Characterisation of CIS- and trans-acting factors that regulate the human alpha 2(1) procollagen gene

The differential expression of the a2(I) procollagen gene in normal and transformed human fibroblasts has been correlated with differential in vitro DNA-protein interactions on the basal promoter region between -100 and -67. A 23 bp region of the a.2(1) procollagen promoter encompassing the G/CBE (CCTCCATTGG) and the Ctv'IE (GGAGGCCCTTTT) has previously been shown to engage in specific DNA protein interactions that determined the transcriptional activity of the promoter. The CME forms two distinct DNA-protein complexes that might be crucial in the regulation of the a2(I) procollagen gene in a cell specific manner. The hypothesis was, therefore, that depending on the protein that participates in complex formation with the CME, the gene would be activated or repressed. The objective of this study was to investigate the role of this 23 bp region in the regulation of expression. of the a2(I) procollagen gene in transformed fibroblasts. In addition, the study sought to establish the role of the proto-oncogene c-fos-in the regulation of expression of the a2(I) procollagen gene. In contrast to previous observations, this study demonstrated that only one DNA protein complex is formed on the CME and the second complex is a specific proteolytic cleavage of the product of the larger complex. Preparation of nuclear extracts in the absence of protease inhibitors, specifically leupeptin, resulted in the formation of a smaller complex, previously shown to bind the CME. The importance of this proteolytic fragment that still retains DNA binding activity is yet to be determined. In addition, the CME binding proteins were fairly ubiquitously expressed in both a.2(1) collagen producing and non-producing cells. CT-1 fibroblasts (transformed by y-irradiation) synthesise over 80% of total a2(I) collagen produced by its untransformed counterpart (WI-38 fibroblasts), whereas the gene, is down regulated in the human embryonic lung fibroblasts transformed with SV40 (SVWI-38 fibroblasts). These cell lines are therefore ideal for studying regulation of a.2(I) procollagen gene. To analyse the importance of the G/CBE and CME regions of the a.2(1) procollagen gene promoter, point mutations were introduced by site-directed mutagenesis. Mutated promoter DNA was cloned into a p8CAT reporter vector, and the activity of the promoter determined in transient transfection experiments. Mutations introduced in the G/CBE region of the a.2(1) procollagen promoter resulted in a 3-12-fold decrease in the activity of the promoter. The decrease was observed with both proximal (-343 bp) and basal (-107 bp) promoter constructs~ a significant reduction in promoter activity was observed in both CT-1 and SVWI-38 fibroblasts. These results imply that the G/CBE region of the promoter is required for the activation of transcription of the a.2(1) procollagen gene and therefore the factor that interacts with the G/CBE functions as a transcriptional activator. Previously, this factor was shown to complex with antibodies raised against the mouse CCAAT binding factor (CBF), suggesting that the protein belongs to the CBF family of transcription factors. Furthermore, these results demonstrate that the adjacent, upstream inverted GGAGG sequence is crucial for activation of the gene through the CCAAT binding element. The inhibition of promoter activity in constructs with a mutated G/CBE element was correlated with lack of protein binding to the mutated sequence as confirmed by electrophoretic mobility shift assays. Transfection of a.2(1) procollagen promoter constructs containing mutations in the CME region, however, resulted in a significant increase in promoter activity in both CT-1 and SVWI-38 fibroblasts. A much higher increase, 3-fold, was observed for the SVWI-38 cell line compared to a 1.5-fold increase observed for CT-1 fibroblasts. These results suggested that the factor that interacts with the CME functions as a repressor of the a.2(1) procollagen gene. Interestingly, the promoter activity in SVWI38 fibroblasts transfected with mutated CME constructs was similar to that observed in CT-1 fibroblasts transfected with the wild type promoter construct. An interesting observation was that repression of the a.2(1) procollagen gene via the CME required upstream elements since transfection of the basal mutated promoter did not result in increased promoter activity. From these results, it can be concluded that the CME binding protein is involved in cell-specific repression of the a2(I) procollagen gene and that the mechanism of repression appears to be dependent on the presence of upstream elements. Mutations in the G/CBE and CME pointed out the significance of these elements in the expression of the a2(I) procollagen gene and since a number of studies have characterised the mouse CCAAT binding protein, this study focused on purification and identification of the CME binding protein(s). Purification was performed by conventional biochemical techniques using heparin-agarose and sequence-specific DNA affinity chromatography, as well as separation on SDS-polyacrylamide gels. Two cycles of DNA affinity chromatography yielded two polypeptides with apparent molecular weights of 50 and 67 kDa. Automated N-terminal sequencing of the polypeptides indicated that they were blocked and therefore no sequence could be obtained. In addition, these polypeptides failed to raise an immune response in mice and rabbits. Subsequently, polypeptides were digested with trypsin in situ in polyacrylamide gels and the eluted peptides were analysed by MAWITOF-mass spectrometry. The mass:charge ratios (mlz ratios) obtained were used to search the database using a mass tolerance of 1.5 Da and only one hit was obtained. The match obtained was that of a mouse zinc finger protein of which not much is known, except that it might be a transcription factor. This result supports previous observations of Collins et al (J Cell Biochem 1998, 70: 455-467) that complex formation requires the presence of zinc. The primary structure of the CME binding protein remains to be determined. Transformation of fibroblasts is normally accompanied by changes in the expression of extracellular proteins, including type I procoUagen. Although CT-I fibroblasts, show very little change in a2(I) procollagen gene expression, the c-f os gene is drastically down-regulated. This study sought to establish if there is any relationship between the unusually high levels of the a2(I) procollagen gene in this transformed cell line and failure of the cells to stimulate c-fos expression in response to serum. CT-1 :fibroblasts that overexpressed wild type Fos were established and changes in the expression of the a,2(1) procollagen gene were measured. Overexpression of Fos down-regulated the a,2(1) procollagen gene, which was not due to increased turnover of the a1(I) procollagen mRNA. Analysis of promoter activity showed that the promoter and first intron, which has been reported to contain negative regulatory elements, did not harbour any Fas-responsive elements. The -343 bp and the -2300 bp promoter constructs were transactivated in cells overexpressing Fos. Thus, although overexpression of Fos resulted in a significant decrease in the levels of the a2(1) procollagen WA, it does not involve the region between -2300 bp and +1800 bp of the a2(1) procollagen gene. Furthermore, there was no change in the stability of the message, indicating that constitutive expression of Fos did not activate a factor that could play a role in altered turnover of the a2(I) procollagen mRNA. It is therefore possible that constitutively high levels of Fos may trigger the expression of a number of other genes, which have a negative impact on the expression of the a2(I) procollagen gene

