Cells respond to changes in their environment, or to mitogenic events at the cell surface by generating signals. Intracellular signals induced as a result, pass from the cell surface to the nucleus via a multitude of mediators, and terminate in cellular proliferation, differentiation or even cell death. Protein phosphorylation is an integral part of signal transduction, and protein kinases are important second messengers in signal transduction pathways. Binding of SV40 to its receptor on CV-1 cells induced signals that led to the upregulation of the primary response genes, c-myc and c-jun. The major histocompatibility complex class I proteins are an essential component of the SV40 receptor. Antibodies against the MHC class I proteins also led to the upregulation of c-myc and c-jun. The serine/threonine kinase Raf and the mitogen activated protein kinases (or MAPK), are highly conserved and play an integral role in signal transduction. Along with the GTP-binding protein p21Ras, the activation of Raf and MAPK form a central paradigm of cell signaling in eukaryotic cell. Activation of protein kinase C, another serine/threonine kinase has also been implied in certain systems. A study of the signaling pathways been elicited by extracellular SV40 imply protein kinase C to be involved in the upregulation of c-myc and c-jun, but provide no evidence for the involvement of either Raf, or MAPK. Antibodies against the MHC class I proteins, on the other hand, indicate the activation of protein kinase C, Raf and MAPK. Moreover, experimental data suggests the anti-class I-induced activation of protein kinase C to be dependent on tyrosine kinases that lie upstream of protein kinase C. Analysis of the signaling pathways being evoked by SV40 and antibodies against class I proteins suggests the existence of separate and distinct signaling pathways being induced by SV4O, and anti-MHC class I antibodies in CV-1 cells.

Programmed cell death (PCD) is an essential component of animal development and serves a variety of functions. The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduco sexta provide a useful model system to study the molecular mechanisms that mediate PCD. The ISMs participate in the emergence behavior of the adult moth at the end of metamorphosis and then die during the subsequent 30 hours. In addition, several populations of interneurons and uniquely identified motor neurons also die following adult emergence. The trigger for this death is a decline in the circulating titer of the molting hormone 20-hydroxyecdysone. Previous work has demonstrated that the ability of the ISMs to die is dependent on new gene expression. Using a differential hybridization cloning strategy, a cDNA library generated from condemned ISMs was screened, and four up-regulated clones were isolated. One of these recombinants was found to encode apolipophorin III (apoLp-III), a component of lipophorin. The expression of apoLp-III was dramatically elevated with the death of the ISMs, some interneurons and identified motor neurons. ApoLp-III was not detectably associated with apolipophorin I and II, required components of lipophorin, or with other molecules in the dying cells, suggesting that apoLp-III has activities independent of lipid transport that may play a role in programmed cell death. Another clone, 18-56, encodes a phylogenetically conserved ATPase domain-containing (CAD) family member related to putative proteasomal subunits and transcriptional regulators. While clone 18-56 was expressed in all tissues examined and during every stage of ISM development, there was a dramatic increase in its expression at both mRNA and protein levels when the ISMs became committed to die. Furthermore, the mouse homolog of 18-56, m56, was cloned and its expression pattern was examined in the mouse. No correlation was detected between enhanced m56 expression and apoptosis in mammalian cells, suggesting that the molecular pathway used by the ISM death may be distinct from that of apoptosis. Rat-1 fibroblast cell lines that over or under express m56 were generated, thus providing tools for further functional study of m56 in mammalian cells.

Loss of regulation of cell cycle events mediated by changes in cytosolic Ca$\sp{2+}$ ion activity has been implicated in the progression of normal cells to neoplasia. In this study, the Ca$\sp{2+}$ buffer 5,5$\sp\prime$-difluoro 1,2-bis(2-aminophenoxy) ethane-N,N,N$\sp\prime N\sp\prime$-tetra-acetic acid (5,5$\sp\prime$-dfBAPTA, abbreviated "dfB") has been used to modulate cell division in transformed and primary mouse keratinocytes. Exogenous application, via the tetra(acetoxymethyl) ester ("AM"), of 18-20 $\mu$M dfB/AM to the growth media of transformed cells inhibits cell division and DNA synthesis, without compromising the cells' viability, as shown by $\sp3$H-thymidine incorporation and flow cytometry. Bulk fluorimetry shows that cells treated with dfB/AM are able to buffer Ca$\sp{2+}$ in proportion to the concentration of dfB/AM applied. Primary cultured cells treated with 18-20 $\mu$M dfB/AM die within 3 hours of treatment. Viable dfB/AM treated cultures of transformed cells have a higher proportion of cells in the G$\sb2$ phase of the cell cycle than do controls, as shown by flow cytometry. This result, in combination with that showing reduced $\sp3$H-thymidine incorporation, suggests that 18-20 $\mu$M dfB/AM inhibits a pre- or mid-mitotic step. Light, electron, and confocal microscopies show 18-20 $\mu$M dfB/AM-treated cells to have prominent, thickened nuclear envelopes along with actin cytoskeletons that are distinguishable from controls. Upon return to medium that does not contain dfB/AM, treated transformed cells gradually resume their pre-treatment growth and division patterns.

