Thesis (Ph.D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves vii, 276-309) Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.) -- University of Maryland, College Park, 2005. / Thesis research directed by: Molecular and Cell Biology. Includes bibliographical references (p. 115-126).

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I. The function, structure and response to environmental factors of a cytoplasmic protrusion found in rat leukemia cells IRC 741 were investigated. A greater rigidity and adhesiveness of the protrusions, as compared to the main cell body, was demonstrated by time-lapse cinematography, and this functional difference was correlated with localized ultrastructural differences in the cytoplasm and on the cell surface, and with higher negative surface-charge density, as shown by cell electrophoresis. The formation or maintenance of the cytoplasmic protrusions depended on adequate nutritional conditions, and was interfered with by diminished intercellular contact, by environmental temperatures below 37°C, by alkaline pH and by calcium-ion depletion. The protrusion appears to represent a type of adhesive organelle not previously described in either cancer cells or normal cells. II. In the course of the above work, a method was developed whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde-fixation. / Science, Faculty of / Zoology, Department of / Graduate

The biosynthesis of purine nucleotides in Ehrlich ascites carcinoma cells was investigated under different conditions. In initial studies the effect of actinomycin D was examined. The nucleotides from the acid soluble fraction of Ehrlich ascites carcinoma cells incubated with actinomycin D and ¹⁴C-formate were adsorbed on charcoal and eluted with a mixture of pyridine and ethanol. The eluted nucleotides were separated by two dimensional paper chromatography using isobutyric acid-ammonia-water in the first direction and aqueaus ammonium acetate-ethanol in the second. .The nucleotides were estimated by ultra violet spectrophotometry and the radioactivity incorporated was determined by liquid scintillation counting. As the results of these studies using small amounts of cells were inconclusive due to variations from experiment to experiment, similar studies were carried out using larger amounts of cell suspensions. The acid soluble nucleotides from these experiments were separated by ion-exchange chromatography on DEAE-cellulose and finally by paper chromatography. It was observed that there was an accumulation of acid soluble nucleotides in Ehrlich ascites carcinoma cells incubated with ¹⁴C-formate and actinomycin D. The specific activities of these nucleotides were not significantly different from those of the control experiments. The incorporation of ¹⁴C-formate into nucleic acids was inhibited by actinomycin D in these cells. From these observations it was concluded that actinomycin D did not inhibit the biosynthesis of purine nucleotides in Ehrlich ascites carcinoma cells in vitro. It is suggested that the effect of actinomycin D on nucleic acid metabolism is therefore, at a stage beyond the synthesis of nucleotides. Further studies on the effect of actinomycin D revealed that the antibiotic inhibited the respiration of Ehrlich ascites carcinoma cells in vitro slightly. The glycolysis in Ehrlich ascites carcinoma cells was unaffected by actinomycin D. In experiments where the effect of glucose on the incorporation of radioactive precursors into nucleotides and nucleic acids of Ehrlich ascites carcinoma cells was examined, contrary to the results of many, a decrease in incorporation was observed. This decrease in incorporation was independent of the presence of Ca⁺⁺ ions in the incubation medium, the buffer and of the radioactive precursors used in these incubations. It was observed that there were two factors controlling the incorporation of radioactive precursors in. vitro into the nucleotides and nucleic acids of Ehrlich ascites carcinoma cells in presence of glucose; the concentration of glucose in the medium and the concentration of cells in suspension. In dilute cell suspensions (packed cell volume less than 5%) glucose at a concentration of 5.5 mM decreased whereas in dense cell suspensions (packed cell volume above 8%) the same concentration of glucose increased the incorporation of labelled precursors into both acid soluble nucleotides and nucleic acids. 