Implementation of clinical guidelines for the management of pre-eclampsia by midwives in uMgungundlovu District of KwaZulu Natal

The purpose of the study is to determine the knowledge of midwives on implementing clinical guidelines for the management of pre-eclampsia with the aim of improving midwifery practice and preventing maternal and neonatal death due to pre-eclampsia. A quantitative, descriptive, cross sectional study design was used for the study. Data was collected using a self-administered questionnaire from 100 midwives working in the maternity section of the clinic referring to the regional hospital in UMgungundlovu District. The Statistical Package for Social Sciences (SPSS) version 23 was used for data analysis. The findings show that respondents to the study have a high mean value (3.6) of knowledge but need support in terms of providing training on the new guidelines. The study also showed that there is need to improve on distribution of guidelines. There was no significant association between demographic factors and the knowledge of midwives on clinical guidelines for the management of pre-eclampsia. A clinical audit of maternity records as well as a quality care project can be developed based on the findingsHealth StudiesM.A. (Health Studies

Epigenetic modification at imprinted loci following alcohol exposure during prenatal development

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy February 2016Fetal alcohol syndrome (FAS) is a devastating developmental disorder resulting from alcohol exposure during fetal development. It is a considerable public health problem worldwide, but in several communities in South Africa, specifically in the Western and Northern Cape, it has an exceptionally high prevalence of 68.0 – 89.2 per 1000 children of school going age. FAS is a developmental disorder characterised by facial dysmorphic features, growth retardation and central nervous system abnormalities. Twin concordance studies and animal models suggest that there are genetic and epigenetic susceptibility factors for developing FAS. Imprinted genes are known to play an important role in growth and development and most of them are located in imprinted clusters. The IGF2/H19, DLK1/MEG3 (GTL2), CDKNIC/ KCNQ1OT1 and PEG3 imprinted loci play a critical role in fetal development. Each of these imprinted loci contain several imprinted genes that are reciprocally imprinted, and their differential expression is controlled by differentially methylated regions (DMR) referred to as imprinting control regions (ICRs). The ICR for IGF2/H19 is called H19 ICR and for DLK1/MEG3 (GTL2) is IG-DMR and they are both marked with DNA methylation on their paternal allele. KvDMR1 and PEG3 DMR are ICRs for CDKNIC/KCNQ1OT1 and PEG3 imprinted loci respectively and they are marked with methylation on their maternal allele. DNA methylation at CpG dinucleotides is an epigenetic modification that is important in regulating gene expression during embryogenesis. It is proposed that alcohol-associated alterations in fetal DNA methylation at the four ICRs may contribute to developmental abnormalities seen in FAS and which persist into adulthood. The aim of the study was to examine the effect of maternal alcohol consumption during pregnancy on DNA methylation profiles at specific ICRs (H19 ICR, IG-DMR, KvDMR1 and PEG3 DMR) between FAS offspring and unaffected controls. The participants were FAS children and controls from the Western and Northern Cape Province. DNA samples extracted from blood and buccal tissues were bisulphite modified and the ICRs were amplified by PCR. The pyrosequencing method was used to derive a quantitative estimate of methylation at selected CpG dinucleotides. Analyses were done for H19 ICR (6 CpG sites; 50 controls and 73 cases); KvDMR1 (7 CpG sites; 55 controls and 86 cases); IG-DMR (10 CpG sites; 56 controls and 84 cases) and PEG3 DMR (7 CpG sites; 50 controls and 79 cases). Age and gender are reported confounders in DNA methylation studies and their effects were investigated in the present study. In this study age was shown to influence methylation at