Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2016. / The early detection of individuals who are at risk of developing type 2 diabetes mellitus (T2DM) would decrease the morbidity and mortality associated with this disease. DNA methylation, the most widely studied epigenetic mechanism, offers unique opportunities in this regard. Aberrant DNA methylation is associated with disease pathogenesis and is observed during the asymptomatic stage of disease. DNA methylation has therefore attracted increasing attention as a potential biomarker for identifying individuals who have an increased risk of developing T2DM. The identification of high risk biomarkers for T2DM could facilitate risk stratification and lifestyle interventions, which could ultimately lead to better ways to prevent, manage and control the T2DM epidemic that is rampant worldwide. The aim of the study was to investigate global DNA methylation as a potential risk factor for T2DM by studying the association between the global DNA methylation levels and hyperglycaemic states. A cross-sectional, quantitative study design, involving 564 individuals of mixed ancestry descent, residing in Bellville South, South Africa was used. Participants were classified as normal, pre-diabetic (impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT)) or diabetic (screen detected diabetic and known diabetics) according to WHO criteria of 1998. DNA was extracted from whole blood using the salt extraction method. The percentage global DNA methylation was measured by an enzyme-linked immunosorbent assay (ELISA). The association between global DNA methylation and hyperglycaemia, as well as other biochemical markers of T2DM was tested in a robust linear regression analysis adjusted for age, gender and smoking.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2016. / The early detection of individuals who are at risk of developing type 2 diabetes mellitus (T2DM) would decrease the morbidity and mortality associated with this disease. DNA methylation, the most widely studied epigenetic mechanism, offers unique opportunities in this regard. Aberrant DNA methylation is associated with disease pathogenesis and is observed during the asymptomatic stage of disease. DNA methylation has therefore attracted increasing attention as a potential biomarker for identifying individuals who have an increased risk of developing T2DM. The identification of high risk biomarkers for T2DM could facilitate risk stratification and lifestyle interventions, which could ultimately lead to better ways to prevent, manage and control the T2DM epidemic that is rampant worldwide. The aim of the study was to investigate global DNA methylation as a potential risk factor for T2DM by studying the association between the global DNA methylation levels and hyperglycaemic states. A cross-sectional, quantitative study design, involving 564 individuals of mixed ancestry descent, residing in Bellville South, South Africa was used. Participants were classified as normal, pre-diabetic (impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT)) or diabetic (screen detected diabetic and known diabetics) according to WHO criteria of 1998. DNA was extracted from whole blood using the salt extraction method. The percentage global DNA methylation was measured by an enzyme-linked immunosorbent assay (ELISA). The association between global DNA methylation and hyperglycaemia, as well as other biochemical markers of T2DM was tested in a robust linear regression analysis adjusted for age, gender and smoking.

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

CRISPR-Cas is an adaptive immune system unique to prokaryotes, which prevents infection by foreign genetic elements. Key to the function of CRISPR-Cas immunity is the ability to adapt to new threats in incorporating short segments, termed spacers, of invading DNA into the clustered regularly interspaced short palindromic repeat (CRISPR) array of the host. Spacers constitute immunological memories, used by CRISPR-associated (Cas) proteins to mount a sequence-specific attack on subsequent infections. The immunisation of the host is called CRISPR adaption. Adaption requires the integration of new spacers at a precise site in the CRISPR array. Two proteins, Cas1 and Cas2, are essential for adaptation; however, the mechanisms of spacer integration remain poorly understood. The work described here focused on understanding adaptation in Sulfolobus solfataricus. Using biochemical assays, I aimed to characterise the activity of the Cas1 and Cas2 proteins in this organism in order to understand their role in the insertion of new spacers. Additionally, I aimed to investigate how the expression of CRISPR-Cas components is regulated in this organism in response to viral infection. The results presented here show that expression of Cas1 was strongly upregulated in response to infection. A Csa3 protein from S. solfataricus was found to bind to the promoter for transcription of cas1, implying a role in the regulation observed. I reconstituted in vitro both the integration reaction performed by Cas1 and Cas2 proteins of S. solfataricus and the reverse of this reaction, disintegration. Cas1 was shown to impose sequence specificity on these reactions, selecting sites similar to the leader-repeat junction of the CRISPR locus. Finally, I demonstrated that, in addition to the intrinsic specificity of Cas1, there was a requirement for an additional host factor for site-specific integration in S. solfataricus.

M.Sc. (Biochemistry) / The vitamin D receptor (VDR) has been implicated in communicable diseases such as tuberculosis (TB), HIV infection and leprosy, as well as development or prognosis of noncommunicable diseases such as multiple sclerosis, lupus, type I diabetes mellitus, and osteoporosis. The burden of these diseases is often higher in specific population groups, for instance the higher TB burden of African as compared to White South Africans. Besides genetics, epigenetic factors play a role in differential disease susceptibility and prognosis between population groups. To uncover the cause of differential susceptibility to VDR-related diseases, it is crucial that population group-specific variations in genetic and epigenetic mechanisms that regulate VDR are identified. Increases in predictive power of in silico tools, and the recent explosion in availability of genome-wide genetic and epigenetic data made bioinformatics an attractive option to preselect regions to study in the VDR...

