3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item De novo production of Taxol intermediates by Saccharomyces Cerevisiae(2022) Rabie, RenéeTaxol is an invaluable anticancer molecule produced by Yew trees and their endophytic fungi. Harvesting taxol is difficult and often has low yields. For these reasons, a method to produce taxol heterologously in a fast-growing, well-studied, safe microbe is desirable. In this study, artificial genes were designed for the expression of two taxol biosynthesis pathway enzymes, as well as an assisting NADPH-cytochrome P450 reductase. The genes were designed to be compatible with the pCut transformation technique, which allows genomic integration into Saccharomyces cerevisiae strain BY4742. The genes were then divided into seven fragments. Two additional DNA fragments were amplified directly from the yeast genome because their complexity made them difficult and expensive to synthesise. These nine DNA fragments were designed to be assembled into three linear fragments of equal length for transformation of S. cerevisiae. Attempts at assembling these nine fragments into three inserts failed for various reasons, which largely came down to the complexity and integrity of the DNA, as well as the size of the fragments.Item The role of the polymerase chain reaction in the routine haematology laboratory.(1993) Gunther, Karen ElizabethThe Polymerase Chain Reaction (PCR) provides a means of amplifying target sequences of DNA exponentially and it is rapidly becoming an indispensable tool in the research laboratory. Many other molecular genetic techniques used for research are far too laborious and expensive to be used for routine diagnostic purposes but PCR has the potential to be different. This Research Report assesses the possible role of PCR as a routine diagnostic tool in the haematology laboratory. In the context of haematology, PCR can be used to detect both "pathological" and "physiological" target sequences present within the genome. Pathological sequences of interest would include mutations, deletions, insertions or translocations not present within the normal genome but which may arise either as a result of an hereditary abnormality or be acquired somatically. Sensitive detection of such sequences is useful for diagnostic purposes and can also be relevant in determining prognosis, evaluating response to therapy and following up minimal residual disease in the context of haematological malignancies. PCR detectable physiological sequences would include the immunoglobulin and T cell receptor gene rearrangements normally present within the genome of cells of the appropriate lineage. These rearrangements differ for each lymphocyte within a polyclonal population but are identical among members of a clone arising by proliferation of a single precursor cell. They can therefore be of value not only in determining cell lineage but also as markers of clonality. In this study the practical aspects of using PCR were assessed by setting up the technique of amplification of immunoglobulin gene rearrangements. The cost of reagents and disposable equipment, as well as that of major items of equipment required which are not usually available in a routine laboratory was also determined. In addition, peripheral blood and bone marrow samples reaching the Haematology Laboratory of the Johannesburg Hospital were analysed to assess the potential demand for such investigations. Once appropriate reaction conditions for the primers used had been established, PCR was found to be quick, technically simple and relatively inexpensive. Sufficient numbers of appropriate samples for which PCR analysis could potentially be of value were received in the Johannesburg Hospital Haematology Laboratory in the periods assessed, to indicate that diagnostic PCR, if available, would be well utilised. Some problems were encountered, particularly with regard to variability in the extent of amplification obtained. Thus for routine diagnostic purposes, extensive research and development of each set of primers to be employed will be necessary to make the technique more reliable and consistent. Adequate quality control will also be essential if PCR is to be used for diagnostic purposes. However, once these issues have been addressed, PCR should definitely find a place as a routine diagnostic tool in the haematology laboratoryItem Molecular methods to determine the presence of hepatitis B virus ( HBV ) genotypes in the serum of a hbv-infected chacma baboon(2004-03-17) Naicker., Tholsi. Jocelyn.In the course of a previous study (Kedda, et al., 2000), it became evident that the baboons had been inoculated with a mixture of HBV genotypes, namely, genotypes A and non-A. Therefore, a follow up study was undertaken to determine whether either A or non-A HBV genotypes predominated over the other during the time course of 52 weeks post-inoculation of HBV into baboons. HBV DNA extracted from sera obtained from one of the four HBV positive baboons, at two time points during a 52 week post-HBV inoculation period, was amplified using primers specific for the HBV core region that is well conserved in all genotypes.Item The defectiveness of the subgroup F adenoviruses in vitro(1992-08-17) Tiemessen., Caroline. T.A distinguishing feature of the human subgroup F adenoviruses, types 40 and 41, is their inability to replicate in cells that permit efficient growth of other adenoviruses. This study was conducted to determine the basis of the viral defect(s) responsible for poor growth in vitroItem Genetic variation in gp 120 V1?V2 and neutralization sensitivity of HIV-1 subtype C isolates from children with rapid and slow disease progression(2003) Choge, Isaac Ang' Ang' AThis project explore genetic variation and antibody neutralization sensitivity in gp 120 V1/V2 and C2-V5 region amongst HIV-1 subtype C pediatric isolates. Isolates from 25 slow progressing children and 16 rapid progressing infants were used in subtyping and quasispecies analysis performed