Malaria is a life-threatening parasitic disease endemic throughout the world. Control methods for malaria are becoming less reliable; thus, efforts to develop a safe and effective vaccine are critical. Immunity to malaria requires both cell- and humoral-mediated immunity, CMI and HMI, respectively. CD4+ T cells play a central role in protection against blood stage Plasmodium infection. Given that clinical features of malaria are caused by blood stages, a vaccine against this stage will be very effective in reducing morbidity and mortality. During the blood stage, purine nucleotides, which are essential for parasites’ survival and proliferation, are in high demand. The inability of the parasite to engage in de novo synthesis of purine nucleotides makes the enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) an essential nutrient salvage enzyme. HGXPRT is located in electron-dense regions in merozoites and in vesicles in the red cell cytoplasm. In contrast to other blood stage antigens, those located on the merozoite surface are targets of HMI. To advance HGXPRT as a malaria vaccine candidate, fermentation and purification of the protein from Plasmodium falciparum (PfHGXPRT) was performed using facilities at Q-Gen, the Queensland Institute of Medical Research (QIMR). Escherichia coli carrying PfHGXPRT gene were a gift in-kind from the University of Queensland (UQ). Recombinant PfHGXPRT expressed in E.coli was purified using anion exchange liquid chromatography and gel filtration techniques. Three methods were used to confirm the Q-Gen PfHGXPRT identity: (1) Western blotting showing identical bands of UQ PfHGXPRT and Q-Gen PfHGXPRT at 26 kDa; (2) N terminal sequencing was compatible with the PfHGXPRT sequence; and (3) mass spectrometry showed homogeneity by giving a subunit molecular mass of 26,231 Da. The purification method used is reproducible and affordable, yielding reasonably pure protein for animal experimentation. Following purification of PfHGXPRT, its efficacy as a subunit vaccine candidate in a rodent model of infection was examined. Multiple rodent models of malaria infection were assessed and it was determined that Plasmodium chabaudi AS (P. chabaudi AS) exhibited the highest cross-reactivity against PfHGXPRT in mice. Hence, P. chabaudi AS was chosen as the appropriate rodent model for study in this thesis. Natural immunity against PfHGXPRT during a blood stage P. chabaudi AS infection was assessed by testing sera and splenocyte responses to PfHGXPRT. IFN- and IL-4, as well as antibodies specific for PfHGXPRT, could be detected after infection, suggesting that PfHGXPRT is a target of natural immunity during the blood stage infection. Therefore, further studies of protective immunity generated by immunisation with PfHGXPRT were conducted, specifically to determine their protective efficacy and to determine immune mechanisms elicited by immunisation. Mice immunised with PfHGXPRT and challenged with P. chabaudi AS developed a slightly reduced parasitaemia. T-cell proliferation, but not antibody responses, was detected after immunisation. Protective mechanism(s) were assessed by adoptively transferring immune CD4+ T cells, B cells or sera to naïve SCID mice followed by parasite challenge. Only recipients of immune CD4+ T cells showed extended survival. Nevertheless, immunisation with PfHGXPRT followed by sub-patent infection induced better protection than immunisation with PfHGXPRT alone, which appeared to be related to CD4+ T cells. Reduction of parasitaemia, as well as augmentation of T cell proliferation and IFN-γ production, was evident in PfHGXPRT and sub-patent infected immunised mice. Recipients of CD4+ T cells from PfHGXPRT and sub-patent infection immune mice also showed some degree of protective immunity. PfHGXPRT was shown to induce natural and acquired immunity to P. chabaudi AS. HGXPRT is highly conserved in parasites and humans; therefore, it is essential to define minimal protective epitopes that could be included in a vaccine. Hence, 22 overlapping peptides (termed P1 P22) corresponding to the entire P. chabaudi AS HGPRT sequence were used to define minimal protective epitopes. Following immunisation of mice with seven pools of peptides (P1 P3, P4 P6, P7 P9, P10 P12, P13 P15, P16 P18 and P19 P22), three immunogenic peptides (P11, P13, and P17), which stimulated significant proliferative and IFN-γ responses were chosen for immunisation studies. Peptide P9 (position 76-95 from N-terminal), which induced the highest IFN- levels during P. chabaudi AS infection was also included in the pool of peptides. Mice immunised with P9, P11, P13 and P17 had significantly decreased parasitaemia. Antibody mediated immunity had a partial effect on suppressing parasite growth. CMI, on the contrary, played a central role in adoptively transferred protection by significantly reducing parasitaemia and prolonging survival of recipient SCID mice. Strong T cell proliferation and IFN- secretion were also detected after stimulation of splenocytes from immune mice with P. chabaudi AS antigen. CMI response was significantly increased after immunisation with the peptides followed by sub-patent infection. The findings that four short epitopes of HG(X)PRT confer strong CMI protection suggest that homologues of such epitopes could be included in a multi-component malaria vaccine.

