Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both cytotoxic assessment assays showed an earlier cytotoxic effect in case of shorter NWs, with longer ones having a more marked toxicity, albeit with a delay in time. These findings demonstrate that different levels of biocompatibility can be obtained with specific doses and properties of Ni NWs and can serve as guideline for future experiments.

Many drugs used in the treatment of various cancers are derived from or influenced by compounds from nature. The soil bacterium Rhodococcus is of interest because of its identified secondary metabolic pathways and the production of novel natural antibiotics from several strains. In this study, a solid agar extraction method was used to collect compounds from strains of Rhodococcus. These bacterial compound extracts were then tested using a MTT assay in order to evaluate their effectiveness in augmenting MCF7 breast cancer cell death. The results of two way ANOVA analyses revealed 18 compound extracts from 15 strains of Rhodococcus that showed significant p-values when assayed with MCF7 breast cancer cells but nonsignificant interaction p-values when assayed with the healthy cell control. These results prompt further identification of specific compounds present in the bacterial extract that caused cell death as well as a mechanism of interaction with the breast cancer cells.

Water is considered the most vital resource on earth and its quality is deteriorating. Not all residents living in South Africa‘s rural areas have access to treated drinking water, and use water from rivers, dams, and wells. The quality of these resources is unknown, as well as the effects of the bacteria in the water on human health. The heterotrophic plate count (HPC) method is a globally used test to evaluate microbial water quality. According to South African water quality guidelines, water of good quality may not contain more than a 1 000 coliforming units (CFU)/mℓ. There is mounting evidence that HPC bacteria may be hazardous to humans with compromised, underdeveloped, and weakened immune systems. In this study the pathogenic potential of HPC bacteria was investigated. Samples were collected from boreholes in the North West Province and HPCs were enumerated with a culture-based method. Standard physico-chemical parameters were measured for the water. Different HPC bacteria were isolated and purified and tested for α- or β-haemolysis, as well as the production of extracellular enzymes such as DNase, proteinase, lecithinase, chondroitinase, hyaluronidase and lipase, as these are pathogenic characteristics. The isolates were identified with 16S rRNA gene sequencing. The model for the human intestine, Hutu-80 cells, were exposed to the potentially pathogenic HPC isolates to determine their effects on the viability of the human cells. The isolates were also exposed to different dilutions of simulated gastric fluid (SGF) to evaluate its effect on the viability of bacteria. Antibiotic resistant potential of each isolate was determined by the Kirby-Bauer disk diffusion method. Three borehole samples did not comply with the physico-chemical guidelines. Half of the samples exceeded the microbial water quality guideline and the greatest CFU was 292 350 CFU/mℓ. 27% of the isolate HPC bacteria were α- or β- haemolytic. Subsequent analysis revealed the production of: DNase in 72%, proteinase in 40%, lipase and lecithinase in 29%, hyaluronidase in 25% and least produced was chondroitinase in 25%. The HPC isolates identified included: Alcaligenes faecalis, Aeromonas hydrophila and A. taiwanesis, Bacillus sp., Bacillus thuringiensis, Bacillus subtilis, Bacillus pumilus, Brevibacillus sp., Bacillus cereus and Pseudomonas sp. All the isolates, except Alcaligenes faecalis, were toxic to the human intestinal cells to varying degrees. Seven isolates survived exposure to the most diluted SGF and of these, four isolates also survived the intermediate dilution but, only one survived the highest SGF concentration. Some isolates were resistant to selected antibiotics, but none to neomycin and