It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Obesity has steadily increased and represents a major public health problem worldwide, reducing quality of life and causing a range of health problems. Obesity has emerged as the fifth leading risk of global deaths. Annually, 2.8 million adults die as a result of being overweight or obese. The increase of obesity remains inexplicable in terms of genetic susceptibility to obesity. The genetic loci identified by genome-wide association studies (GWASs) explains about 2% of the heritability for obesity. Perhaps other factors such as epigenetics may be involved in the increase of obesity and may offer solutions for the management of obesity. Epigenetics is defined as a heritable change in gene expression without altering the genome sequences. It may help in providing a logical explanation between the genome and environment which shapes obesity risk and may help to explain the "missing heritability". Epigenetics may affect two mechanisms, namely: i) DNA methylation,and ii) histone modifications. DNA methylation might give scientists a link to the rise in obesity.The study aimed to investigate the effect of sugars used as sweeteners in sugar-sweetened beverages (SSB) on DNA methylation in a Caco-2 cell line in vitro. Four major objectives were pursued in the study which were to:(1) stimulate the Caco-2 cells with varying concentrations of sugar sweeteners and assess the morphological changes of the cells; (2) evaluate the cytotoxicity of different concentrations of the sugar sweetener on the Caco-2 cell line using the Alamar blue and LDH assay; (3) obtain genomic DNA from the treated Caco-2 cell line and perform bisulfite conversion and rest; and (4) amplify the WT1, MEG3, TNFRSF9, ATP10A, and CD44 obesity-associated genes and ascertain their degree of methylation. Caco-2 cells were stimulated with sugar sweeteners at varying concentrations (low, medium and high) for an incubation period of 62 days,and images of the cells were captured for morphological characterisation. The incubation condition entailed cells plated in a 12 or 96 well plate, incubated in a humidified 5% CO2 incubator at 37 °C and there is nutrient renewal every three days.Alamar blue, a cell proliferation colourimetric assay and lactate dehydrogenase assays (LDH), a homogenous membrane fluorimetric assay were used for the cytotoxicity studies. The results of the characterisation showed that different concentrations of sugar sweeteners affected the morphology of the cells as the incubation period progressed. The cytotoxicity results of both LDH and Alamar blue depicted low concentration of sweeteners that had low-to-moderate toxicity and the medium and high concentration of the sweeteners had a moderate to high toxicity on the Caco-2 cells. DNA from the Caco-2 cells was extracted. Techniques used to study DNA methylation such as bisulfite conversion, PCR amplification and restriction enzymes that have differential sensitivity to 5-methyl-cytosine were performed. The quality of DNA extracted was good. The bisulfite conversion was conducted andno amplification was observed, as a contingency plan Normal PCR was performed to amplify the CpG islands, and there was amplification. In conclusion, the study showed that a low concentration of a sugar sweetener (fructose: glucose) used in beverages had low toxicity to the Caco-2 cell line and prolonged exposure of the low concentration might have an adverse effect on the cells' morphology. At medium concentrations, the sugar sweetener used in beverages had medium toxicity to Caco-2 cells; prolonged exposure may lead to morphological changes. These findings indicated that control of dietary glucose intake is an important strategy in combating the development of obesity and type-2 diabetes. DNA methylation could not be established.