with a novel V1/V2 heteroduplex mobility assay (HMA)Item Development of the routine laboratory diagnosis of activated protein c resistance and its evaluation in a population of pregnant women(1997-10) Munster, MarionVenous thromboembolic disease is a common health problem. It contributes considerably to morbidity as well as to mortality. Thrombosis usually occurs due to an underlying risk factor which may be environmental or genetic in origin. The recently described activated Protein C (APC) resistance is the commonest cause of familial thrombophilia documented to date. The molecular lesion is a single point mutation in the factor V (FV) gene which abolishes a cleavage site whereby it is normally inactivated by APC. This defect, termed the FV Leiden mutation, is highly prevalent in normal Caucasian populations. Although it would appear to have arisen due to a founder effect, there is a paucity of data concerning non-Caucasian populations.Item Functional analysis of the cydDC encoded ABC-type transporter in mycobacterium smegmatis(2017) Moeketsi, Moseki RaymondElectron transport and respiration in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) occurs through the use of the aa3 type cytochrome c oxidase (CcO) under normoxic conditions or cytochrome bd oxidase (CbdO) under microaerophillia. Using these oxidases, Mtb couples substrate level oxidation to generation of metabolic energy in the form of adenosine triphosphate (ATP) under aerobic conditions. The presence of the CbdO is expected to allow for growth and survival of Mtb in the oxygen restricted environment of the human TB granuloma, thus rendering it an important enzyme for further study. CbdO is comprised of two structural subunits, CydA (subunit I) and CydB (subunit II), which are encoded by the cydAB operon. Both subunits span the cytoplasmic membrane to form part of the mycobacterial electron transport chain. Notably, two other genes that are transcribed separately from the cydAB operon, the cydDC operon, have been proposed to be required for the synthesis of a functional CbdO. Based on amino acid sequence and structural predictions, the cydDC encode heterodimeric members of the ATP Binding Cassette ABC-type transporters. In other organisms, the cydDC functions to transport reducing agents to the periplasm, thus contributing to periplasmic redox homeostasis. In this study we aimed to analyze the function of the cydDC genes in Mycobacterium smegmatis. Through bioinformatics analyses, it was demonstrated that the CydDC subunits retain conserved residues associated with the ABC domain and adopt a three-dimensional fold that is similar to their counterparts in Escherichia coli. However, the published sequence of M. smegmatis suggests that cydC is a pseudogene, which was inconsistent with the demonstrated evidence of a functional CbdO in this organism. In this study, using standard DNA sequencing, it was demonstrated that the CBTBR laboratory strain of M. smegmatis does not harbor a cydC pseudogene but rather has a functional cydC gene. Next, we interrogated the function of the M. smegmatis and Mtb cydDC genes by heterologous expression in an E. coli cydD mutant. Heterologous expression of the Mtb cydDC genes restored CbdO biogenesis in the E. coli mutant. Using various microbiological approaches, we demonstrated that the mycobacterial cydDC was able to revert the stationary phase exit defect, high temperature sensitivity and increased oxidative stress susceptibility defects of the E. coli cydD mutant. Collectively, these data provided strong evidence that the mycobacterial cydDC genes encode a functional transporter that contributes to periplasmic redox homeostasis. Following this, we generated a double deletion mutant of the cydDC operon in M smegmatis. We confirmed the genetic integrity of the ΔcydDC strain by Southern Blot analysis and proved by absorption difference spectroscopy that this strain is defective in the ability to synthesize a functional CbdO, as measured by the lack of a heme d peak in membrane preparations from the mutant. In addition, the ΔcydDC mutant displayed increased sensitivity to oxidative stress and a reduced ability to exit stationary phase, phenotypic defects that were consistent with the lack of CbdO. In summary, this study provides the first evidence that loss of the M. smegmatis cydDC genes affects CbdO biogenesis. These data also confirm that the CydDC ABC-type transporter most likely transports reducing equivalents that allow for maturation of CbdO in the periplasm. Collectively, our observations advance the understanding of the mycobacterial electron transport chain and provide new evidence to assist in the development of CbdO as a TB drug target.Item Phylogeography of Y chromosome haplogroups A & B in Africa(2015-04-22) Naidoo, ThijessenEvolution and historical events over the past 300 000 years have contributed in shaping the gene pool of sub-Saharan African populations. By examining patterns of Y chromosome variation, through the screening of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs), the present study aimed to characterise the phylogeography of ancient African Y chromosome haplogroups found in populations across sub-Saharan Africa, as well as understand the genetic affinities of these populations. In order to screen the large number of the markers required, seven multiplex single base extension assays were developed. These were used to refine the resolution of Y chromosomes commonly found in Africa, but also included a few markers to delineate the common non-African Y chromosome haplogroups, following a hierarchical screening process. In total, 1667 males were screened, and these data were compiled together with comparative published data. The resultant SNP and STR dataset was used in illustrating, more specifically, the phylogeographies of haplogroups A and B. The wide geographic distribution of haplogroup A, together with its position at the root of the phylogeny and high diversity, support an early diversification