Extracts of the African potato have long been believed to have anti-cancer properties. The aim of the current research was to isolate hypoxoside (HYP) from Hypoxis hemerocallidea (African potato) and synthesize the dimethyl (DMH) and decaacetyl (DAH) derivatives and to test their selective cytotoxicity on a model consisting of a normal (MCF10A) and premalignant, invasive breast epithelial cells (MCF10A-NeoT). Hypoxoside was extracted from the H. hemerocallidea corms using ethanol, purified using a C-18 reverse phase column and the compound examined by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry and found to be of high purity. This was also the case for the synthesized compounds. To assess possible selective effects (cytotoxicity) of derivatized and underivatized hypoxoside, effects on the metabolism of premalignant and normal cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects on cell number (total counts) and cell death [trypan blue and propidium iodide (PI) staining for dead cells versus a lack of staining for live cells] were, thereafter, assessed. Imaging of live adherent cells was also carried out using acridine orange (AO) and PI for live and dead cells (respectively). Propidium iodide staining of detached cells was carried out for flow cytometric determination of cell death (PI indicating early apoptotic or late apoptotic/necrotic cells). After treatment of normal (MCF10A) breast epithelial cells and premalignant cHa-rastransfected (MCF10A-NeoT) derivative breast epithelial cells with HYP, DMH and the DAH derivative, the MTS assay and the Duncan‟s multiple range, analysis of variance (ANOVA) post hoc analysis of the MTS results revealed that only the 150 and 300 µM DAH derivative had a statistically significant effect on the metabolic activity of the abnormal cell line relative to the dimethyl sulfoxide (DMSO) and revealed no significant effect on the normal MCF- 10A cell line after treatment with any of the test compounds. Supravital PI staining of adherent cells seemed to indicate a far higher rate of induction of cell death in abnormal cells than evident in the MTS assay and the PI-based flow cytometry or the trypan blue exclusion assays and need re-investigating, though result trends were similar. Total cell counts, show that HYP and its derivatives appear to increase both cancer and normal cell proliferation significantly, except in the case of DAH at 150 and 300 μM in the MCF10A-NeoT, without affecting the MCF-10A cell line. The trypan blue method for detection of cell death, together with total cell counts, the Duncan‟s analysis of MTS results and a 24 hour exposure to test compounds, seems to constitute an optimal system for drug screening and indicates the statistically significant selective toxicity of the DAH compound at 150 and 300 μM in the MCF10A-NeoT, suggesting that the DAH derivative at 150 and 300 µM would have significant, selective therapeutic potential on Ras-related malignancies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Black wattle (Acacia mearnsii) is one of South Africa's leading commercial exotic species comprising nearly seven percent of South African forestry plantations. The planting of black wattle has become increasingly popular, initially for its high quality tannin content and in more recent times, for its wood and wood products. The industry also provides jobs for more than 36 000 people. Despite the commercial value of black wattle, if left unmanaged, it is one of South Africa's top invader species that aggressively colonise and rapidly out-competes indigenous vegetation. Thus, both plant breeders and environmentalists alike are faced with an interesting paradox of balancing the commercial significance of black wattle on the one hand with increasing environmental concern on the other. At the Institute for Commercial Forestry Research (ICFR), black wattle breeding programmes are being designed and implemented in order to reduce invasiveness whilst still maintaining product quality. One way of minimising invasiveness is to decrease fertility through the introduction of semi-sterility; while at the same time leaving product yield and quality unaffected. A method of achieving semi-sterility is by the induction of autopolyploidy that results in unviable gametes. Autopolyploidy, tetraploidy, is induced chemically through doubling of the chromosomes of diploids. These induced tetraploids may then be crossed with diploids to produce triploids. Thus, an effective method to identify polyploids at the seedling stage would greatly facilitate the success of the abovementioned breeding programmes in the black wattle industry. Polyploidy in plants is often associated with physiological and biochemical changes that become apparent as gigantism of organs which include fruits, flowers and leaves. Polyploidy is also associated with an increase in the number of organelles such as the number of