Magister Scientiae - MSc (Biochemistry)

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The adenomatous polyposis coli (APC) and p53 gene status in South African oesophageal cancer patients
(University of the Western Cape, 1996) Skelton, Michelle; Hendricks, Denver; Smith, Arlene
Oesophageal cancer is the most conmon cause of cancer-related deaths in black males in South Africa. In an effort to understand the molecular nature of oesophageal carcinogenesis in South Africa, two tumour suppressor genes, Adenomatous Polyposis Coli (APC) and p53, were examined in normal and tumour tissue obtained from 33 oesophageal cancer patients. Several studies have shown that alterations of the APC and p53 genes are associated with the development of cancer. Allelic loss at the APC gene locus was examined using two polymorphic markers within the coding region of the APC gene. Single stranded conformation polymorphism, heteroduplex and DNA sequencing analyses were used to detect mutations in the mutation susceptible regions of the APC and p53 genes. The "Mutation Cluster Region" (MCR) in exon 15 of the APC gene was examined. Only exons 5 and 6 in the "hot spot" region of the p53 gene were examined. An allelic loss of 21% (4 of 19 informative cases) and an informativity of 59Yo (19 of 32) of the APC gene was demonstrated in patients analysed. No somatic mutations were detected in the MCR in exon 15 of the APC gene. Three putative mutations were detected in the p53 gene using SSCP and HD analysis, two of which were confirmed by DNA sequencing. Analysis of one patient revealed a TCA to TGA base substitution at codon 183 in exon 5 of the p53 gene, resulting in a stop codon at that position. An eleven base pair deletion in exon 6 of the p5 3 gene was detected in another patient. This deletion caused a frame shift mutation and culminated in a premature stop codon 13 codons downstream. Overall, a mutation frequency of 8% (2 of 25 patients analysed) was detected for the p53 gene with exons 7 and 8 still pending further study. These results suggest that the APC gene may not be involved in oesophageal cancer in South Africa and further studies are necessary in order to examine the role of thep53 gene in this disease in South Africa.
Isolation and Partial Characterization Serine Protease Inhibitors from Triticum aestivum cv' Witwol
(University of the Western Cape, 2001) Morse, Monique; Bradley, Graeme; Klaasen, Jeremy
Plant diseases and plant pests are major constraints to plant growth and development, resulting in severe crop losses annually. Plant pathogens can be a variety of things, living and non-living. Non-living pathogens exist as physical conditions that plants are exposed to- climatic conditions can cause damage to plants and there are various agricultural practices that can also be harmful to the plant. Living pathogens are called parasitic or infectious diseases and are extremely contagious and can spread from plant to plant very quickly. Insects, nematodes, mites and higher animals can be considered as pathogens, as are slime mold, bacteria, fungi and parasitic higher plants. Viruses and viroids are also considered pathogens (URL ). Fungal diseases, in particular, severely limit the production of major crops, as do insect crop pests. Effective control of pathogens has led to widespread use of chemical fungicides and insecticides, but with potentially deleterious environmental and human health consequences associated with this practice, strategies to utilize natural host plant resistance mechanisms for disease control are being pursued (Yun e/ a1.,1997). The agrochemical industry has been actively looking for less damaging ways to control insect pests, and has introduced a number of more environmentally friendly pesticides. In addition, alternative strategies for pathogen and pest control have been pursued, such as biological control, and the use of plant varieties with inherent resistance
Genetic variation of rana fuscigula in Southern Africa
(University of the Western Cape, 1994) Arieff, Zainunisha; Channing, Alan
