The three South African crane species, namely, the Wattled Crane (Bugeranus carunculatus), the Blue Crane (Anthropoides paradisea) and the Grey Crowned Crane (Balearica regulorum regulorum) are all threatened. South African legislation protects the cranes, however eggs and/or fledglings are sometimes illegally collected from the wild. These are then sold, often by registered breeders, who falsely claim them as the offspring of their captive breeding pair. DNA fingerprinting is one method to detect this crime. Fifteen RAPD primers were screened for polymorphism in the three species. Seven primers produced polymorphic profiles in the Blue Crane and eight each in the Grey Crowned Crane and Wattled Crane, with an average of 14.57, 12.38 and 5.88 scorable loci per primer, respectively. The Band Sharing Coefficient for unrelated individuals was found to be 0.665, 0.745 and 0.736 for the Blue, Grey Crowned and Wattled Crane respectively. Five microsatellite primers, originally developed for use in Whooping Cranes (Grus american), had previously been shown to be polymorphic in the Wattled Crane. This was also the case in this study with an average of 3.6 alleles per primer. Although all primers cross amplified, only a single primer each showed polymorphism in the Blue Crane (showing 6 alleles) and the Grey Crowned Crane (showing 5 alleles). The RAPDs were found to be irreproducible, show high numbers of novel bands and had parent: offspring BSC values that were not significantly higher than those of unrelated individuals. Statistics showed that, in the Blue Crane, the probability that misassigned parents would be detected was low whilst there was an almost certainty that true parents would be incorrectly excluded. The five microsatellite primers examined gave exclusionary powers of 0.869 and 0.641 where one or two parents were unknown in the Wattled Crane. The exclusionary powers for the Blue Crane and Grey Crowned Crane calculated at only one locus were much lower. It was concluded that RAPDs were totally inappropriate for parentage analyses, however, microsatellites are a suitable technique and recommendations are made that other microsatellites, developed for other species of crane, should be examined for their potential in this respect. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

La susceptibilité ou la résistance aux cancers peuvent impliquer plusieurs mécanismes, incluant l’apoptose, la croissance cellulaire et la différenciation, la réplication et la réparation de l’ADN. Mon projet porte plus particulièrement sur l’apoptose. Une dérégulation dans les voies d’activation de l’apoptose entraîne une accumulation de cellules déréglées, créant ainsi un environnement propice à l’instabilité génétique et au développement du cancer. Comme l’apoptose est une voie biologique hautement régulée, nous proposons l’hypothèse que des polymorphismes « fonctionnels » dans les régions de régulations des gènes (rSNPs) perturberaient cette voie à cause de taux variables de transcrits et des protéines correspondantes dû à la modification des sites de reconnaissances des facteurs de transcription. Les principaux objectifs de mon projet sont : (i) identifier les SNPs présents dans la région promotrice des gènes d’apoptose; (ii) déterminer les haplotypes de promoteurs les plus fréquents présents dans la population générale; (rHaps) (iii) vérifier leurs impacts fonctionnels sur l’expression génique par des essais in vitro (gène rapporteur et retard sur gel). Cette étude permettra d’identifier des rSNPs et rHaps ayant un impact sur le niveau d’expression des gènes d’apoptose, au moins dans un contexte in vitro. Ces différences alléliques au niveau de l’expression de ces gènes d’apoptose pourraient contribuer à la susceptibilité interindividuelle de développer un cancer. / Cancer susceptibility can involve many different mechanisms, including apoptosis, cell growth, cell differentiation, DNA replication and DNA repair. My project focuses on the apoptosis pathway. Decreased apoptosis level can lead to the accumulation of dysregulated cells, creating a favourable environment for genetic instability and tumorigenesis. Since apoptosis is a highly regulated pathway, the hypothesis of this project is that functional polymorphisms (rSNPs) in the regulatory regions of apoptosis genes such as promoters could create or disrupt transcription factor binding sites and modify their transcription levels. The main objectives of my project are: (i) Identify rSNPs in promoter regions of apoptosis genes; (ii) Calculate the frequent promoter haplotypes (rHaps) in general population; (iii) Validate their functional impact on gene expression with in vitro assays (gene reporter and gel shift). This study will allow identification of rSNPs and rHaps that could influence apoptosis gene expression levels, at least in the in vitro context. These allelic differences of expression in apoptosis genes could contribute to interindividual cancer susceptibility.