Ph.D. / Please refer to full text to view abstract

The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.

This study consisted of an evaluation of different processes of change observed during the implementation of a treatment programme for incarcerated rapists that was developed by the researcher for a unique South African context. As no treatment programme was readily available for rapists, such a programme for incarcerated rapists first had to be developed and then implemented before the evaluation process could proceed. In order to do this, the researcher utilized a processual approach as well as two models of change to guide and evaluate the different processes within the broad research process. The Total Transformation Management Process (Mink, Esterhuysen, Mink&Owen, 1993) focused on the process to develop the treatment programme, while the Interchange Cycle (Brock&Salerno, 1994) focused on the changes that took place in the rapists during and after the implementation of the treatment programme. The information obtained from the results suggests that the two models that were used to evaluate the different change processes complemented each other as both models indicated that change took place on different levels. The findings determined that the treatment programme for incarcerated rapists was effective for first-time rapists and recommendations were made for future research. Copyright 2002, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Bergh, LB 2002, Evaluation of a treatment programme for incarcerated rapists, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-09302005-100826 / > / Thesis (PhD (Psychology))--University of Pretoria, 2006. / Psychology / unrestricted

Autism spectrum disorders (ASD) are a group of related neurodevelopmental diseases displaying significant genetic and phenotypic heterogeneity. Despite recent progress in ASD genetics, the nature of phenotypic heterogeneity across probands is not well understood. Notably, likely gene-disrupting (LGD) de novo mutations affecting the same gene often result in substantially different ASD phenotypes. We find that truncating mutations in a gene can result in a range of relatively mild decreases (15-30%) in gene expression due to nonsense-mediated decay (NMD), and show that more severe autism phenotypes are associated with greater decreases in expression. We also find that each gene with recurrent ASD mutations can be described by a parameter, phenotype dosage sensitivity (PDS), which characterizes the relationship between changes in a gene’s dosage and changes in a given phenotype. Using simple linear models, we show that changes in gene dosage account for a substantial fraction of phenotypic variability in ASD. We further observe that LGD mutations affecting the same exon frequently lead to strikingly similar phenotypes in unrelated ASD probands. These patterns are observed for two independent proband cohorts and multiple important ASD-associated phenotypes. The observed phenotypic similarities are likely mediated by similar changes in gene dosage and similar perturbations to the relative expression of splicing isoforms. We also identify patterns of developmental and cell type-specific expression that additionally contribute to the variability of several autism phenotypes.

X-disease, caused by phytoplasmas, is one of the destructive diseases in stone fruit trees, causing yield loss and poor fruit quality. So far no effective methods are available to control X-disease. X-disease resistance has been first discovered in chokecherry (Prunus virginiana, 2n=4x=32), which is a native woody species of North America. To identify molecular markers linked to X-disease resistance, simple sequence repeat (SSR) markers were used to construct genetic linkage maps for chokecherry and to identify markers associated with X-disease resistance in chokecherry. In this research, three segregating populations of chokecherry were developed by crossing one X-disease resistant (CL) with three susceptible chokecherry lines (a, c, and d), of which the progenies were 101, 177, and 82, respectively. In order to construct a genetic map for chokecherry, 108 pairs of SSR primers were employed from other Prunus species. Additionally, a set of 246 SSRs were developed from chokecherry sequencing by Roche 454 sequencing technology. A total of 354 pairs of SSR primers were used to screen individuals of all three populations. Two software programs, TetraploidMap and JoinMap, were used to construct linkage map based on single-dose restriction fragments (SDRFs) and two parental linkage maps were generated for each population from both software programs. Bulked segregant analysis (BSA) was applied for identification of X-disease resistance markers. As a result, one SSR marker was found to be linked to the X-disease resistance. The set of 246 chokecherry SSRs was later used to test transferability among another 11 rosaceous species (sour cherry, sweet cherry, wild cherry, peach, apricot, plum, apple, crabapple, pear, june berry, and raspberry). As a result, chokecherry SSR primers can be transferable in Prunus species or other rosaceous species. An average of 63.2% and 58.7% of amplifiable chokecherry primers amplified DNA from cherry and other Prunus species, respectively, while 47.2% of amplifiable chokecherry primers can be transferable to other rosaceous species. The genetic information, including genetic map, disease linked marker, chokecherry sequence, and confirmed transferability of the identified chokecherry SSRs to other species, will benefit the genetic research in Prunus and other rosaceous species.