Naturalists have long been engaged in describing and explaining diversity in the biological world. The discovery of the molecular basis of inheritance has led to rapid increase in the use of biological macromolecules in these investigations. Scientists now routinely investigate the DNA of a range of organisms. The elationships between taxa and the phylogeny of groups is determined by examining the differences and similarities between them. These differences are then appropriately analyzed. lt is important to understand the natural variation within a group, before the differences between groups can be established. This study aims to determine the molecular differences between individuals at the extreme edges of the distribution of a species. This will serve as a molecular baseline, from which other studies can proceed. The experimental species is the trog Rana fuscigula, which has a range restricted to southern Namibia and South Africa. lt was thus possible to collect material from the edge of the distribution assuming that maximum genetic difference would be found between individuals at the edge of the range
"Genetic variation between two subspecies of reedfrogs in the genus Hyperolius (Anura: Hyperoliidae)"
(University of the Western Cape, 1993) Hess, Anthony Jacobus; Hendricks, Denver
H. m. broadleyi and H. m. verrucosus are not only different in terms of colour pattern, but distinct genetic differences were detected in restriction site maps of their ribosomal DNA. A sequence divergence value of 13,8% was found between the subspecies. This value exceeds the range recorded between separate species of the genus Rana, ie. 2,2 % between Rana pustulosa and Rana tarahumarae, and 10, 1 % between Rana pustulosa and Rana pipiens. The value of 13,8% between the subspecies is also in the same range as that found between was detected between samples from three different localities within the distribution range of H. m. verrucosus. The genetic data associated with the different colour patterns, suggests that H. m. broadleyi and H. m. verrucosus can be regarded as distinct species. However, a similar study should be performed to examine the genetic status of the subspecies forming the gradient along the east coast of southern Africa. Although the effect of concerted evolution (as discussed in Chapter 2) allows for small sample sizes it would be useful to examine a large number of individuals especially from the overlap zones to determine the extent of genetic heterozygosity and to determine if similar genetic differences (found between H. m. broadleyi and H. m. verrucosus) exist between the rest of the subspecies. The current study has shown that genetic evaluation of all the southern African H. marmoratus subspecies could have a positive impact on the taxonomy of this group of frogs which is still unresolved. This study has identified at least two species within the H. marmoratus complex and it is possible that more species exist within the group. Because of morphological homogeneity it will be difficult to use morphological characters, but more than one molecular technique can be utilized to verify results obtained with one technique.
Identification and partial characterisation of a putative novel plant stress response gene from Arabidopsis thaliana.
(University of the Western Cape, 2000) Ludidi, Ndiko Ndomelele; Gehring, C. A.
Exposure of plants to stress results in the expression of plant genes whose products play a role in defence responses against the stress. Stress stimuli to which plants may be exposed are wounding, drought, salinity, excessive light intensity, heat stress, pathogens, biotic and abiotic factors that lead to the accumulation of H2O2, salicylic acid and plant hormones e.g absiscic acid. This study describes the identification and partial characterisation of a putative novel gene from Arabidopsis thaliana, named the DIINN gene. The sequence of the predicted protein encoded by the DWNN gene shows similarity to a gene isolated from Chinese hamster ovary cells that were resistant to chemically induced programmed cell death. Since programmed cell death is one of the processes involved in plant defence responses to stress, it is hypothesised that the DWNN gene may also play a role in plant programmed cell death. The protein product encoded by the DWNN gene, DWNN, shows homology to proteins from diverse species and phyla. Plants transformed to overexpress DWNN show severely stunted growth and abnormal developmental patterns while plants in which DWNN has been knocked out show an accelerated growth rate. Analysis of the expression pattem of the DLI/NN gene using the GUS gene reporter system suggests that the DWNN gene is expressed in secondary lignification during xylogenesis and in wounded plant tissue. Both xylogenesis and wounding are processes known to involve progralnmed cell death and the regulation of protein turnover.