vancomycin. Amoxillin and oxytetracycline were the least effective of the antibiotics tested. A pathogen score was calculated for each isolate based on the results of this study. Bacillus cereus had the highest pathogen index with declining pathogenicity as follows: Alcaligenes faecalis > B. thuringiensis > Bacillus pumilus > Pseudomonas sp. > Brevibacillus > Aeromonas taiwanesis > Aeromonas hydrophila > Bacillus subtilis > Bacillus sp. The results of this study prove that standard water quality tests such as the physico-chemical and the HPC methods are insufficient to provide protection against the effects of certain pathogenic HPC bacteria. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Water is considered the most vital resource on earth and its quality is deteriorating. Not all residents living in South Africa‘s rural areas have access to treated drinking water, and use water from rivers, dams, and wells. The quality of these resources is unknown, as well as the effects of the bacteria in the water on human health. The heterotrophic plate count (HPC) method is a globally used test to evaluate microbial water quality. According to South African water quality guidelines, water of good quality may not contain more than a 1 000 coliforming units (CFU)/mℓ. There is mounting evidence that HPC bacteria may be hazardous to humans with compromised, underdeveloped, and weakened immune systems. In this study the pathogenic potential of HPC bacteria was investigated. Samples were collected from boreholes in the North West Province and HPCs were enumerated with a culture-based method. Standard physico-chemical parameters were measured for the water. Different HPC bacteria were isolated and purified and tested for α- or β-haemolysis, as well as the production of extracellular enzymes such as DNase, proteinase, lecithinase, chondroitinase, hyaluronidase and lipase, as these are pathogenic characteristics. The isolates were identified with 16S rRNA gene sequencing. The model for the human intestine, Hutu-80 cells, were exposed to the potentially pathogenic HPC isolates to determine their effects on the viability of the human cells. The isolates were also exposed to different dilutions of simulated gastric fluid (SGF) to evaluate its effect on the viability of bacteria. Antibiotic resistant potential of each isolate was determined by the Kirby-Bauer disk diffusion method. Three borehole samples did not comply with the physico-chemical guidelines. Half of the samples exceeded the microbial water quality guideline and the greatest CFU was 292 350 CFU/mℓ. 27% of the isolate HPC bacteria were α- or β- haemolytic. Subsequent analysis revealed the production of: DNase in 72%, proteinase in 40%, lipase and lecithinase in 29%, hyaluronidase in 25% and least produced was chondroitinase in 25%. The HPC isolates identified included: Alcaligenes faecalis, Aeromonas hydrophila and A. taiwanesis, Bacillus sp., Bacillus thuringiensis, Bacillus subtilis, Bacillus pumilus, Brevibacillus sp., Bacillus cereus and Pseudomonas sp. All the isolates, except Alcaligenes faecalis, were toxic to the human intestinal cells to varying degrees. Seven isolates survived exposure to the most diluted SGF and of these, four isolates also survived the intermediate dilution but, only one survived the highest SGF concentration. Some isolates were resistant to selected antibiotics, but none to neomycin and vancomycin. Amoxillin and oxytetracycline were the least effective of the antibiotics tested. A pathogen score was calculated for each isolate based on the results of this study. Bacillus cereus had the highest pathogen index with declining pathogenicity as follows: Alcaligenes faecalis > B. thuringiensis > Bacillus pumilus > Pseudomonas sp. > Brevibacillus > Aeromonas taiwanesis > Aeromonas hydrophila > Bacillus subtilis > Bacillus sp. The results of this study prove that standard water quality tests such as the physico-chemical and the HPC methods are insufficient to provide protection against the effects of certain pathogenic HPC bacteria. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