Thesis (M. Phil.)--University of Hong Kong, 1992.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (MSc (Paraclinical Sciences))--University of Pretoria, 2007. / Includes bibliographical references. Also available in print format.

Thesis submitted in fulfilment of the requirements for the Degree of Master's in Chemistry, Durban University of Technology, 2017. / Quinoline and its derivatives represent an important class of nitrogen-containing heterocylces as they are useful intermediates in organic synthesis and possess a broad spectrum of biological activities, such as anti-asthmatic, anti-inflammatory and anti-malarial activity. Hence, synthesis of novel compounds with potent biological activities is important in medicine. Significant research is directed into the development of new quinoline based structures and new methods for their preparations. In the past, synthesis of complex molecules was accomplished by step-wise reaction. This was time consuming and yield was generally low. Nowadays, multi-component reactions (MCRs) are being used since three or more substrates can be reacted in a one-pot reaction. Therefore yields are higher and the reaction is more efficient. In this research investigation novel quinoline derivatives, using the multi-component reaction protocol, were synthesized. After characterization of the product by several spectroscopic techniques, the biological potential of these compounds were assessed using lung cancer cell lines, bacteria and molecular modeling in an enzymatic system. In the synthetic part of this study, the first step was the preparation of the starting compound 2- chloro-3-formyl quinoline for which the Vilsmeier-Haack cyclisation protocol was used. The cyclisation was carried out by combining DMF and POCl3 at 5°C to form an electrophile which then reacted in situ with N-phenylacetamide at 100ºC to afford 2-chloro-3-formyl quinoline in high yield (95%). This was followed by the synthesis of a series of novel quinoline derivatives in a MCR system comprising 2- chloro-3-formyl quinoline, malononitrile, aromatic amines and dimethyl acetylenedicarboxylate in the presence of a catalytic amount of triethylamine. Valuable features of this routine included high yields, extensive substrate range and straight forward procedures. Eight novel poly-functionalised dihydropyridine quinoline derivatives were synthesized, purified and characterized. The outline for the synthesis of poly-functionalised dihydropyridine quinoline derivatives is presented graphically in Scheme 1. Scheme 2 shows the eight compounds synthesized and used subsequently for further studies. . Step 1 CH3 a N O H CHO N Cl Step 2 CHO CN N Cl CN NH2 R O OCH3 b OCH3 O MeO2C MeO2C N Cl CN N NH2 R = m-CH3, o-OCH3, p-Cl, m,p-Cl, o-F, m-F, p-F R Reaction Conditions: a. DMF, POCl3 b. Et3N, EtOH Scheme 1: Graphical representation for the synthesis of poly-functionalised dihydropyridine quinoline derivatives The novel eight compounds were screened for their potential activity in lung cancer cell lines. A549 cells were incubated for 24 hours with a range of concentrations of each compound, in triplicate, in a micro-titre plate together with an untreated control. Each experiment was conducted twice on separate occasions; the results from the first set matched the repeated experiment. The cells were then incubated (37ºC, 5% CO2) with the MTT substrate for 4 hours. Thereafter all supernatants were aspirated and DMSO was added to the wells. Finally the optical density was measured at 570 nm at a reference wavelength of 690 nm with an ELISA plate reader. The net MTT dependant absorbance (optical density) of each sample was calculated by subtracting the average absorbance of the blank from the average absorbance of each sample. Data were represented as mean optical density plus or minus the standard deviation. Four of the synthesized compounds (A1-A8) were evaluated for their cytotoxicity activities. The anti-cancer assay indicated that poly-functionalised dihydropyridine quinoline compounds, A2, A3 and A4 have good potential as anti-cancer drugs. Among them, A2 and A4 proved to be dose dependent with A4 having the highest toxicity at 250 µM and A8 having the highest toxicity at 125, 250 and 500 µM, whereas A1, A5, A6 and A7 were not cytotoxic. O H3CO H3CO O N Cl CN NH2 O H3CO H3CO O N Cl CN N NH2 OCH3 O H3CO H3CO O N Cl CN N NH2 O H3CO H3CO O N Cl CN NH2 CH3 Cl A1 A2 A3 A4 O H3CO H3CO O N Cl CN N NH2 F O H3CO H3CO O N Cl CN N NH2 O H3CO H3CO O N Cl CN NH2 O H3CO H3CO O N Cl CN N NH2 F Cl F Cl A5 A6 A7 A8 Scheme 2: Structures of novel