Bioactivity guided fractionation of Sutherlandia frutescens extracts for the induction of apoptosis
(University of the Western Cape, 2003) Chinkwo, C.A; Gehring, C.A
Sutherlandia frutescens popularly known as "cancer bush" or "kankerbos" is indigenous to South Africa and claimed by traditional healers to have wide therapeutic potential, most specifically against cancer. To verify these traditional claims we used apoptosis-based bioassays, organic solvent extraction, TLC (thin layer chromatography) and HPLC (high perforrnance liquid chromatography) to evaluate extracts from Sutherlandia frutescens microphylla from different geographical populations and from selected S. frutescezs subspecies. The data demonstrate that a specific Sutherlandia frutescens extract has the ability to induce apoptosis in cultured cells. This investigation has suggested that the induction of apoptosis by the extract shows some specificity for transformed cultured cells. In addition, biological activity was traced by chemical fractionation of the crude extract to the chloroform and ethyl acetate fractions. Extracts obtained from S. frutescens microphylla from different geographical regions and subspecies were compared, there was variation in apoptotic activity between the extracts. This suggests that the apoptotic activity and hence possible anti-cancer activity of this plant depends on external environmental factors. In summary, the presented findings are supportive of the claims made by traditional healers that S. frutescens has anti-cancer activity. However we have found that apoptotic activity was not present in all the plants, even of the same species, and hence further investigations are required to identify which factors cause certain plants to have greater apoptotic activity than others. Furthermore the extract analyzed in this study must be further characterized to identify compounds with anti-cancer activities.
Aberrations in the Retinoblastoma susceptibility gene in tumours from South Africa oesophageal cancer patients
(University of the Western Cape, 1996) Gamieldien, Junaid; Hendricks, D.T; Smith, A; Parker, M.I
Little is known about the genetic events occurring in oesophageal cancer and very few studies have been undertaken to analyse oesophageal tumours from South African patients in this regard. Inactivation of numerous tumour suppressor genes, including the Rb gene, has been implicated in oesophageal tumourigenesis in different populations. This study had two objectives. The first was to develop a procedure for the simultaneous extraction of DNA and RNA from small (ca. 25mg) oesophageal biopsy samples. The procedure developed here has proven to be rapid, cost effective and consistently produced excellent yields of high-quality DNA and RNA. It has to be determined, however, whether long-term storage affects the integrity of the isolated RNA. The second and primary objective of this study was to determine whether the Rb gene is involved in oesophageal tumourigenesis in South African patients. Loss of Heterozygosity analysis using a VNTR marker in intron 20 and a microsatellite marker in intron 4 of the Rb gene revealed that Rb-allelic loss had occurred n 50% of the thirty-three patients analysed. Furthermore, microsatellite instability was demonstrated at the intron 4 marker in 15% of the patients analysed. Mutation screening of exons 17 and 21 of the Rb gene, frequently mutated in oesophageal tumours from Chinese patients, in twenty samples using the mutation screening techniques of SSCP and heteroduplex analysis, followed by DNA sequencing of putative positives, revealed no positive mutations. However, the high percentage of allelic loss found suggests that the /lb gene is inactivated in the progression of South African oesophageal tumours. Furthermore, the microsatellite instability suggests that defective DNA repair may also play a role in oesophageal tumourigenesis.
Genetic variation between two subspecies of Reedfrogs in the genus Hyperolius (Anura: Hyperoliidae)
(University of the Western Cape, 1993) Hess, Anthony Jacobus; Hendricks, Denver
The frog genus Hyperolius is the largest of 19 genera in the Hyperoliidae (Channing, 1939). The members of the genus display considerable morphological homogeneity with a diversity of dorsal colour patterns. The genus is endemic to Africa and is distributed throughout the wetlands south of the Sahara excluding the western plateau slopes and South African plateau. They are found in close proximity to water, on reeds, sedges and also on water-lily leaves (Iambiris, 1989). The South African forms are commonly referred to as reedfrogs (Passmore and Camrthers, 1979). The members of the genus bury themselves in the ground and under leaves during the dry season and emerge after the first rains (Iambiris, 1989). The eggs are laid in water, attached to waterplants, but certain species deposit eggs above the water level or between water-lily leaves (Lambiris, 1989). Characters that disqinguish the genus from other African genera are as follows: The pupil is horizontal to round; Vomerine teeth are absent; The fingers and toes are webbed; The oral disc is ventrally situated (Passmore and Camrthers, 1979).