β-sitosterol/poly(ethylene glycol)-block-poly(lactic acid) (PLA-b-PEG) complexes were prepared by solution blending in purified water and ethanol. The mixture of water and ethanol is a suitable solvent system for the two components. The complex was studied by using Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DSC). β-sitosterol is a drug that may reduce the swelling of benign prostatic hyperplasia (BPH) and diminishing inflammation. However, it is hydrophobic and difficult to deliver in aqueous solution. Since PLA-b-PEG has amphiphilic properties, the complex described here may enhance delivery of this drug for treatment of BPH. Proton NMR (1HNMR) of the complexes shows that the methylene (CH2) protons of the PEG, the (-O-CH-) of PLA, and (CH3) of PLA are slightly shifted because of its non-covalent interaction with β-sitosterol. The complex formation was supported by 2-D NMR (NOESY) spectroscopy. NOESY spectra show cross peaks, indicating the interaction between the two components. DSC of the complexes shows thermal characteristics that are different from the individual components. In particular, the PEG in the complex shows a lower melting point and decreased crystallinity compared to the pure PEG. The melting point is lowered from 57°C to 55.3 °C for the PEG-b-PLA/β-sitosterol (5%) complex. Under the same condition, the melting point of PLA dropped from 170 °C to 130 °C. Atomic force microscopy shows changes in the surface morphology of the copolymer from crystalline to amorphous when incorporated with the drug. NMR, DSC, AFM, and MTT assay studies suggest the formation of a relatively stable β-sitosterol/poly(lactic acid)-block-poly(ethylene glycol) complex. The cell proliferation assay (MTT assay) suggests significant inhibition of the stimulation of growth of prostate cancer cells upon addition the complex.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in women and fourth in men globally. While expansions in preventative measures have increased the detection of CRC at the early stages of disease, only 40% of CRC patients are diagnosed when the disease is at a local stage. Moreover, many anti-cancer drugs fail to significantly improve the life expectancy of patients due to innate and acquired resistance, underscoring a need for better diagnostic and therapeutic strategies for CRC. Immunoglobulin-containing and proline-rich receptor-1 (IGPR-1) is a novel cell adhesion molecule (CAM) that was recently identified in our laboratory. IGPR-1 is expressed in epithelial and endothelial cells and promotes cell-cell adhesion. Expression of IGPR-1 in endothelial cells regulates angiogenesis; however, its role in epithelial cells, particularly cancer cells with an epithelial origin, remains unknown. The overall goal of this study was to investigate the possible function of IGPR-1 in CRC tumor cell growth and response to chemotherapeutic agents. Specifically, we aimed to test the hypothesis that increased expression of IGPR-1 in CRC tumor cells promotes cell survival and contributes to the resistance of tumor cells to doxorubicin. Human CRC tumor cell lines, HCT116 and HT29, were transduced via a retroviral system to express IGPR-1 or empty retroviral vector pQCXIP. The effect of overexpression of IGPR-1 in HCT116 and HT29 cells was measured by MTT assay in non-adherent 24-well plates. In addition, cells were viewed under a light microscope, and images were taken to assess multicellular aggregation. Results demonstrated that expression of IGPR-1 in HCT116 and HT29 tumor cells promoted CRC tumor cell growth, increased multicellular aggregation, and stimulated resistance to the conventional chemotherapeutic agent doxorubicin in non-adherent cell culture conditions in vitro. Intriguingly, treatment of cells with doxorubicin promoted phosphorylation of IGPR-1 at serine 220 (Ser220), suggesting a critical role for phosphorylation of IGPR-1 in the development of resistance to chemotherapeutics. In addition, non-adherent cell culture conditions promoted activation of the key pro-apoptotic kinase, p38 MAPK in CRC tumor cells. Ectopic expression of IGPR-1 reversed this activation. This data suggests that IGPR-1, by suppressing p38 activity, in part, promotes tumor cell survival and increases the resistance of tumor cells to the killing effects of doxorubicin. Our findings are the first to demonstrate that IGPR-1 promotes CRC tumor cell growth and increases the resistance of CRC tumor cells to the cytotoxic effects of chemotherapeutic agents. The data suggests that IGPR-1 plays an important role in CRC by inhibiting the cellular apoptotic response and promoting chemotherapeutic resistance. Finally, IGPR-1 phosphorylation at Ser220 in response to doxorubicin may account for the IGPR-1-mediated development of resistance to doxorubicin in CRC.