stomatal chloroplasts and nucleoli, as well as an increased production of some proteins and pigments such as chlorophyll. These ploidy-related manifestations are often utilised in breeding programmes to increase the size and quality of plant products as well as a tool to discriminate between polyploids and diploids. Two putative diagnostic procedures to differentiate between diploid and tetraploid black wattle were developed in this investigation. The study focused on the discriminating power of stomatal chloroplast numbers and arrangements as well as the chlorophyll content in the two different ploids. A number of associated experiments were initially conducted to establish the optimal conditions for chlorophyll content analyses such as the type of leaf material and storage conditions. Stomatal chloroplast frequencies were determined in diploid and tetraploid black wattle and comprised three lines per ploidy level with five plants per line. A thin epidermal layer from the abaxial surface of a pinnule was stripped, stained, mounted and 15 stomatal guard cells per plant were viewed at 40X magnification. The mean number of chloroplasts per cell in diploids (9.89 ± 0.222) was found to be statistically different (p < 0.001) to that of tetraploids (22.43 ± 0.222) with no overlapping of the mean chloroplast values between the two ploidy levels. The ratio of diploid and tetraploid stomatal chloroplast numbers was roughly 1:2. An analysis of the least significant difference (LSD) was performed and indicated significant differences between plants within lines, between lines of different ploids (LSD =0.6266), as well as between the different ploids (LSD =0.2802). Furthermore, stomatal chloroplasts spatial arrangements were distinctly different in diploids and tetraploids. In diploids, chloroplasts were clustered into two regions, each towards the extreme ends of the kidney shaped stomatal cells. In the tetraploids, no clustering of chloroplasts could be identified, with an even distribution around the convex curvature/perimeter of the cells. There are a number of factors that influence chlorophyll content and degradation, which are either environmental or genetic in nature. Environmental factors that were considered are sample age and sample storage conditions. Genetic factors include genetic composition and, specifically, the number of sets of chromosomes, that is, the ploidy. Chlorophyll content was investigated by chemically extracting chlorophyll from leaf material and obtaining absorbance spectra with a PerkinElmer UV/vis spectrometer for wavelengths from 400 nm to 700 nm. Chlorophyll absorbance spectra were generated in terms of leaves stored prior to chlorophyll extraction, leaves of different ages, trees of different ages and ploidy. The effects of storage of leaves on chlorophyll content were determined in five non-identical two year-old nursery diploid black wattle genotypes. Fifteen leaf samples from each genotype were either oven dried and then stored for one week or one month at room temperature, or frozen for one week or one month at -4 °C, before chlorophyll was extracted and absorbance spectra determined. Chlorophyll absorbance values of chlorophyll extracted from leaf material on the day of collection (day-0) was used as the control. An analysis of variance (ANOVA) revealed that the chlorophyll absorbance values of the different storage treatments were all significantly lower than the chlorophyll absorbance values of the control (p < 0.001). Assessment of the mean chlorophyll absorbance (TĀ), sum of the three peak absorbance values at three wavelengths, namely, 433 nm, 456 nm and 663 nm, revealed significant differences (p < 0.001) from the control (TĀ = 1.275) for all treatments. Dried leaves that were stored for seven days (TĀ = 1.132) resulted in the least amount of chlorophyll degradation followed by 28 day ice storage (TĀ = 1.114), seven day ice storage (TĀ = 1.103) and lastly 28 day dried storage (TĀ = 1.093). An analysis of least significant differences (LSD) revealed that chlorophyll absorbance values within lines and between wavelengths were significantly different (LSD = 0.005). Furthermore, LSD analysis revealed significant differences between all treatments (LSD =0.003) which also supported the ANOVA findings. Chlorophyll absorbance values within dried and frozen treatments were compared with respect to storage time periods of one week and one month. It was noted that whilst all treatments decreased from the control (day-0), dried samples responded differently to storage periods as compared to frozen samples. Chlorophyll absorbance values of dried material decreased steadily over time from control to seven-day storage to one-month storage, whereas, in the case of frozen material, a similar trend could not be identified. A greater decrease from the control to seven day ice storage was recorded than for the decrease from the