The effect of kinetin and salt stress on pnp-a expression in erucastrum strigosum and arabidopsis thaliana
(University of the Western Cape, 2002) Makgoke, Gile Dineo; Gehring, C. A.
In agriculture crop productivity is greatly affected by stresses such as salinity, drought, temperature and honnonal changes of crop plants and responses to these stresses. Studies have shown that a natriuretic peptide based regulatory system responsible for water and ion homeostasis in animals has a hctcrologous equivalent in plants. Plant natriuretic peptide immunoreactants (irPNPs) have been reported to be involved in K+, Na+ and er ions fluxes in plants. Previously, an Arabidopsis thaliana transcript (AtPNP-A) encoding an irPNP (AtPNP-A) has been identified and isolated (Ludidi et al., 2002). The AtPNP-A a novel protein and part of its physiological role is presented here.
Tungsten Telluride Quantum dot-based Biosensor for Alpha-Methylacyl CoA Racemase – An Emerging Prostate Cancer Biomarker
(University of the Western Cape, 2019) Sampson, Zaiyaan Begum; Iwuoha, Emmanuel
Prostate cancer, commonly referred to as adenocarcinoma of the prostate, is the leading cause of cancer death in men in 46 countries, and it was estimated that by the end of 2018 there would approximately be 1.3 million new cases of prostate cancer worldwide. Currently, the Food and Drug Administration (FDA) approved biomarker for prostate cancer disease diagnostics Prostate Specific Antigen (PSA) is not specific to the disease itself but extends to other cases such as Benign Prostate Hyperplasia (BPH) a condition in which the prostate grows uncontrollably. This biomarker is then detected in blood samples via conventional methods which require a qualified individual to operate and are often time consuming. Examples of these methods are spectrophotometry and High Performance Liquid Chromatography (HPLC). Hence, a more efficient biomarker and method of detection is needed for prostate cancer disease diagnostics, as early detection of the disease means early treatment, which could ultimately save lives. Currently, an emerging biomarker for prostate cancer known as Alpha-Methyl CoA Racemase (AMACR) has shown to be more specific to the disease with advantages such as being a non-invasive biomarker. AMACR has been reported to be present in urine, and thus may be detected via a non-invasive method. This study proposed an economical, non-invasive electrochemical biosensor for the rapid detection of AMACR based on mercaptosuccinic acid capped tungsten telluride (MSA-WTe3) quantum dots (QDs). Nanomaterial has shown promise in terms of increasing the sensitivity and specificity of sensors. MSA-WTe3 QDs was successfully synthesized using easy, inexpensive method and was studied by various techniques such as High Resolution Transmission Electron Microscopy (HR-TEM) where the size was confirmed to be within the nanometer scale and was reported to be 2.65 nm with a good crystallinity. X-ray diffraction (XRD) confirmed the structural properties and chemical composition of the QDs and it is reported that the QDs are rich in both tellurium and tungsten and comprise of a hexagonal structure. Scanning Electron Microscopy (SEM) confirmed the successful immobilization of aptamer sequence specific to AMACR onto the electrode surface by showing a distinct conformational change when aptamers were introduced to the QDs under study. This study reports the successful detection of AMACR using an MSA-WTe3 QDs based aptasensor immobilized onto a screen printed glassy carbon electrode, with a detection limit of 0.35651 ng/mL and a limit of quantification calculated to be 1.08033 ng/mL.