This study was focused on antiviral effects of stilbenoids against a virus transmitted by ticks. The cell viability of selected cell line cultures in the presence of various concentrations of stilbenoids was determined using the MTT assay. Similarly, the mixed effect of other known antiviral substances and stilbenoids was studied using the MTT assay. Both the prophylactic effects of stilbenoids on the infected culture cell lines and the effect on viral replication were examined. The viral titres from samples were determined using plaque assay. Some of the experiments were performed also in vivo using laboratory mice.

There is an increasing interest for the use of nanostructures as potential tools in areas that include biology and medicine, for applications spanning from cell separation to treatments of diseases. Magnetic nanoparticles (MNPs) have been the most widely studied and utilized nanostructures in biomedical applications. Despite their popularity, the regular shape of MNPs limits their potential for certain applications. Studies have shown that magnetic nanowires (MNWs), due to their high-­-aspect ratio and specific magnetic properties, might provide improved performance for some biomedical applications. As a consequence, MNWs have received increasing attention from researchers in the last years. However, as with any other nanostructure intended for biomedical applications, rigorous studies must be carried out to determine their potential toxicity and adverse effects before they can be successfully incorporated in clinical applications. This work attempts to elucidate the cytotoxic effects of nickel NWs (Ni NWs) in human fibroblasts by measuring cell viability under different parameters. Ni NWs of three different lengths (0.86 ± 0.02 μm, 1.1 ± 0.1 μm and 6.1 ± 0.6 μm) were fabricated by electrodeposition using porous aluminum oxide (PAO) membranes as templates. Energy dispersive X-­-Ray analysis (EDAX) and X-­-Ray diffraction (XRD) were used for the chemical characterization of the Ni NWs. Their physical characterization was done using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging. MTT assays were performed to assess cell viability of human fibroblasts in the presence of Ni NWs. NW length, NW/cell ratio and exposure time were changed throughout the experiments to elucidate their effects on cell viability. The results showed that NWs length has a strong effect on internalization and cytotoxicity. Smaller NWs showed higher toxicity levels at earlier times while longer NWs had stronger effects on cell viability at later times. NW/cell ratio did not seem to have a very strong effect at low concentrations. However, at high concentration (1000 NW/cell) significant loss of cell viability was observed, with the effects becoming stronger at later times. Other factors such as cell surface area, presence of oxide layer on NWs, and the cytotoxicity of Ni salts, were also studied and found to affect cell viability. For our knowledge, this is the first systematic study done in human fibroblasts wi-­-38 using ferromagnetic NWs; where the toxic effects of equivalent amounts of Ni in its salt and in its NW form are compared. It is also the first study to provide insights of the interaction between wi-­-38 cells and Ni NWs. The results of this study complement and enrich previous cytotoxicity studies of Ni NWs. This work aims at providing a more comprehensive understanding of the interaction between NWs and biological systems. Despite the advancements, further studies will be required to fully understand the factors affecting NW cytotoxicity. Only when we understand the underlying mechanisms, will we be able to design suitable nanostructures for biomedical applications.

Pyrrolobenzodiazepines (PBDs) are a group of naturally occurring compounds that were discovered in the cultures of Streptomyces in the 1960s. Some natural PBDs discovered in these cultures, such as anthramycin and sibiromycin, were shown to possess a broad spectrum of anti-tumor activity. Since cancer is still a leading cause of death globally, the development of novel anti-proliferative derivatives of PBDs is essential for human welfare worldwide. Further synthesis and structure-activity relationship (SAR) studies of the parent natural products and their tetracyclic analogs will lead to the discovery of drug candidates. In this work, thirteen PBD analogues were synthesized using no more than three to four synthetic steps, beginning with commercially obtainable L-proline and isatoic anhydride. The MTT assay, which is a colorimetric assay that uses 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) to assess cell metabolic activity, was initially implimented to test the in vitro cytotoxicity of the compounds using multiple cell lines, namely: SKBR-3, MCF-7, SKMEL-2, CaCo 2, HCT 116, and Mia Paca. Nearly all of the compounds decreased the cell viability of MCF-7 by roughly 20%. Additionally, the anti-proliferative activity of the PBD products were further evaluated by the NCI-60 Human Tumor Cell Lines Screen, which is a part of the National Cancer Institute’s Development Therapeutics Program - Drug Synthesis and Chemistry Branch.

The natural product 2 crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxy-cyclohex-2-enone (COTC) was isolated from the microorganism Streptomyces griseosporeus in 1975. It was shown to exhibit 'cytotoxic and cancerostatic activity'. The simplified synthetic analogue 2-crotonyl-oxymethyl-cyclohex-2-enone (COMC) has been shown to exhibit potent anti tumour activity against murine and human tumours in cell culture. For several years, the Whitehead research group at the University of Manchester have focused on the synthesis of COTC and COMC analogues in an attempt to produce compounds with enhanced cytotoxicity. In this thesis, the syntheses of several polyfunctionalised cyclohexenones are described. These compounds are analogues of COTC and COMC which also bear structural resemblance to the antheminone and carvotacetone natural products. Initially, the syntheses of six novel compounds from the chiral pool starting material (-)-quinic acid are described. The first four synthetic steps of each sequence were carried out by slight modification of procedures previously reported by the Whitehead research group. As part of the synthetic strategy, the diastereoselective conjugate addition of carbon nucleophiles to several polyfunctionalised cyclohexenones was investigated. The cytotoxicity of four of the synthetic analogues towards A549 non small cell lung cancer cells was investigated by use of an MTT assay. Two of the analogues were found to be more cytotoxic then COMC. The most effective synthetic analogue had an IC50 value of 2.2 μM. This analogue was more cytotoxic than similar molecules that had previously been synthesised by members of the Whitehead research group. Based on the results of the MTT assay, another two analogues were designed and their synthesis from (-)-quinic acid is described. The cytotoxicity of these analogues has yet to be assessed. In summary, the general synthetic strategies developed in this thesis will provide easy access to new analogues of the natural products, enabling the development of new cytotoxic compounds.