat meer as 75% konservering getoon het. Hierdie gekonserveerde area (GA) is 140 bp in lengte en is geïdentifiseer in elk van die promotors van die nege gene. Die GA was verder bloodgestel aan analises, met die hulp van spesifieke sgateware, wat gebruik maak van vergelykende algoritmes vir motief karakterisering. Vier cis-motiewe is identifiseer en kom voor in dieselfde volgorde en oriëntasie in elk van die gene. In silico voorspelling van moontlike transkripsie faktor bindingsplekke het getoon dat daar talle bindingsmotiewe van belang teenwoordig is en dié motiewe kan gekoppel word aan biologiese funksies in hierdie padweg, insluitend ‘n nuwe MTF-1 bindingsplek in vyf van die gene van belang. Die bioinformatiese analises is verder gevalideer om die relevansie van die resultate in ‘n in vitro sisteem ten volle te assesseer. Luciferase rapporteerder konstrukte is vir die nege gene ontwerp wat die volgende bevat: 1) die 2 kb promotor, 2) ‘n 1.86 kb promotor met die GA verwyder en 3) die 140 bp GA element. Die vlakke van uitdrukking van hierdie drie rapporteerder konstrukte was genormaliseer met ‘n dubbele-luciferase rapporteerder assay onder standaard kultuur kondisies en gesimuleerde ysteroorlading kondisies in twee verskillende soogdier sellyne. Resultate van die luciferase assays dui aan dat die GA promotor konstrukte statisties betekenisvolle variasie toon in vergelyking met die onbehandelde kontrole konstrukte. Verder, die GA blyk om transkipsionele regulatoriese effekte te medieer via ‘n yster-onafhanklike meganisme. Dit blyk duidelik dat die bioinformatiese voorspellings ook funksioneel getoon kon word en was dus relevant in dié studie en regverdig verdere ondersoek. Hierdie eksperimentele ontwerp verteenwoordig ‘n unieke en omvattende oorsig van nuwe transkripsionele beheer elemente wat voorkom in die yster metaboliese padweg. Die resultate van dié studie versterk die hipotese dat gene met soortgelyke promotor argitektuur en wat betrokke is in ‘n gemene padweg saam gereguleer kan word. Daarbenewens, die gekombineerde strategie wat in hierdie studie gebruik is het toepassings in alternatiewe metaboliese paaie, en kan dien as ‘n verfynde benadering vir die voorspelling en studie van die regulerende teikens in nie-koderende genomiese DNS. / National Research Foundation (Thuthuka) / Stellenbosch University

Objective.Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and disease progression. This heterogeneity is attributed to the interplay between the environment, viral diversity, immune response and host genetics. This study focused on host genetics. We studied the association of single nucleotide polymorphisms (SNPs) in peptidyl prolyl isomerase A (PPIA), transportin 3 (TNPO3) and PC4 or SFRS1 interacting protein 1 (PSIP1) genes with HIV-1 infection and disease progression. These genes code for Cyclophilin A (CypA), Transportin-SR2 (TRN-SR2) and Lens epithelium derived growth factor/p75 (LEDGF/p75) proteins respectively, which are all validated HIV replication cofactors in vitro. Methods. One SNP A1650G in the PPIA gene was genotyped in 168 HIV-1 negative and 47 acutely infected individuals using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 6 intronic and 2 exonic haplotype tagging (ht) SNPs (rs13242262; rs2305325; rs11768572; rs1154330; rs35060568; rs8043; rs6957529; rs10229001) in the TNPO3 gene, 4 intronic ht SNPs (rs2277191, rs1033056, rs12339417 and rs10283923) and 1 exonic SNP (rs61744944, Q472L) in the PSIP1 gene were genotyped in 195 HIV-1 negative and 52 acutely infected individuals using TaqMan assays. The rs1154330, rs2277191, rs12339417 and rs61744944 were further genotyped in 403 chronically infected individuals. CypA and LEDGF/p75 messenger RNA (mRNA) expression levels in peripheral blood mononuclear cells (PBMCs) were quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The impact of the Q472L mutation on the interaction of LEDGF/p75 with HIV-1 integrase (IN) was measured by AlphaScreen. Results. The minor allele (G) of SNP A1650G (1650G) in the promoter region of PPIA was significantly associated with higher viral load (p<0.01), lower CD4+ T cell counts (p<0.01) and showed a possible association with rapid CD4+ T cell decline (p=0.05). The 1650G was further associated with higher CypA expression post HIV-1 infection. The minor allele (G) of rs1154330 in the intron region of TNPO3 was associated with faster HIV-1 acquisition (p<0.01), lower CD4+ T cell counts, higher