Electrochemical Aptasensing of B-Type Natriuretic Peptide-A Biomarker for Myocardial Infarction
(University of the Western Cape, 2019) Oranzie, Marlon; Iwuoha, Emmanuel
infarction (MI) affects many parts of the western world and in South Africa alone it is estimated that MI is responsible for 1 in 6 deaths (17.3%). Traditional diagnostic methods for MI include an electrocardiograms and blood tests. The problem with these diagnostic methods are that they are time consuming, require large sample volumes, expensive equipment and complicated machinery. To achieve early detection of MI the discovery of specific, sensitive and reliable biomarkers are required. Brain natriuretic peptide (BNP) has been identified as a reliable biomarker for MI due to the fact that it has a defined cutoff of 100 pg/ml and it is not susceptible to patient‘s age which could make early detection of BNP complicated. Early detection methods for BNP has been based on immunoradiometric assays but problems associated with immunoradiometric assays are that there is a restricted availability of antigens and incubation of the labeled antibody could take up to two weeks which affects the patients waiting time on results. Electrochemical biosensors are emerging as early detection method for MI because they can be designed to be sensitive, specific to BNP at a low cost. This research study reported for the first the successful fabrication and implementation of highly sensitive mercaptosuccinic acid capped nickel selenide quantum dots (MSA-NiSe2 QDs) aptasensor for the detection of BNP. The poly-dispersed MSA-NiSe2 QDs were synthesized via an inexpensive, simple and reproducible aqueous microwave assisted irradiation method. The prepared MSA-NiSe2 QDs were characterized by Ultraviolet spectroscopy (UV-Vis), X-ray Diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR), High Resolution Transmission/Scanning Electron Microscopy (HR TEM/SEM) and Small Angle X-ray Scattering (SAXSpace). The electrochemical properties of the MSA-NiSe2 QDs were investigated by Cylic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). HR-TEM revealed the formation of small sized MSA-NiSe2 QDs about 4 nm in diameter which was complemented by SAXSpace. UV-Vis studies showed absorption peaks in the ultraviolet region (100-400 nm) confirming the small size of these QDs as well confirming the direct and indirect bandgap of the QDs. XRD confirmed that the QDs are crystalline and belong to the bulk cubic MSA-NiSe2 QDs phase. FTIR studies confirmed the successful capping of MSA on the QDs due to the disappearance of the thiol peak at 2652 cm-1. Electrochemical studies revealed that the MSA-NiSe2 QDs showed good electrochemical properties on screen printed carbon electrodes (SPCE) which allowed them to be used as a mediating platform between the aptamer and SPCE. The successful detection of BNP was achieved by an incubation process between the aptamer drop coated on the MSA-NiSe2 QDs/SPCE surface overnight. The response of the MSA-NiSe2 QDs based aptasensor towards different concentrations of BNP was studied by differential pulse voltammetry (DPV). DPV showed a good linearly with correlation coefficient of R2 = 0.98. DPV also showed a high sensitivity (0.4513 μA/ pg/mL) towards detecting BNP with a detection limit of 11.93 pg/ml. The value of 11.93 pg/ml falls within the negative predictive value range of 10-100 pg/ml for early-stage diagnosis of BNP.
Green synthesis and characterization of silver nanoparticles (AgNPs) from Bulbine frutescens leaf extract and their antimicrobial effects.
(University of the Western Cape, 2020) Lucas, Shakeela; Madiehe, Madimabe
Combating antimicrobial resistant infections caused by nosocomial pathogens poses a major public health problem globally. The widespread use of broad-spectrum antibiotics for the treatment of wound infections has led to the appearance of multidrug-resistant (MDR) microbes which further exacerbates the growth of microbes amongst patients. It may result in prolonged debility of the patient and an increase in healthcare costs due to prolonged hospital stays and expensive treatment regimens to avoid patient-patient transmission. Therefore, it is imperative that alternative sources of treatment to antimicrobial use in wound infections needs to be developed in order to inhibit or kill resistant microbes and to provide point of care medical treatment to the less fortunate at an affordable cost.