three of the four loci investigated, IG-DMR, KvDMR1 and PEG3 DMR. The effect of gender on methylation was shown to be significant at only one locus, PEG3 DMR. After adjusting for gender and age, there was a significant difference in methylation (CpG specific and locus averaged) at KvDMR1 and PEG3 DMR but not at the H19 ICR, with only a small effect on average methylation (0.84% lower in cases; p=0.035) at IG-DMR. The two maternally imprinted loci, KvDMR1 and PEG3 DMR, showed significantly lower locus averaged methylation in the FAS cases (1.49%; p<0.001 and 7.09%; p=0.001, respectively). Hypomethylation at the KvDMR1 was unexpected since reduced methylation at KvDMR1 has been associated with Beckwith Wiedemann Syndrome, an overgrowth syndrome. The largest effect was observed at the PEG3 DMR, which regulates the paternal PEG3 gene expression in the brain, but we are yet to understand its impact on the FAS phenotype. This study provides supportive evidence for the role of epigenetic modulation as a mechanism for the teratogenic effect of alcohol by altering the methylation profiles of ICRs of imprinted loci in a locus-specific manner.MT201

Assessing the conditions and ingridients of political clientelism within the South African context

The study examines the conditions, associated with political clientelism, as well as the ingredients of clientelism within the South African context. The study brings the understanding about the relationship between politicians who go out on a periodic basis to canvass for votes and then not much happens in the lives of ordinary people. Despite this, a large number of the same politicians is re-elected to another five-year term. The broad argument in this study is that there seems to be an observable patronage type of relationship between elected parties (politicians) and citizens, and that no matter what service delivery challenges citizens confront (coupled with violent protest in some cases), voting patterns do not change much come election time. The study adopted a conceptual approach, relying on secondary data. Clientelism refers to the relationship that exists between citizens/voters (clients) and politicians (patrons) who make electoral promises in order to gain office. The first section describes what clientelism entails, followed by a discussion of clientelism’s relevance to the present study. Then, anecdotal evidence from the research site will be examined to demonstrate some elements of clientelism, as well as its significance and utility in examining service delivery challenges in local government. Following that, essential components of democratic states will be explored because they have an impact on available research evidence, showing clientelistic elements are prevalent in democracies around the world. The chapter's closing will shed some light on how leadership will be approached in this study, which stems from the fact that leadership is a key concept in this research and is implied in clientelis

An analysis and appraisal of restructuring in SADCC/SADC since 1990

This dissertation analyses and appraises factors that lead SADCC/SADC to restructure in 1992 and in 1999 respectively. Regime theory is used as an analytical tool of these factors throughout this study. The restructuring of regional organisation in the world is often associated with a decision that is taken by regional leaders, only to hide failures of these organisations to deliver. Studies of this phenomenon, however, frequently fail to research the underlying causes. In the case of SADCC/SADC, apart from the fact that the organisation failed to achieve its intended objectives, such as regional integration, economic independence, regional security, and more, this study argues that there were a lot of elements that influenced the pace and the operations of SADCC/C in achieving regional integration and other objectives. The basic debate in this study thus revolves around the fact that the restructuring exercise in SADCC/SADC was a result of many factors and this argument is supported by the regime theory.Political ScienceM.A. (Political Science