La régulation de l’apoptose est importante dans le maintient de l’homéostasie cellulaire et l’intégrité du matériel génétique. L’apoptose est un mécanisme cellulaire qui élimine les cellules endommagées. Le bon fonctionnement de cette voie biologique est crucial pour contrer la propagation des cellules avec leurs anomalies génétiques. La dérégulation des gènes codants pour des composantes de la voie intrinsèque de l’apoptose est fréquemment observée chez divers types de cancers, incluant la leucémie. Nous proposons que des polymor¬phis¬mes fonctionnels localisés dans la région régulatrice (rSNP) des gènes impliqués dans la voie d’apoptose intrinsèque auraient un impact significatif dans l’oncogenèse en modifiant le taux d’expression de ces gènes. Dans cette étude, nous avons validé, à l’aide d’une combinaison d’approches in silico et in vitro, l’impact fonctionnel de la variabilité génétique sous la forme d’haplotypes (rHAPs), au niveau du promoteur proximal, de 11 gènes codant pour des composantes de la voie intrinsèque de l’apoptose. Pour ce faire, nous avons sous-cloné les rHAPs majeurs dans un vecteur contenant le gène rapporteur luciférase (pGL3b). Ces constructions furent utilisées dans des essais de transfections transitoires dans 3 lignées cellulaires (Hela, Jeg3 et Jurkat). Nous avons observé qu’au moins 2 rHAPs influencent significativement l’activité transcriptionelle de façon allèle spécifique. Ces rHAPs sont associés aux gènes YWHAB et YWHAQ. Les analyses de retard sur gel d’électrophorèse (EMSA) ont permis d’identifier 2 sites de liaison ADN-protéine différentielles dans les rHAPs du gène YWHAB. La variabilité du niveau d’expression des gènes étudiés pourrait contribuer à la susceptibilité interindividuelle de développer un cancer, tel que la leucémie de l’enfant. / The regulation of apoptosis is important in maintaining cellular homeostasis and the integrity of the genetic material. Apoptosis is a cellular mechanism that eliminates damaged cells. The operation of this biological pathway is crucial to counter the spread of cells and their genetic abnormalities. Deregulation of genes encoding components of the intrinsic pathway of apoptosis is frequently observed in various types of cancers, including leukemia. We propose that functional polymorphism located in the regulatory region (rSNP) of genes involved in the intrinsic apoptosis pathway would have a significant impact in oncogenesis by altering the expression levels of these genes. In this study, we validated, using a combination of in silico and in vitro approaches, the functional impact of genetic variability (analyzed as haplotypes, rHap) at the proximal promoters of 11 genes encoding components of the intrinsic apoptosis pathway. To do this, we subcloned the major rHaps in a vector containing the luciferase reporter gene (pGL3b). These constructs were used in transient transfection assays in three human cell lines (HeLa, Jurkat and JEG3). We observed that at least 2 rHaps significantly influence the transcriptional activity of a specific allele. These rHaps are associated with genes YWHAB and YWHAQ. Electrophoretic mobility shift assays (EMSA) have identified 2 sites for differential DNA-protein binding with the rHaps of YWHAB. Variability in the level of expression of the genes studied could contribute to interindividual susceptibility to developing cancer, such as childhood leukemia.

