Pollen tube elongation is a polarized cell growth process that directionally transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actin cytoskeleton is known to support this growth process and Rac-like GTPases have been shown recently to be important to regulate actin organization in elongating pollen tubes. Actin depolymerizing factor/cofilins (ADF/cofilins) are actinbinding proteins that increase actin dynamics by enhancing actin depolymerization. They are also responsible to regulate actin organization in Rac-mediated signaling. My thesis research focuses on establishing a signaling pathway from Rac GTPase to the actin cytoskeleton via the regulation of ADF/cofilins in tobacco pollen tubes. I have isolated and characterized cDNAs for tobacco pollen ADF, NtADFs, to study their function in pollen tube growth. First, I showed the activity of Rac-like GTPase is essential for pollen germination. Tobacco pollen germination and early pollen tube growth stimulates the activation of these small GTPases, the phosphorylation of NtADFs and an increase in the ratio of F- to G-actin. Moreover, over-production of a pollen-expressed Rac-like GTPase, NtRac1 from tobacco, induces increased ADF phosphorylation in transformed pollen and diminished the binding of GFP-tagged NtADF1 (GFP-NtADF1) to actin filaments in growing pollen tubes. These observations are consistent with the presence of a signaling pathway in pollen whereby Rac-like GTPase are stimulated by germination to activate a phosphorylation cascade that down regulates the activity of ADFs. This ultimately affects actin dynamics and growth characteristics in pollen tubes. Second, I also showed that NtADF activity is important for actin organization in pollen tube growth. When expressed in a moderate level in pollen tubes, GFP-NtADF1 associated prominently with a sub-apical actin mesh comprised of short dynamic actin filaments and with long dynamic actin cables in the shank. Over-producing NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes. Pollen tube growth was also inhibited by over-expressed NtADF1 in a dosage-dependent manner, suggesting proper regulation of actin turnover by NtADF1 is critical for the pollen tube growth process. In addition, NtADF1 activity is regulated by phosphorylation and pH. By creating mutants on the serine 6 residue on NtADF1, the result showed that the charge characteristics on serine 6 is important for NtADF1 interaction with actin and for its activity on pollen tube growth. By an in vitro depolymerization assay, recombinant NtADF1 depolymerizes actin more efficiently at pH 8 than pH6. This and localization of a NtADF1-rich actin mesh in the sub-apical region of elongating pollen tube, which is known to have a more alkaline cytoplasmic condition relative to the apex, suggest that interaction between NtADF1 and actin in this vicinity maybe critical for the pollen tube growth process. Finally, to examine a signaling pathway from Rac to ADF, I showed that overexpression of NtADF1 suppressed Rac-induced isotropic pollen tube growth. These observations demonstrating biologically that pollen ADFs mediate signaling activated by Rac-like GTPase to the actin cytoskeleton in pollen tube growth.* *This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.

Bibliography: leaves 60-72. / The stress response protein Hsp 12 is induced in S. cerevisiae cells upon exposure to salt stress, heat shock, ethanol, and upon entry to stationary phase (Mtwisha et aI., 1998). In this study, the occurrence of proteins related to Hsp12 was investigated in a number of yeasts (namely, Saccharomyces cerevisiae S288C, Schizosaccharomyces pombe, Debaromyces hansenii, Lipomyces starkeyi Y-2024, Saccharomyces cerevisiae IFO 23X7 (Kaokai), Zygosaccharomyces rouxii and Pichia sorbitophila. This was performed by selective protein extraction followed by SDS-P AGE and western blotting using a S. cerevisiae anti-Hsp 12 antibody. The results showed that almost all the yeasts investigated possessed a protein that had an identical migration to that of Hsp 12 with the exception of S. pombe, which contained a 9 kDa protein. Western blotting using the antiHsp 12 antibody cross-reacted only with the two S. cerevisiae species in addition to the 12 kDa protein from Lipomyces starkeyi of all the species investigated. MALDI-TOF peptide mass analysis after tryptic digestion of the L. starkeyi 12 kDa protein showed that a close sequence similarity existed to that of S. cerevisiae Hsp 12 and none to rest of the 12 kDa proteins isolated from all the other species investigated. In order to determine the sequence of the Hsp 12 protein, the L. starkeyi Hsp 12 gene was amplified using S. cerevisiae Hsp 12 primers. Gene sequencing of both S. cerevisiae and L. starkeyi Hsp 12 genes revealed three nucleotide differences existed between them. L. starkeyi Hsp 12 was found to be present in relatively small amounts during early growth stages but increased during log phase with a slight further increase during stationary phase. Increasing the salt concentration in the growth medium was found to induce Hsp 12. Increased levels of Hsp 12 appeared to confer a degree of protection during desiccation and subsequent rehydration of both L. starkeyi and S. cerevisiae.

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.

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.

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.

Includes abstract. / Includes bibliographical references (leaves 89-94). / Eusocial insects, in particular ants, demonstrate great variability in their sociogenetic structure with regards to colony organization, queen number, queen mating frequency, levels of relatedness and worker reproduction. Within this study I perform an analysis on two groups of ant nests of the species Campo notus klugii, in order to investigate how the genetic structure may inform us of the sociogenetic structure of the species.