2-Deoxyglucose, an analogue of glucose at a similar concentration decreased the incorporation of ¹⁴C-formate in dilute as well as in dense cell suspension. Dinitrophenol, an uncoupler of oxidateive phosphorylation, also decreased the incorporation of ¹⁴C-formate in these cells which was more marked in dilute cell suspensions in presence of glucose. It was concluded from these observations that the main factor controlling the incorporation in vitro of radioactive precursors into nucleotides and nucleic acids of Ehrlich ascites carcinoma cells was the transient depletion and regeneration of ATP in these cells in presence of glucose. Glucose increased the incorporation of ¹⁴C-formate into the serine of the acid soluble fraction of Ehrlich ascites carcinoma cells. A marked increase in the incorporation of ¹⁴C-formate into serine was observed in presence of 2-deoxyglucose. These effects were independent of the concentration of cells in suspension. It was suggested that when the availability of the common precursor, N⁵-N¹º methylenetetrahydrofolic acid was increased particularly due to a decrease in incorporation into nucleic acids, more of the labelled precursor may be incorporated into serine. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / The exposure of organisms to environmental stresses and pathogens results in rapid activation of a range of defensive pathways that act as part of the innate immune system. The most common innate immunity response is the activation of cationic amphiphilic peptides in response to microbial infection. Moreover, cationic amphiphilic peptides possess desirable attributes for the pharmaceutical development of cancer-selective drugs. They selectively and rapidly kill cancer cells without killing normal mammalian cells and have a broad spectrum of mechanisms of action. The aim of this exploratory study was to screen for cationic amphiphilic peptides with anti-proliferative activity that is induced by genotoxicity. GeneFishing® technology, 2-D gel analysis and bioassays were used to identify and analyse molecules induced in response to genotoxic stress in an embryonic cell line originating from the dung beetle Euoniticellus intermedius. Bioassay results revealed that the cell line has constitutive expression of probable cationic amphiphilic proteins that are further induced by camptothecin treatment. GeneFishing® and 2-D gel analysis showed changes in gene expression at both transcriptional and translational levels, respectively. Overall, the study failed to identify the involvement or induction of cationic amphiphilic peptides in response to genotoxic stress. However, gene expression analyses revealed changes in the expression of classes of proteins involved in stress response, oxidative phosphorylation, mitochondrial maintenance, protein translation, cytoskeletal proteins and immunophilins. The results show that the cell line constitutively expresses probable cationic amphiphilic peptides which are further induced by camptothecin.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2013 / Cancer is a hypernym used to describe a group of diseases characterised by the uninhibited growth and spread of abnormal cells in the body. An estimated 7.6 million annual deaths are attributed to the disease while 12.7 million new cases are reported every year. The severity of this disease demonstrates the urgent requirement of novel anti-cancer therapeutic agents. The non-integrin laminin receptor, here designated the 37 kDa/67 kDa laminin receptor (LRP/LR), is a multifunctional protein located on the surface, in the cytoplasm, in the perinuclear compartment and in the nucleus of cells. While this receptor is imperative for normal cellular functioning, it has also been implicated in many diseases – it serves as a cell surface receptor for numerous viruses, infectious prion proteins as well as certain respiratory tract pathogens. Additionally, LRP/LR has been found to have some involvement in zoonotic diseases and those involving neurodegeneration, such as Alzheimer’s disease. Most importantly for this study, LRP/LR has been implicated in cancer progression, where it was found to be overexpressed on the surface of various cancer cell lines, this overexpression correlating to increased metastasis. The aim of this study was to investigate the effect of the siRNA-mediated downregulation of LRP expression on the viability of tumorigenic lung and cervical cancer cells (A549 and HeLa, respectively). The cell surface LRP/LR and total LRP levels were investigated using flow cytometry and western blotting, respectively, in A549 and HeLa cells, the results revealing high percentages of both cell lines expressing LRP/LR on their surface. Additionally, A549 and HeLa cells express similar total levels LRP. The transfectability of these cells was confirmed and siRNA-LAMR1 was shown to significantly downregulate LRP expression (80% and 60% in A549 and HeLa cells, respectively). MTT assays revealed that the significant 13% and 18% reduction in cellular viability in A549 and HeLa cells, respectively, was as a consequence of LRP downregulation. This reduction in cellular viability was found to be a consequence of induced apoptosis (identified by the visualisation of the loss in nuclear integrity, as well as the significantly increased activity of the apoptosis-associated protein caspase- 3) and inhibited cellular proliferation in the aforementioned cells. These findings suggest that siRNA targeting LRP mRNA may act as a potential alternative therapeutic tool for the teatment of cancer.

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