Host genetic factors may partially account for the uneven distribution of HIV infection worldwide. In addition to influencing relative susceptibility to HIV, host genetic factors may also affect the rate of disease progression in persons who are already HIV infected. J.L-10 was previously identified as an AIDS restricting gene (ARG), i.e. human genes with polymorphic variants that influence the outcome of HIV-1 exposure or infection. IL-10 is a Th2 cytokine, with anti-inflammatory properties, and plays a significant role in the regulation of immune responses; this cytokine may also directly influence viral replication. This study focused on the role of genetic polymorphisms in the proximal promoter region of the IL-10 gene on HIV-:eptibility and primary HIV-1 pathogenesis in a South African comprising of women at high risk of HIV-1 infection In this study 228 black females from the CAPRISA Acute Infection cohort were genotyped for two polymorphisms that naturally occur within the proximal region of the IL-10 promoter, at positions -.1082 and -592 (tracking -819) relative to the transcription start site. DNA samples from study participants were genotyped using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, which utilises specifically designed primers to detect single nucleotide polymorphisms. The allele frequencies for the mutant -1082G and -592A variants were 0.3203 and 0.333 respectively.Individuals homozygous for the mutation at the -392 position (AA genotype) were 2.78 times more likely to become HIV infected, compared to those who were homozygous wild type (CC genotype) at the same position (p-value=0.0237). Among those who became HIV infected, we found a hierarchical association between IL-10 promoter variants and HIV-1 plasma viral load or CD4+ T cell counts over the course year of HIV-1 infection. At earlier time points, i.e. 0-3 months post-te -1082GG group had significantly higher median viral loads than the -AA or -1082AG groups (pvalues= <0.0001 and 0.0003 respectively); and the -1082AA group had the highest median CD4'' T cell count compared to the -1082AG or -1082GG groups and this was significant (p-values= 0.0194 and 0.0122 respectively). At 6-12 months post-infection the median viral load of the -1082GG group was lower than -1082AA group, however this was not significant (p-value=0.6767). Analysis of the effect of the -592 polymorphism showed that the -592AA group had a lower median viral load at 0-3 months post-infection compared to the homozygous wild-type group (i.e. -592CC p~value=0.0093); and the median CD4+ T cell count for the -592AA group was significantly higher than the -592CC group (p~ value= 0.0198). At 6-12 months post-infection, the median viral load as well as the median CD4+ T cell count of the -592 A A group were both no longer significantly different to the -592CC group (p-values= 0.644land 0.6461 respectively). Plasma IL-10 expression was not significantly different between the IL-I0 genotypes for any of the polymorphic positions.Overall, these results suggest that polymorphisms within the IL-10 promoter may influence the risk of HIV infection and that they may affect primary HIV-1 pathogenesis. Interestingly, our data suggests that the effect of these polymorphic variants on viral and CD4+ T cell counts may vary according to time post-infection. To our knowledge, this is the first study to suggest that an ARG may have a differential effect on markers of disease progression depending on the phase of infection studied. The mechanisms underlying these observations require further studies and may have important implications for HIV/AIDS pathogenesis and the development of effective vaccine and immunotherapeutic strategies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Genome-wide association studies (GWAS)s provide an unbiased means of exploring the landscape of complex genetic disease. As such, these studies have identified genetic variants that are robustly associated with a multitude of conditions. I hypothesize that these genetic variants serve as excellent tools for evaluation of the genetic interface between epidemiologically related conditions. Herein, I test the association between SNPs associated with either (i) plasma lipids, (ii) rheumatoid arthritis (RA) or (iii) diabetes mellitus (DM) and late-onset Alzheimer’s disease (AD) to identify shared genetic variants. Regarding the most significantly AD-associated variants, I have also attempted to elucidate their molecular function. Only cholesterol-associated SNPs, as a group, are significantly associated with AD. This association remains after excluding APOE SNPs and suggests that peripheral and or central cholesterol metabolism contribute to AD risk. The general lack of association between RA-associated SNPs and AD is also significant in that these data challenge the hypothesis that genetic variants that increase risk of RA confer protection against AD. Functional studies of variants exhibiting novel associations with AD reveal that the lipid-associated SNP rs3846662 modulates HMGCR exon 13 splicing differentially in different cell types. Although less clear, trends were also observed between the RA-associated rs2837960 and the expression of several BACE2 isoforms, and between the DM-associated rs7804356 and expression of a rare SKAP2 isoform, respectively. In conclusion, the overlap of lipid-, RA- or DM-associated SNPs with AD is modest but in several instances significant. Continued analysis of the interface between GWAS of separate conditions will likely facilitate novel associations missed by conventional GWAS. Furthermore, the identification of functional variants associated with multiple conditions should provide insight into novel mechanisms of disease and may lead to the identification of new therapeutic targets in an era of personalized genomic medicine.