control to 28 day ice storage. The effects of tree and leaf ages of diploid black wattle on chlorophyll content were determined. Two types of leaf flushes namely, old and new flush, were examined in relation to different tree ages; two, four, six, eight and nine year-old; in order to assess whether the choice of material impacts on chlorophyll absorbance values. Five leaf samples from each tree were collected, bagged and chlorophyll extracted within two hours of collection. These chlorophyll absorbance values were compared to young diploid seedling material as a base-value and as a control value. An analysis of variance (ANOVA), revealed significant differences between tree ages and between leaf ages (p < 0.001). An analysis of least significant differences (LSD) revealed that new flush of all tree age groups were significantly different from the control (LSD = 0.006). This was mostly true for old flush, except that of six year-old old flush which was not significantly different from the control (LSD =0.006). The chlorophyll absorbance values of both old and new flush of different age groups produced spectral graphs for which no specific trends could be ascertained. Therefore, the data from the two flush types were pooled and revealed a marked increase in chlorophyll absorbance as trees became older. Moreover, this increase was more apparent in new flush than in old flush. Interestingly, juvenile characteristics were identified in two year-old black wattle trees, where a marked increase in chlorophyll content was noted. The effects of the number of chromosome sets on chlorophyll content were assessed for diploid and tetraploid black wattle. Seedlings, bagged juveniles as well as two year-old field trees were analysed. Three genetic lines per ploidy level comprising of ten plants per line were used in the analysis. An analysis of variance (ANOVA) revealed significant increases of chlorophyll absorbance values (p < 0.001) for diploid seedlings (TĀ = 1.1086) to bagged trees (TĀ = 1.149) to field trees (TĀ = 1.224). Similar significant increases were recorded for the tetraploid seedlings (TĀ = 1.886) to bagged trees (TĀ = 1.931) to field trees (TĀ = 2.059). There were distinct differences in chlorophyll absorbance between the two levels of ploidy (LSD =0.002). Furthermore, chlorophyll absorbance within lines, between wavelengths were found not to be significant (p = 0.984), which was supported by an analysis of least significant differences (LSD = 0.004). Moreover, the ratio of diploid to tetraploid chlorophyll absorbance was roughly 2:3. Additionally, the increase of chlorophyll content from seedlings to bagged juveniles to field material of both diploid and tetraploid black wattle further supported the findings in the previous age study that there was an increase in chlorophyll content as the tree matures. Stomatal chloroplast frequencies and chlorophyll content have been identified as two methods that are able to effectively, and with ease, discern between diploid and tetraploid black wattle. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently one of the leading causes of human food poisoning in the world. It is believed that contaminated poultry products, especially eggs and egg products, have been responsible for the dramatic increase in the incidence of this Salmonella serotype. Detection of S. entertidis has conventionally involved bacteriological examination of samples, yet these procedures are time-consuming which could lead to the rapid spread of S. enteritidis through commercial flocks and potentially cause a human health risk. A number of alternative detection techniques, mostly based on serological methods, have been reported as effective diagnostic assays. However, some of these reports have not been supported by representations of SDS-PAGE gels or Western blots. The objective of this study was the evaluation of these serological techniques as well as a PCR amplification technique, which has been reported to show promising results as a diagnostic method. The techniques discussed in these reports were evaluated with regards to how rapid they were, their specificity and their potential for use in local diagnostic laboratories. Antigens from the outer surface of S. enteritidis were purified by several methods and their antigenicity was tested by separating the antigens by means of SDS-PAGE, followed by Western blotting using sera of chickens infected with S. enteritidis. A high degree of cross reactivity was observed with many of the antigens tested, especially the lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) which had previously been reported as containing antigens which could be used for specific detection of S. enteritidis. This cross-reactivity could be explained by the conserved nature of many of the LPS and OMP antigens among the Salmonella serotypes tested. A fimbrial antigen, SEF14, which has been reported as a novel