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Thesis (Ph. D.), Faculty of Health Sciences, University of the Witwatersrand, 2009 / With the high prevalence and high mortality rate of cancer in the global community, it is increasingly essential to accelerate our understanding of the disease, to identify new genetic targets for therapy, and to pursue avenues for improving on the therapies in development and in current use. The aim of this study is to identify cell cycle-related genes whose expression is influenced by the chemotherapeutic drugs curcumin, SAHA, lycopene and thalidomide in breast and colon cancer and normal cell lines. These drugs are currently not in clinical use for cancer in South Africa, and while there have been investigative studies of these chemotherapeutic agents, this study aims to identify the specific genes that are influenced by the drugs. The result of this is that several genes that were not previously documented as targets of these drugs are highlighted. The cell cycle pathway is the area of focus as loss of regulation in the cell cycle is one of the important factors involved in promoting cancer initiation and progression. In the first instance, flow cytometry was used to identify optimal drug concentrations relative to the cell cycle stages. Following this, alterations in gene expression were assessed using a PCR-based differential display after each drug treatment. Subsequently, a more focussed approach was taken in a PCR-array analysis of panels of cell cyclerelated genes. A subset of genes is identified that is implicated in oncogenic transformation in breast cancer. This has the potential to inhibit the genetic pathways involved in breast malignancy by providing targets that perhaps may not be manipulated in current therapies. The gene expression studies here suggest that lycopene and thalidomide function in inhibiting this transformation, and play significant roles in suppressing the oncogenic state of breast cancer. Curcumin and SAHA also exhibit important functions in inhibiting tumourigenesis in colon cancer. While the results propose that the drugs have clear roles in inhibiting breast and colon cancer, they are also implicated in promoting cancer. This research has defined the genes that must be carefully monitored during drug administering as they may promote these and other cancers. The availability of these results to researchers will aid in selecting the criteria for assessing the success rate of these drugs.

Doctor of Philosophy / Department of Chemistry / Sundeep Rayat / Sundeep Rayat / This dissertation focused on the synthesis of 1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione derivatives 1 and evaluating their potential as a new class of photoactivated DNA cleaving prodrugs. We hypothesized that light activation of 1 would cause the decomposition of the tetrazolethione ring system to enyne-carbodiimide 2 with simultaneous loss of dinitrogen and sulfur via 1,3-triplet biradicals 1′, which would be spontaneously followed by Schmittel cyclization to indoloquinolines 3 via benzofulvene type biradicals 2′. The biradicals 1′ and 2′ would have the potential to cause DNA cleavage by abstracting hydrogens from its sugar phosphate backbone, analogous to the mechanism of action of naturally occurring enediyne antitumor antibiotics. Note that our proposed prodrugs contained a 1,4-diaryl tetrazolethione functionality and a direct synthetic route for their construction was lacking in the literature despite their wide spread applications. Therefore, our initial efforts were directed towards developing a general strategy to obtain these ring systems. We employed a highly versatile and efficient copper mediated N-arylation to first obtain a series of 1,4-diaryl tetrazol-5-ones which were thionated with Lawesson’s reagent to afford the corresponding 1,4-diaryl tetrazole-5-thiones in moderate yields. Specifically, the synthesis of 1 involved Sonogashira coupling of the obtained 1-(2-bromophenyl)-4-phenyl-1H-tetrazole-5(4H)-thione with the appropriate ethynyl compounds (Chapter 2). Since the tetrazole ring is an important structural component in many biologically and medicinally relevant compounds, we were interested in evaluating the anticancer activity of these compounds in the absence of photochemical activation. The moderate IC50 values against leukemia and breast cancer cell lines showed that the anticancer activity of these compounds prior to photoirradiation was minimal (Chapter 3). Independent studies have shown that the photodecomposition of tetrazolethiones gives carbodiimides via biradicals, and photocyclization of enyne-carbodiimide forms indoloquinoline also via biradicals. However, it was not known whether these two photoreactions could happen sequentially in one pot with one light source from a substrate like 1, generating biradicals 1′ and 2′ which could later be employed for DNA photocleavage as hypothesized. Therefere, we photolysed 1 in acetonitrile, and our results show clean formation of a mixture of enyne-carbodiimides and indoloquinolines via biradicals 1′ and 2′ (Chapter 4). Finally, we investigated DNA photocleavage by 1 at 350 nm and our results showed significant DNA cleavage in concentrations as low as 100 μM (Chapter 5).

Aim: To determine and compare the prevalence of CCR5-Δ32 and CCRV64I genes in HIV positive and HIV negative population of pregnant women from Harare, in Zimbabwe.Results: The proportion of pregnant women with the homozygous CCR2V64I gene was 24.38% and this gene was two times more associated with HIV infection than in those without it ( RR= 2.32, 95% CI-1.38-3.92). No CCR5-Δ32 deletion was detected in the studied population. Conclusion: The homozygous CCR2V64I gene and STIs were more prevalent in HIV infected pregnant women than in uninfected pregnant women and no homozygous CCR5-Δ32 gene was detected in this study.