viral load during primary infection (p<0.05) and rapid CD4+ T cells decline (p<0.01). The minor allele (A) of rs2277191 (rs2277191A) in the intron region of PSIP1 was more frequent among seropositives (p=0.06). Among individuals followed longitudinally, rs2277191A was associated with higher likelihood of HIV-1 acquisition (p=0.08) and rapid CD4+T cell decline (p=0.04) in the recently infected (primary infection) cohort. In contrast, the minor allele (C) of rs12339417 (rs12339417C) also in the intron region of PSIP1 was associated with higher CD4+ T cell counts during primary infection. The rs12339417C was also associated with slower rate of CD4+ T cell decline (p=0.02) and lower mRNA levels of LEDGF/p75 (p<0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 compared to nonseroconverters (p<0.01) and these levels decreased after HIV-1 infection (p=0.02). The Q472L mutation showed approximately 2-fold decrease in the association constant (Kd), suggesting stronger binding to HIV-1 integrase. Our findings demonstrate, for the first time, that genetic polymorphisms in the TNPO3 and PSIP1 genes may be associated with susceptibility to HIV-1 infection and the disease progression. These data provide in vivo evidence that TRN-SR2 and LEDGF/p75 are important host cofactors for HIV-1 replication. This is also the first study to show the association of genetic polymorphisms in the PPIA gene with disease outcome in a population (South African) with high burden of HIV-1 infection. Conclusions. Genetic variation in HIV-1 replication cofactors may be associated with disease outcome in a South African population. These data strongly support the role of these HIV replication cofactors in disease pathogenesis in vivo and suggest that these factors are possible targets for therapeutic interventions. However, these data will need to be replicated in larger cohorts to confirm the effect of these genetic variants. Further studies on how to target these factors in antiviral strategies are needed. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

The development of a genetic engineering system for soya bean (Glycine max L.) is described in this thesis. Routine tissue culture regeneration systems were developed for South African cultivars of soya bean despite the recalcitrant nature of this plant to in vitro manipulation. Regeneration of shoots was obtained when cotyledons were excised from seeds germinated for two days and cultured on B5 BA 20 medium containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting were overcome by culturing cotyledons in the dark for four weeks to produce shoots with unusually long stems. This was followed by one week of culture under conditions of high light intensity to obtain healthy green shoots which could be rooted , either in sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of rooted soya bean regenerants was essential for the recovery of fertile soya bean plants. Molecular techniques for the cloning of foreign genes into binary vectors suitable for plant genetic engineering were also studied and are described in the thesis. The Basta herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121 which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct, pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains and these strains, along with other binary vector-containing strains, were used to transform soya bean plant material. Although a protocol for the routine transformation of soya bean was not developed, transgenic soya bean material resistant to kanamycin and showing GUS activity was obtained. Transformation of wound sites on cotyledons was obtained in several experiments and transgenic shoots were regenerated from inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin resistant soya bean shoots were produced. Transgenic soya bean plants resistant to the herbicide Basta were not produced due to the recalcitrant nature of the crop to genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a model system for plant genetic engineering was achieved. The binary pat gene containing vector constructed in th is study, as well as vectors obtained from AgrEvo, were tested. The transgenic Basta resistant tobacco plants obtained were used to optimize assay systems for the analysis of transformed plant material containing the pat gene. These assay systems included the use of the polymerase chain reaction as well as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.