antigen, was seen as a prominent band at 14.3 kDa and was found to react with antibodies against S. enteritidis, yet not to the specificity levels described in previous reports. PCR amplification of the sefA gene sequence, which encodes for the SEF14 fimbrial antigen, was found to give a predicted product of 310 bp when using a previously described oligonucleotide primer pair. This amplified product was found to be specific for S. enteritidis and other serogroup D Salmonella serotypes that are not poultry pathogens The cross-reactivity observed with many of the serological techniques used in this study, meant that detection of S. enteritidis infection in chickens was considerably hindered. However, the identification of further novel antigens by serological means, could result in the development of new vaccines. The specificity and speed afforded by PCR amplification indicated that this technique showed excellent potential for use in local diagnostic laboratories. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

The mammalian cell cycle and its points-of-entry are well characterized pathways. These points-of-entry are normally regulated via mitogens and include, amongst others, the ERK, JNK and p38 mitogen-activated protein kinase (MAPK) pathways. However, while the restriction point(R-point), the temporal switch-point at which a cell becomes irrevocably committed to division irrespective of mitogenic stimulus, is known among other cell types, its position within the murine myoblast line C2C12 is currently unknown. Similarly, while MAPK pathways, such as JNK and ERK, have been modeled computationally, no model yet exists of p38 MAPK as stimulated by mitogens. The aims of this dissertation, then, were to determine the R-point within the C2C12 cell cycle and construct a computational mitogen-stimulated p38 MAPK model. It was found that a synchronous C2C12 population, when stimulated to divide, took 7 to 9 hours to reach S-phase from G0, consistent with data from the literature. The R-point was determined to lie between 6 and 7 hours post G1-re-entry stimulation,which was consistent with studies in other cell types. Core modeling of the p38 MAPK pathway revealed that ultrasensitivitywas inherent within the pathway structure. Further, a branching/re-converging structure within the pathway imparted greater responsiveness to signal upon the pathway. A realistic p38 MAPK model demonstrated good responsiveness to signal, its output matched that of several other MAPK models, and it was capable of replicating previous in vitro data. This model can be used as a tool for further investigation of the mammalian cell cycle by linking it to other cell cycle models. The predictions by an expanded model may be better suited for understanding the effects of mitogen stimulus on the cell cycle in situ. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Mammalian skeletal muscle can regenerate after injury and this response is primarily mediated by the satellite cell, a muscle stem cell. Following injury, satellite cells are activated to myoblasts, undergo rapid proliferation, migrate towards the injury site, and subsequently differentiate into myotubes in order to facilitate functional muscle repair. Fibrosis, caused by the secretion of structural extracellular matrix (ECM) proteins such as collagen I and fibronectin, by fibroblasts, impairs complete functional repair of the muscle. In this study, the role of the microenvironment during wound conditions was assessed by analysing the effect of specific extracellular matrix and growth factors on myoblast migration. The role of the Rho/ROCK pathway as a possible mechanism in mediating the effects seen was investigated. In order to analyse wound repair in an in vitro setting, we optimised and improved a wound healing model specifically designed for skeletal muscle repair. To this end we also developed a co-culture assay using primary myoblasts and fibroblasts isolated from the same animal. The studies showed that collagen I and fibronectin both increased myoblast migration in a dose-dependent manner. Decorin displayed opposing effects on cellular movement, significantly increasing collagen I-stimulated, but not fibronectin-stimulated, migration of myoblasts. ROCK inhibitor studies revealed a significant increase in migration on uncoated plates following inhibition with Y-27632 compared to untreated control. When cells were cultured on ECM components (Matrigel, collagen I, or fibronectin), the inhibitory effect of Y-27632 on migration was reduced. Analysis of ROCK and vinculin expression, and localization at the leading front, showed that ROCK inhibition resulted in loosely packed focal adhesion complexes (matrix dependent). A reduced adhesion to the ECM could explain the increased migration rates observed upon inhibition with Y-27632. We also investigated the role of TGF-β and decorin during wound repair, as TGF-β