Representation of male infertility in newspaper reports

Abstract: Infertility is a global issue. It is defined by the World Health Organization as the inability for sexually active couple who are not on contraceptives to achieve pregnancy over a period of a year. Infertility affects both men and women and has been found to result in feelings of anger, shock, denial, frustration, loss of control, isolation, guilt, lowered self-worth, and poor life quality. Although infertility affects men and women, it has throughout history been viewed as a woman’s problem. The media is frequently identified as being the central platform in the production and reproduction of rules of masculinity. The media shape the social representations in public spaces. Newspapers provide insight into community discussions and debates. Male infertility forms part of these discussions and debates. Traditionally for men, the ability to have children is associated with masculinity more than the role of being a father. This study used thematic analysis that was informed by procedures from grounded theory to explore the representations of male infertility in South African news reports. In the 43 news reports that were analysed, three global themes were identified. The first global theme was Risks and it was divided into two subthemes, environment and lifestyle. The second global theme was Stigma. The third global theme was Remedies and it was divided into two subthemes, supplements and biotechnology. The themes identified are important in adding to the literature on the psychosocial aspects of male infertility.M.A. (Psychology

The familial reconfiguration of the subject of cultural discourse in Tsitsi Dangarembga's Nervous conditions.

Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.Abstract available in PDF.Quality of scanned pdf has been compromised owing to poor condition of original document

Systematics, diversity and forage value of indigenous legumes of South Africa, Lesotho and Swaziland

The diversity of legumes, indigenous to South Africa, Lesotho and Swaziland is reported using recorded descriptive and distribution data. A total of 24 tribes, 118 genera and 1662 species were documented with the majority of genera belonging to subfamily Faboideae, tribe Phaseoleae, and the majority of species to tribe Crotalarieae. In terms of distribution patterns, most species were present in the Savanna Biome and Central Bushveld Bioregion. Even though most tribes contain species with secondary metabolites (mainly non-protein amino acids), tribes containing poisonous species are far fewer. Herbs are the key growth form, followed by shrubs, dwarf shrubs and trees. The majority of species are perennials. A map representing the collection intensity for the study area showed that the majority of legumes species were collected in the Fynbos, Savanna and Grassland Biome. It is concluded that indigenous South African legumes are extremely diverse and this denotes the importance of further investigating their forage potential or use in other agricultural practices.Key words: Biomes, bioregions, Fabaceae, fodder, genebank, Leguminosae, pastures, tribe

Salim “Slim” Abdool Karim: Attacking AIDS in South Africa

Slim Abdool Karim joined the anti-apartheid movement as a boy in South Africa and has had the pleasure of watching the regime fall. Now he's set his sights on taking down AIDS

The role of the Standard Bank Joy of Jazz Festival in the career development of six local musicians, especially jazz musicians

M.A., Faculty of Humanities, University of the Witwatersrand, 2011Most previous academic studies undertaken on music festivals in South Africa have been largely done through economic lenses. They have dealt with growing the festival; financial benefits for the festival organisers; management styles and models of festivals. This research interrogates the impact of music festivals on the participating artists’ social, creative, intellectual and sustainable growth. The research studied these aspects through the analysis of the Standard Bank Joy of Jazz festival, which in 2010 was celebrating its diamond jubilee, and following six musicians who have participated in the festivals at different levels. Being a jazz artist myself, I became interested in what constitutes the development of a musician who participates in a jazz festival. This research seeks to depart from the socio-economic based studies by analysing the role of the Standard Bank Joy of Jazz festival in the lives of six South African jazz musicians and the perceived benefits. It also draws on a wide range of literature that covers issues ranging from socio-political scenarios to arts and culture policies from around the 1920s to the present day. What the research found is that music festivals had varying impact on developmental bands in comparison to established bands and musicians. It also looked at the effectiveness of arts and culture policies, legislations and their implementation in the South African music industry. Due to the width and breath of this research topic it must be stated that there are other areas that the research did not delve into due to the researcher’s limited tools - such as the psychological analysis capacity