Research Doctorate - Doctor of Philosophy (PhD) / Endometrial cancer is one of the most common female cancers in industrialized countries. Traditional risk factors associated with endometrial cancer are well understood and include excessive exposure to estrogen or estrogen unopposed by progesterone. However, variations in the genes that influence these hormones and their association with endometrial cancer have not been well investigated. By studying genetic variation in endometrial cancer, novel markers of risk may be discovered that can be used to identify women at high risk and for the implementation of specialised treatments. Polymorphisms in the genes involved in the following pathways; hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control, have been suggested to be involved in the initiation and development of endometrial cancer. The focus of this study was to examine genetic variants in these pathways to assess the existence of an association with the risk of endometrial cancer. In the first part of this study, the COMT V158M polymorphism was examined in a hereditary non-polyposis colorectal cancer (HNPCC) cohort to determine its association with disease expression. The heterozygous genotype was over-represented in women with endometrial/ovarian cancer that did not harbour mismatch repair (MMR) gene mutations. This result suggested that the COMT V158M polymorphism may alter the risk of developing HNPCC related endometrial/ovarian cancer in MMR mutation negative women. Since COMT is involved in the metabolism of estrogen and that estrogen is the main risk factor for endometrial cancer development, closer examination was warranted to determine the association of genetic variation involved in hormone-related pathways and endometrial cancer risk, outside of the context of an inherited predisposition to disease. In the second part of this study, a cohort of 191 women with endometrial cancer and 291 healthy control women were genotyped for polymorphisms in genes involved in hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control. The results revealed that variations in estrogen receptor alpha (ESR1) and beta (ESR2), and the androgen receptor (AR), were associated with an increase and decrease in endometrial cancer risk, respectively. Additionally, polymorphisms in CYP1A1, CYP1B1, GSTM1 and GSTP1 were related to a decrease in endometrial cancer risk. A trend was observed for the cyclin D1 870 G>A polymorphism and an increase in endometrial cancer risk, however, this result did not reach significance. Taken together, these results revealed that perturbations in the hormone receptors and estrogen metabolism genes, may aid in the identification of women at high risk of developing endometrial cancer. Interestingly, stratification of the women with endometrial cancer revealed that combinations of polymorphisms in TP53 and MDM2 were associated with higher grades of cancer. This finding may possibly have significant implications as women with reduced apoptotic ability, due to combinations of polymorphisms in these genes, have an increased risk of presenting with higher grades of endometrial cancer, that are associated with lower survival rates. In summary, the results of this thesis showed that variation in the estrogen and androgen receptors, and estrogen metabolism genes, may alter the risk of developing endometrial cancer. Moreover, polymorphisms in the cell cycle control genes, TP53 and MDM2, appear to be associated with higher grades of endometrial cancer. This study of polymorphisms may help explain genetic differences in individual susceptibility to endometrial cancer and are markers of risk that aid in the development of effective and personalised strategies to prevent disease development. This study has improved the understanding of genetic variation associated with endometrial cancer risk. It has the potential to enhance our ability to treat women with endometrial cancer through improved identification and treatment strategies, by virtue of the genetic variation identified, that appears to predispose to disease.

Thesis (M.Sc.). / Written for the Institute of Parasitology. Title from title page of PDF (viewed 2008/07/29). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Institute of Parasitology. Title from title page of PDF (viewed 2008/07/29). Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Aug 16.