of the haplogroup into its subclades, which subsequently spread across Africa. The distribution of major haplogroup B subclades, however, are possibly due to post-glacial migrations in the case of haplogroup B-M112, and recent population expansions, leading to the common presence of haplogroup B-M152 across sub-Saharan Africa. The spread of haplogroup E, however, created the biggest impact on African populations; with its expansion likely resulting in the diminished presence of many of the subclades of haplogroups A and B. The Y chromosome compositions of present sub-Saharan African populations are, thus, the result of several diversification events, followed by migration, and mixing of population groups, over the course of modern human existence.Item Effect of alcohol on global and locus specific DNA methylation in spermatozoa: implications for fetal alcohol spectrum disorders (FASD)(2013-04-24) Patel, SanamFetal alcohol spectrum disorders (FASD) is an umbrella term that describes a range of symptoms associated with prenatal alcohol exposure. Fetal Alcohol Syndrome (FAS) is the most severe disorder in the spectrum and is a major health problem in South Africa, with a prevalence rate of 68.0-89.2 per 1000 children of school-going age. The primary cause of FAS is in utero alcohol exposure. However, secondary factors that contribute to the syndrome include various genetic, epigenetic and additional environmental factors. The proposal that paternal preconception alcohol exposure has adverse effects on offspring development is supported by children born with FASD-like characteristics whose mothers did not drink but whose fathers were alcoholics. Mouse models further support these findings. One of the main epigenetic factors that have been shown to be affected by alcohol is DNA methylation. This chemical modification of DNA is associated with developmentally important genes known as imprinted genes. Imprinted genes are expressed in a parent of origin specific manner. Methylation occurs at specific regions in these genes known as differentially methylated regions (DMRs) or imprinting control regions (ICRs). Alcohol’s ability to alter DNA methylation at imprinted genes raises the possibility that epigenetic disruption could contribute to the clinical features seen in FASD. The main aim of this research was to examine global DNA methylation and locus specific H19 ICR DNA methylation in spermatozoa, related to alcohol exposure. This was done using the luminometric methylation assay (LUMA) and bisulfite based quantitative pyrosequencing, respectively. In this study there was no significant correlation between alcohol exposure and global DNA methylation (p = 0.17), nor was there a significant correlation with drinking frequency (p = 0.31). Although not significant, a slight trend towards decreased global DNA methylation in alcohol-exposed spermatozoa was observed. This suggests that either alcohol does not affect global sperm DNA methylation or that the technique used in this study was not sensitive enough to detect minor changes in global DNA methylation percentage. There was also no significant correlation between alcohol exposure and average H19 ICR DNA methylation (p = 0.051), nor was there a significant correlation with drinking frequency (p = 0.47). There was no significant correlation between alcohol exposure and DNA methylation at individual CpG sites except for CpG 3, where there was a significant increase in DNA methylation in the drinking group (p = 0.03). The findings of this study together with the findings of significant selective demethylation at individual CpG sites within the IG-DMR from another study on the same sperm samples, suggest that alcohol may have the ability to affect DNA methylation levels in spermatozoa at certain loci within the sperm genome. However, these loci-specific effects are not reflected in global DNA methylation levels. These findings do not disprove the hypothesis that there is an epigenetic mechanism responsible for the paternal effects seen in FASD. Instead they suggest that the techniques used in this study were not sensitive enough to detect these changes in DNA methylation or alternatively, alcohol may be exerting its effects through other epigenetic mechanisms.Item Elucidation of the aerobic respiratory chains in mycobacteria(2006-10-27T07:51:11Z) Matsoso, LimenakoThe aerobic respiratory chain of mycobacteria consists of at least two branches, a cytochrome c branch terminating in an aa3-type cytochrome c oxidase, and a quinol branch terminating in cytochrome bd oxidase. The structure and function of the former branch, leading from the menaquinone-menaquinol pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase, was characterized in Mycobacterium smegmatis. Allelic exchange mutants of M. smegmatis in the bc1 complex (ΔqcrCAB::hyg) and in subunit II of the aa3-type cytochome c oxidase (ΔctaC::hyg) were constructed and analyzed for growth, and gene expression using lacZ reporter assays and genome expression profiling by DNA microarray. Both mutants were found to be profoundly growth impaired. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less-efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Other adaptations to re-routing of the electron flux through the branch terminating in the bd-type oxidase were revealed by comparative expression profiling of the bc1-deficient mutant and its parental wild type strain using a partialgenome microarray of M. smegmatis that is enriched in essential genes. The majority of the genes up-regulated in the mutant are involved in intermediary metabolism and respiration. Also induced were several genes including, uspL and a homologue of Rv1592c, which were previously shown to be up-regulated by hypoxia in M. smegmatis (uspL) and M. tuberculosis (uspL and Rv1592). The cytochrome bc1-aa3 branch is required for growth of M. smegmatis under aerobic conditions and its disruption results in growth attenuation and up-regulation of cytochrome bd oxidase.