is a known pro-fibrotic agent. TGF-β treatment decreased wound closure rates; however, the addition of decorin with TGF-β significantly increased wound closure. The addition of ECM components, Matrigel and collagen I enhanced the effect seen in response to TGF-β and decorin; however, fibronectin negated this effect, with no increase in migration seen compared to the controls. In conclusion, the importance of extracellular matrix components in regulating myoblast migration and therefore skeletal muscle wound repair was demonstrated. We emphasize that, in order to gain a better understanding of skeletal muscle wound repair, the combination of ECM and growth factors released during wounding need to be utilised in assays which mimic the in vivo environment more closely. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and thought to be relevant in invasion and inflammation, had been established while that of their inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not. Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly located in a distinct oval, electron translucent organelle, a little larger than azurophil granules. A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored proteins, established using granule fractionation and immunolabelling to be markers for the secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal nature of this organelle. Density gradient cofractionation with the least dense secretory vesicle population and some pleiomorphism of the organelle suggested that it is a "vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and termination differentiation, but before secretory vesicle synthesis. Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that specific and azurophil granules and a small number of proMMP-8-containing granules (a specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium regulated. However, studies using the calcium ionophore, ionomycin, and monitoring extracellular granule marker protein release upon addition of increasing levels of extracellular calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated. This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be studied further, but results achieved to date may explain the observed differential mode of release of TIMP-1 relative to proMMP-9. The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a novel reverse zymography method. The role and relevance of this form remains unknown as does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Plasmodium falciparum is a protozoan parasite responsible for the most severe form of human malaria, with infection often resulting in death. Efforts to control malaria have been hindered by an increased spread of parasite resistance to previously effective antimalarial drugs, leading to an intensified search for novel antimalarial drug targets. A group of proteins suggested as potentially effective targets are the integral membrane transport proteins, since they play key roles in Plasmodium parasite growth and replication. One such membrane protein recently characterised was the P. falciparum copper efflux transporter. Treatment of cultured P. falciparum parasites with the intracellular copper chelator neocuproine inhibited parasite growth, suggesting that additional mechanisms for malaria parasite copper homoeostasis are likely to be present. Copper is an essential trace element involved in enzymatic processes requiring redox-chemistry. In higher eukaryotes copper is transported across the plasma membrane via the copper transport protein, Ctr1, and distributed intracellularly by copper metallochaperones. The mechanisms for copper acquisition and distribution in the Plasmodium parasite are, however, yet to be characterised. An in silico Basic Local Alignment Search Tool for protein (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified sequences corresponding to a putative copper transporter, and associated copper metallochaperones, in eight species of the Plasmodium parasite. Each of the Plasmodium copper transport protein sequences was found to contain features common to the well characterised copper transporters. These features included predicted copper-binding motifs in the protein's amino terminus, three membrane spanning domains and the characteristic MxxxM and GxxxG motifs located in the second and third transmembrane domains, respectively. Affinity purified anti-peptide antibodies, generated against an immunogenic peptide (CSDKQSGDDECKPILD) in the amino terminus of a putative malaria parasite copper transporter (PY00413), detected the target protein in murine malaria parasites in association with a parasite membrane. The open reading frames corresponding to the amino terminal domains of one P. berghei [PBANKA_130290 (447 bp)] and two P. falciparum [PF14_0211 (132 bp) and PF14_0369 (282 bp)] putative copper transport proteins were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with