In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1983.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1983.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1989.