During the course of HIV pathogenesis, the virus induces multiple defects in immune cells, altering their functional ability to efficiently control HIV itself and other infections. Whilst the widespread implementation of antiretroviral therapy (ART) has led to reduced morbidity and mortality in most HIV-infected individuals having access to treatment, we still do not know whether full restoration of immune function occurs. The aim of this study was to assess the extent to which ART restores both phenotypic and functional T and B cell immunity. HIV-infected women were studied before and 1 year after ART initiation. In Chapter 2, the effect of ART on T cell activation and differentiation profiles was evaluated in HIV-infected individuals (n=28; pre- and post-ART), and compared to HIVuninfected age- and sex-matched controls (n=23). In Chapter 3, the restoration of copathogen specific CD4+ T cells was determined by comparing their cytokine secretion ability and memory differentiation profiles in response to Mycobacterium tuberculosis and cytomegalovirus in HIV-infected (n=15; pre- and post-ART), compared to uninfected (n=9) individuals. Finally, Chapter 4 examined changes in B cell activation and memory profiles in HIV-infected persons (n=19; pre- and post- ART), and compared profiles to HIV-uninfected individuals (n=19). Multiparameter flow cytometry was performed to address the study objectives. T cell activation, as measured by CD38 and HLA-DR expression, was significantly reduced one year after ART for both CD4+ and CD8+ T cells, but normalisation to levels in HIV-uninfected individuals did not occur, despite suppression of viral load. In addition, skewed CD4+ and CD8+ T cell memory profiles were not completely restored. Furthermore, no change in the cytokine production capacity and memory profile of pathogen-specific CD4+ T cells was found before and after ART, but pathogen-specific CD4+ T cells exhibiting a late differentiated profile (CD27- CD45RO+) had a lower ability to replenish (p=0.02; r = -0.5) compared to cells with an early differentiated profile (CD27+CD45RO+; p=0.04; r = 0.45) prior to ART. Similar to T cells, activated B cells (CD40+CD86+) were only partially normalised post-ART, and remained significantly higher than B cells of HIV-uninfected individuals. The frequency of all B cell memory subsets were comparable between HIV-treated and uninfected individuals, with the exception of plasmablasts, whose frequency was still significantly higher than in HIV-uninfected subjects. In summary, these results demonstrate that HIV-infected women on suppressive ART show a substantial but only partial normalisation of T cell and B cell memory subsets, and lower levels of T cell and B cell activation. In addition, restoration of co-pathogen specific memory CD4+ T cells upon treatment was dependent on their memory profile before ART. Understanding the impact of HIV on T and B cell dysfunction and restoration upon ART may provide important insights into the mechanisms of HIV pathogenesis in the era of ART.

Bibliography: leaves 167-195. / The primary aim of the project was, therefore, to analyse carbohydrate metabolism for the identification of and/or the development of prebiotic substrates, and to provide a molecular characterisation for their utilisation. Several carbohydrates were tested for their ability to support the growth of bifidobacteria as a sole carbohydrate source. The four bifidobacterial strains, B. breve, B. bifidum, B. longum and B. lactis were able to utilise a wide variety of substrates.

Bibliography: leaves 122-139. / This thesis describes investigations into the genetic effects of long term UV-B exposure in Namaqualand daisies (Dimorphotheea sinuata) grown for several generations under ambient and enhanced UV-B levels. Enhanced UV-B radiation was found to have a major effect on the biochemical composition of the chloroplast accompanied by impairment of photosynthetic function, involving a down-regulation of photosynthetic genes and an up-regulation of flavonoid biosynthesis.

Bibliography: p. 201-216.

Includes abstract. / Includes bibliographical references. / There is a growing interest in legumes and legume based foods because of the health claims associated with their consumption. The aim of the current study was to explore the nutraceutical potential of bambara groundnuts (Vigna subterranea L. Verdc) and common beans (Phaseolus vulgaris L.) commonly grown in Zambia based on the antioxidant properties and phenolic phytochemical profiles. Two market classes of bambara groundnuts (red and brown) and four of common beans (red, grey mottled, brown and white) were screened in raw dry form. Effects of cooking and sprouting on the antioxidant activities and phenolic phytochemicals of the promising market classes were assessed. The study employed in vitro antioxidant assays (DPPH and FRAP) to screen for antioxidant properties, HPLC-PDA-ESI-MS and Folin Ciocalteu assay to screen for phenolic phytochemical profiles.

No description available.

Fish farming is rapidly becoming the world's fastest growing production sector, achieving an annual growth rate of approximately 8.9% since the early 1970s. However, high stocking densities result in elevated stress levels in farmed fish, leading to increased susceptibility to infection by opportunistic pathogens and parasites. Antibody phage display is a method that allows foreign peptides or proteins to be expressed on the phage surface through translational fusion with phage coat proteins. Consequently, antibodies expressed by a diverse repertoire of genes coding for the single chain variable fragment (scFv) of immunoglobulin M can be isolated and screened for affinity to a specific infectious agent or parasite. In this study, a phage display library displaying scFvs derived from combination pairings of Dusky kob (Argyrosomus japonicas) variable heavy and light chain fragments, sourced from the splenic B cells of healthy Dusky kob, was constructed. The library was subjected to two rounds of biopanning against brome mosaic virus (BMV), a grass virus to which Dusky kob would have no prior exposure that served as a proxy for an emerging fish pathogen. Five clones were identified as having high affinity and specificity to BMV, as determined by phage enzymelinked immunosorbent assay (ELISA) and phage western blot analysis, respectively. To validate the diagnostic and therapeutic potential of antibody fragments isolated from this phage display library, the gene encoding the antibody fragment of the clone displaying the highest affinity to BMV was selected and expressed using a yeast surface display system. ELISA analysis of serum sampled from Dusky kob exposed to BMV by injection demonstrated that the yeast displayed anti-BMV antibody could successfully detect BMV in the blood serum of BMV-infected Dusky kob with similar sensitivity to a commercially available counterpart. Similarly, this study demonstrated the neutralising effect of yeast displayed anti-BMV antibodies which were found to successfully reduce BMV infection in barley. Overall, these findings demonstrate the feasibility of a Dusky kob phage display library as a source of diagnostically and therapeutically important antibodies against emerging fish pathogens or parasites that threaten the fish farming industry of South Africa.