Bacillus licheniformis isolated from Mozambican soil capable of producing 2, 3-butanediol
(The University of the Western Cpae, 2018) Hendricks, Layla; Trindade, I.M Prof
Due to the current fossil fuel sources rapidly depleting as a result of the increased global need, alternative, sustainable and renewable sources are required. Biofuels, which are environmentally friendly, meet all the requirements as they can be generated from the biomass of biodegradable waste. Selected species of yeast, bacteria and algae are capable of producing biofuels from a host of substrates. Microorganisms, historically used to manufacture valuable products ranging from the pharmaceutical to food industry, are now employed to generate biofuels. Several bacteria are native producers of biofuels and do so without requiring any manipulation. Some of the most effective biofuel producers are pathogenic organisms, therefore industrialisation is complicated due to the associated health risks. Generally regarded as safe (GRAS) microorganisms are preferred vehicles for the production of biofuels as they do not pose any risks when manufactured at industrial levels. 2, 3-Butanediol (2, 3-BD) was identified as being a favourable biofuel due its heating value being more favourable than methanol and ethanol. This compound exists in three stereoisomeric forms and organisms often produce a mixture thereof depending on the fermentation conditions. In this study, five bacterial samples isolated from a hot spring were screened for the production of acetoin, a 2, 3-BD precursor. As mesophilic industrial processes often result in contamination, the hot spring was an ideal screening environment to by-pass the contamination issue. The isolates were identified as Bacillus licheniformis with two isolates being closely related to Bacillus licheniformis ATCC 14580. The B. licheniformis isolate is a GRAS organism known to produce a mixture of meso and dextro-2, 3-BD at a wide range of temperatures while using several different substrates and carbon sources. Nutrient broth (NB), Luria Bertani (LB), Beef extract (BE) and Zymobilis media (ZM), an in-house media, were compared to determine which yielded the highest growth rate. Based on the literature and the results generated in the comparative analysis, LB was selected to determine the effect of various carbon sources on the growth rate of the isolates. Unsupplemented Luria Bertani was compared to LB supplemented with either sucrose, fructose, starch and glucose. A marked increase in cellular density was detected in the carbon-supplemented media. High performance liquid chromatography was used to determine the compounds produced in the glucose-enriched media. We were able to identify 2, 3-BD at 37°C in cultures of all five isolates. Four of the isolates produced only meso-2, 3-BD, which is significant and of great industrial importance as no downstream applications would be required to separate the two isoforms. Further work can be performed to examine production of 2, 3-BD at elevated temperatures.
Design and synthesis of polycyclic amine derivatives for sigma receptor activity
(2013) Strydom, Natasha; Malan, S.F.; Joubert, J.
New therapeutic strategies are needed for a diverse array of poorly understood neurological impairments. These include neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, and the psychiatric disorders such as depression, anxiety and drug dependence. Popular neuropharmacotherapies have focused on dopamine (DA), serotonin (5HT), γ-aminobutric acid (GABA) and glutamate systems (Jupp & Lawrence, 2010). However recent research points to the sigma receptor (σR) as a possible neuromodulatory system. Due to its multi-receptor action, the σR can trigger several significant biological pathways. This indicates its ideal potential as a drug target to effectively minimise drug dosage and potential side effects. Currently there are a limited number of σR ligands available and few possess the selectivity to significantly show σR’s role in neurological processes. Polycyclic amines have shown notable sigma activity and provide an advantageous scaffold for drug design that can improve pharmacodynamic and pharmacokinetic properties (Banister et al., 2010; Geldenhuys et al., 2005). Aryl-heterocycle amine groups were also shown to improve σR activity (Piergentili et al., 2009).
Seasonal dynamics of edaphic bacterial communities in the hyper-arid namib desert
(2012) Armstrong, Alacia; Tuffin, Marla; Ramond, J.B.; Cowan, D.A.