Aim: To determine and compare the prevalence of CCR5-Δ32 and CCRV64I genes in HIV positive and HIV negative population of pregnant women from Harare, in Zimbabwe.Results: The proportion of pregnant women with the homozygous CCR2V64I gene was 24.38% and this gene was two times more associated with HIV infection than in those without it ( RR= 2.32, 95% CI-1.38-3.92). No CCR5-Δ32 deletion was detected in the studied population. Conclusion: The homozygous CCR2V64I gene and STIs were more prevalent in HIV infected pregnant women than in uninfected pregnant women and no homozygous CCR5-Δ32 gene was detected in this study.

OBJECTIVE: Low level light therapy (LLLT) is being widely used in wound healing and pain relief, and its use is expected to be expanded rapidly to treatment of other disorders as well in a foreseeable future. However, before its expansion, the fear that LLLT could initiate or promote metastasis must be addressed thoroughly. As an initial effort towards this end, the current study evaluates the safety of LLLT in vitro using two different human cancer cell lines (Michigan Cancer Foundation-7 (MCF-7) and Jurkat E6-1) by determining the viability of cells after low level light (LLL) application while treatment under anti-cancer chemotherapeutic drugs (5-fluorouracil (5-FU) and cisplatin) separately on each cell line. METHODS: Two human cancer cell lines (MCF-7 and Jurkat E6-1) were cultured throughout the experiments. Two different anti-cancer chemotherapeutic drugs (5-FU and cisplatin) were used to treat both cell lines. The half maximal inhibitory concentration (IC50) of each drug on each cell line was determined by treating each cell line with varying concentrations of each drug. A total of 3 or 4 trials were done for each cell line with each drug, and the range of concentration was narrowed closer to the IC50 value at each successive trial. Once the IC50 concentrations were determined, each cell line was treated with 808 nm LLL at varying energy densities in a single dose using a light emitting diode (LED) source both in the absence and the presence of each drug at one IC50. A total of 3 or 5 trials were done for each cell line with each drug, and for each trial, six different energy densities ranging from 0 J/cm2 (control) to 10 J/cm2 were applied. The energy densities were varied for each trial with control always being used. After application of LLL, the viability of cells was determined, and three different 1-way ANOVA (Analysis of Variance) analyses were done to compare the viability of cells at each energy density to that of control. RESULTS: The IC50 of 5-FU in MCF-7 and Jurkat E6-1 cells was determined as 70 µM and 20 µM respectively. The IC50 of cisplatin in MCF-7 and Jurkat E6-1 cells was determined as 17 µM and 7 µM respectively. No significant difference (P > 0.05) in the viability of MCF-7 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of 5-FU at IC50 (70 µM). No significant difference (P > 0.05) in the viability of MCF-7 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of cisplatin at IC50 (17 µM). No significant difference (P > 0.05) in the viability of Jurkat E6-1 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of 5-FU at IC50 (20 µM). However, a significant increase (0.01 < P < 0.05) in the viability of cells was observed when treating Jurkat E6-1 cells with 10 J/cm2 of LLL in the presence of cisplatin at IC50 (7 µM) compared to control group (0 J/cm2). Except for the comparison mentioned previously, no significant difference in the viability of Jurkat E6-1 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of cisplatin at IC50 (7 µM). No definite trend in the viability of cells was observed with increasing energy density of LLL for each cell line either in the absence of the presence of each drug at IC50. CONCLUSIONS: The application of LLL at 808 nm with energy densities ranging from 0.1 J/cm2 to 10 J/cm2 under an LED source did not induce cell proliferation or death compared to control (0 J/cm2) for each cell line in the absence or the presence of each drug, and no definite trend was observed with increasing energy density. The study suggests that LLLT at these parameters may be safe to use on cancer patients, but further studies on different cancer cell lines and animal models with different parameters (wavelength, energy density, dosage) of LLL are warranted.

Magister Public Health - MPH / Aim: To determine and compare the prevalence of CCR5-Δ32 and CCRV64I genes in HIV positive and HIV negative population of pregnant women from Harare, in Zimbabwe. Results: The proportion of pregnant women with the homozygous CCR2V64I gene was 24.38% and this gene was two times more associated with HIV infection than in those without it ( RR= 2.32, 95% CI-1.38-3.92). No CCR5-Δ32 deletion was detected in the studied population. Conclusion: The homozygous CCR2V64I gene and STIs were more prevalent in HIV infected pregnant women than in uninfected pregnant women and no homozygousCCR5-Δ32 gene was detected in this study. / South Africa