maltose binding protein (MBP). The resulting sizes for the recombinant proteins were 61kDa for MBP-PbCtrNt, 48kDa for MBP-PfCtr211Ntᵀᴰ and 55kDa for MBP-PfCtr369Ntᵀᴰ, with each protein being recognised by a corresponding anti-peptide antibody. All three recombinant proteins bound copper in vitro and in vivo, with each having a binding preference for the reduced cuprous ion. This preference has been similarly established for the characterised copper transporters. Although the results supported the expression and copper binding ability of a Plasmodium parasite copper transport protein, the directional transport of copper, by this protein, requires experimental confirmation as does its specific location. The identification of a P. falciparum copper transporter, and other copper dependent proteins, implies a parasite metabolic requirement for copper. Mammalian and yeast cells require a Cox17 metallochaperone for copper delivery to cytochrome-c oxidase. Identification of P. falciparum orthologs for Cox17 (PF10_0252) and a number of cytochrome-c oxidase subunits (PF13_0327; PF14_0288; mal_mito_1; mal_mito_2; PFI1365w; PFI1375w), suggests the existence of similar parasite mechanisms for copper delivery. Analysis of the Plasmodium Cox17-like sequences identified essential amino acids conserved in the well characterised yeast and mammalian Cox17. This included the identification of six cysteine residues essential for Cox17 function. A homology model of P. falciparum Cox17, with human Cox17 as the template [PDB ID: 2RN9 (apoCox17); 2RN8 (Cu⁺-Cox17)], suggested that Plasmodium Cox17 orthologs would adopt a similar structural conformation. The open reading frames for full-length P. yoelii [PY03823 (192 bp)] and P. falciparum [PF10_0252 (195 bp)] Cox17 were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with either a His₆-tag or glutathione S-transferase (GST)-tag, respectively. The resulting sizes for the recombinant proteins were 11.6kDa for His₆-PyCox17 and 33.5kDa for GST-PfCox17, with each protein being recognised by a corresponding anti-peptide antibody. Both recombinant Cox17 proteins bound the cuprous ion in vitro and in vivo, similar to mammalian and yeast Cox17. This supported the likely existence of a mitochondrial copper metallochaperone pathway within the malaria parasite; however, this requires further experimental confirmation. Identification of a parasite copper transport protein, and associated metallochaperones, could provide novel targets for drug-based inhibition of parasite growth. Alternatively, the copper transporter may provide a novel mechanism for drug delivery into the Plasmodium parasite. The potential of these malaria parasite proteins being effective drug targets does, however, remain to be confirmed. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Kinases play a crucial role in regulating cellular signaling cascades, making them therapeutic targets for several human diseases. In human cancers, mis-regulation and mutations of kinases such as EGFR (epidermal growth factor receptor) have been found to drive malignant transformation. Due to the conserved structural elements of protein kinases, the majority of kinase inhibitors available have a tendency to inhibit multiple targets. The biological impact of this promiscuity is insufficiently defined and the prevalence of cellular compensatory mechanisms additionally varies the clinical responses to drug treatment. In order to understand the relationship between selectivity and efficacy, prior to clinical trials, it is essential to characterize how inhibitors interact with the kinome within a cellular context. Monitoring inhibitor-target interactions generally involves in vitro assaying with purified proteins or protein domains, which compromises the native integrity of the kinases. Cellbased assays either gain outcomes from bulk populations that average out cell variance or phenotypic assays that lack molecular resolution. To obtain information on drug interactions on a single cell level, we have developed a method to measure the direct binding of kinase inhibitors to their targets in situ and in vivo. Kinase inhibitors are chemically tagged with fluorophores that serve as acceptors to genetically tagged donor fluorophores on the enzyme and the interaction is measured using FRET-FLIM. With epidermal growth factor receptor (EGFR) and irreversible EGFR inhibitors as the model system, this approach has been applied to image inhibitor-kinase interactions in live and fixed cells. Using this method, a small panel of tyrosine kinase targets, and labeled inhibitors, we were able to investigate the cross-specificity within the panel. Additionally it was found that the specificity of inhibitors for specific kinase conformations enables the distinction between EGFR in the active and inactive conformation by the inhibitor-probes. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.