The Namib Desert is a hyper-arid, coastal desert with limited bioavailable water and nutrients; characteristics which collectively impose constraints on edaphic microbial communities. Several studies in the Namib Desert have investigated changes in soil microbial communities across space. However, the temporal variation of edaphic bacterial community in response to seasonal microenvironmental variation in the Namib Desert gravel plains has never been investigated in situ.The edaphic bacterial community dynamics were evaluated over short (57 days) and long-term (1 year) sampling intervals using an extensive sampling strategy in combination with community fingerprinting by T-RFLP analyses and microenvironmental characterization. The short-term study was conducted on three distinct locations in the Namib Desert gravel plains. Soil bacterial communities were found to be more similar within habitats than between habitats, with the differences likely shaped by soil pH. These findings are consistent with the concept of habitat filtering.Investigation of edaphic bacterial communities over 1 year in an 8100 m2 sampling site revealed seasonal patterns of variation in community structure. Soil moisture,phosphorus, potassium and magnesium were identified as significant abiotic drivers of community temporal dynamics. β diversity was found to increase over time, while the environment remained relatively static. These findings support previous observations that desert communities are likely structured by stochastic and deterministic processes.Taken together, these findings advance understanding of temporal variation of edaphic communities in the Namib Desert.
An in vitro study on the immunotoxicity of sewage effluents discharged into the Eerste River-Kuils river water catchment system
(2008) Magcwebeba, Tandeka; Pool, Edmund J.
"The aim of the study was to use in vitro human whole blood cultures to screen the water samples collected from the Eerste/Plankenbrug river system for cytotoxicity and inflammatory activity and for the first time investigate the impact on the cell- mediated and humoral immune pathways. Water samples were collected fronm the sites during the dry summer season and rainy winter season. Blood was collected from the healthy male volunteers and diluted with RPMI 1640. For cytotoxicity and inflammatory activity 2.5ul of blood for 18-20 hrs at 37 C... This study shows that waster from the Plankenbrug River is heavily polluted by contaminants from both the agricultural area and informal settlement of Kayamandi. These contaminants can be potentially immunotoxic during the summer season and they can result in inflammatory diarrheal disease and immunosuppression in exposed individuals..."
Genomic instability in South African breast cancer patients
(2013) Langa, Bridget Cebisile; Abdul-Rasool, Sahar; Hiss, Donavon
Breast cancer (BC) is one of the most common malignancies in women. Death results from treatment failure and metastatic disease. Thousands of lives might be saved if it was possible to detect and eliminate occult metastatic cells before they become clinically evident. Therefore, there is a critical need to identify new markers to improve treatment options for these patients. Genomic instability is the earliest indication of breast cancer and the use of genomic methodologies is a progress towards early detection and treatment, through the identification of biomarkers that can be translated into novel therapy targets. The interferon regulatory factor-1(IRF-1) gene, localized on chromosome 5q31.1, is believed to act as a tumor suppressor gene in breast cancer. The IRF-1 was found to be inactivated by single nucleotide polymorphism (SNP) in breast cancer suggesting that the loss of its function might be critical to the development of the disease. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions and alterations of genes in this pathway, including PIK3CA, serine-threonine protein kinases (AKT1and AKT2), phosphatase and tensin homolog (PTEN), fibroblast growth factor receptor 2 (FGFR2) and ERBB2, whose expression have been demonstrated to be altered in breast cancer patients. In addition, these genes are linked to treatment resistance. vi In this study, we have investigated allelic loss of IRF-1 gene in primary tumors obtained from patients undergoing mastectomy at Groote Schuur hospital (Cape Town, South Africa). These samples were then further analyzed for the DNA copy number changes of specific genes involved in the PIK3/AKT signaling pathway. Statistical analysis has been performed in order to correlate genomic findings with clinical-histopathological and follow up information from the patients and to establish whether these genes can predict prognosis. Our data analysis has indicated that 46 cases (45.5%) out of 101 cases were informative for the IRF-1 dinucleotide marker used for LOH analysis (Figure 3.1). LOH was detected in 23 of these informative cases (23/46; 50%). No statistical significance was found between LOH at the IRF-1 locus and age (≤50 years or >50 years) (P value = 1.0000) and earlier stage (Stages I and II) (P value= 0.4982) based on Fisher’s exact test. Patients presented a high level of DNA copy number changes in genes involved in the PIK3/AKT pathway. The most frequent changes were observed in the PIK3CA and PTEN genes. PIK3CA presented high copy number in 36.8% of the cases. PTEN was observed with low copy number in 47.5% of the cases. This dissertation shows the effectiveness of genomic methodologies as means for the detection of early breast cancer progression in South African women. The PIK 3/AKT genes can validate the usefulness of breast cancer therapies.
Investigating novel cis-acting regulatory elements involved in the regulation of heat shock response in cardiomyocytes
(2013) Fortuin, Ira; Meyer, Mervin
Ischemic heart disease is a disease which is characterized by the reduced blood supply to the heart. According to WHO 2013, ischemic heart disease is one of the major causes of death globally. For this reason, it is imperative to search for methods whereby heart cells can be protected from cell death. The upregulation of heat shock proteins (Hsps) is one of the major techniques which can be used to protect the heart cells from Hsps cell death and improve the tolerance to ischemic stresses in various models. The increased expression of Hsps during heat shock pre-conditioning is regulated by heat shock transcription factors (HSFs). HSFs orchestrate the initiation of gene expression by binding to sequence motifs, known as cis-acting regulatory elements (CAREs). Since gene expression is regulated at a transcriptional level, it is expected that functionally related genes (e.g. heat shock response genes) might also be regulated by the same transcription factors (TFs). In this study an in silico approach was performed to identify the promoter sequences of 50 known heat shock responsive genes using Genomatix Software. This software was also used to identify transcription factor binding sites that are statistically over represented in the promoter sequences of these genes. The use of the Electrophoretic Mobility Shift Assay was included to confirm that protein cell lysates of stressed cells contain proteins (TFs) that bind to this sequence (SP1F_KLFS_01). Luciferase promoter reporter assay were also used to iii investigate the transcriptional activity of mutant promoter constructs in which the SP1F_KLFS_01 was mutated. SP1F_KLFS_01 is a ±25 base pair sequence that was identified in the promoter sequences of 19 heat shock responsive genes, including the well-known Hsp70 and Hsp90. This sequence is a potential binding site for two TFs, Specificity Protein-1 and Krueppel like TFs. Consequently, the aim of this study is to identify CAREs that are statistically over-represented in the promoter regions of heat shock response genes. In conclusion, in vitro experiments of this study did not support the findings of the in silico experiments, therefore additional methods should be implemented to expand the investigation for the involvement of cis-acting regulatory elements in the regulation of heat shock proteins in cardiomyocytes, prior to heat shock.
Low detection of exon skipping in mouse genes orthologous to human genes on chromosome 22
(University of the Western Cape, 2002) Chern, Tzu-Ming; Hide, Winston; Faculty of Science
Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes. Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves.
The development and application of informatics-based systems for the analysis of the human transcriptome
(University of the Western Cape, 2003) Kelso, Janet; Hide, Winston; Faculty of Science
Despite the fact that the sequence of the human genome is now complete it has become clear that the elucidation of the transcriptome is more complicated than previously expected. There is mounting evidence for unexpected and previously underestimated phenomena such as alternative splicing in the transcriptome. As a result, the identification of novel transcripts arising from the genome continues. Furthermore, as the volume of transcript data grows it is becoming increasingly difficult to integrate expression information which is from different sources, is stored in disparate locations, and is described using differing terminologies. Determining the function of translated transcripts also remains a complex task. Information about the expression profile – the location and timing of transcript expression – provides evidence that can be used in understanding the role of the expressed transcript in the organ or tissue under study, or in developmental pathways or disease phenotype observed. In this dissertation I present novel computational approaches with direct biological applications to two distinct but increasingly important areas of research in gene expression research. The first addresses detection and characterisation of alternatively spliced transcripts. The second is the construction of an hierarchical controlled vocabulary for gene expression data and the annotation of expression libraries with controlled terms from the hierarchies. In the final chapter the biological questions that can be approached, and the discoveries that can be made using these systems are illustrated with a view to demonstrating how the application of informatics can both enable and accelerate biological insight into the human transcriptome.

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