poly-functionalised dihydropyridine quinoline derivatives by MCRs Since molecular docking is a key tool in structural molecular biology and computer-assisted drug design, these compounds were subjected to molecular docking and the binding mode for the compounds, within the active site of the protein, was analyzed. Docking of A1 to Human mdm2 protein provided insights into the binding regions. Three hydrogen bonds were formed between GLU 25 (2.7 Å distance), LEU 27 (3.2 Å distance) and LEU 54 (3.2 Å distance) atoms with binding energy of -8.91 kcal/mol. Docking of A1 with Human mdm2 indicated the lowest binding energy thereby showing strong affinity of the ligand molecule with the receptor which has been stabilized by strong hydrogen bond interactions in the binding pocket. This confirms that A1 is a better inhibitor for E3 ubiquitin-protein ligase mdm2 than all the other compounds tested (A2-A8). Further, the eight novel poly-functionalised dihydropyridine quinoline derivatives were evaluated for their antibacterial activity. This was performed using the MABA method against three strains i.e. Gram negative; Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Gram positive; Staphylococcus aureus (ATCC 29213) using the broth micro dilution method. Standard antibiotics (ciprofloxacin and nalidixic acid) were used as positive controls and DMSO was used as a negative control. The results obtained from the anti-bacterial assay showed that compounds A4, A7 and A8 have high activity, whereas A2 and A3 showed poor activity against all the tested bacterial strains. Compound A6 showed no activity against S. aureus and E. coli. / M

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Natural killer (NK) cell deficiency (NKD) occurs when an individual’s major clinical immunodeficiency derives from abnormal NK cells and is associated with several genetic etiologies. Three categories of β actin-related diseases with over 60 ACTB variants have previously been identified, none with a distinct NK cell phenotype. An individual with mild developmental delay, macrothrombocytopenia, susceptibility to infections, molluscum, and EBV-associated lymphoma had functional NK cell deficiency for over a decade. A de novo ACTB variant encoding G342D β actin was identified and was consistent with the individual’s developmental and platelet phenotype. This novel variant also was found to have a direct impact in NK cells, as its expression in YTS (YTS-NKD) cells caused increased cell spreading in lytic immune synapses created on activating surfaces. YTS-NKD cells were able to degranulate and perform cytotoxicity, but demonstrated defective serial killing owing to prolonged conjugation to the killed target cell and thus were effectively unable to terminate lytic synapses. G342D β actin results in a novel mechanism of functional NKD via increased synaptic spreading and defective lytic synapse termination with resulting impaired serial killing leading to overall reductions in NK cell cytotoxicity.

Abelson murine leukemia virus (A-MuLV)-induced transformation was investigated to determine whether cells not sensitive to TNF-α could be made sensitive to the cytolytic action of TNF-α when infected with this retrovirus. Mouse embryonic fibroblast cell line CL.7 was found to be relatively insensitive to TNF-α. Upon transformation with A-MuLV, these cells gave rise to a clone (3R.1) which was found to be insensitive to TNF-α and another clone (6R.1) which had an increased sensitivity to TNF-α. The differential cytotoxicity was observed when cells were treated with TNF-α, for 18 hr, at 0 to 100 units/ml, at 37°C. The mechanism of this differential cytotoxicity was further investigated. Thus, TNF-R levels on the cell surface were found to be not correlated with the differential TNF-α response. The A-MuLV transformation suppressed the epidermal growth factor-receptor (EGF-R) in 3R.1 clone and induced its levels significantly in the 6R.1 clone (p<0.05). Cell surface EGF-receptor (EGF-R) levels in CL.7 and 3R.1 clones were lower than the 6R.1 clone (p<0.05). Although the EGF-R levels in all the clones were induced with TNF-α, the expression of EGF-R correlated with the susceptibility to TNF-α. The role of antioxidants, such as α-tocopherol and β-carotene, (known anti-cancer agents) in modulating TNF-α-induced EGF-R expression was investigated. In both the untransformed and the transformed clones, f-carotene suppressed the constitutive and the TNF-α induced EGF-R levels whereas α-tocopherol was found to have an enhancing effects. Studies with metabolic inhibitors on TNF-R and EGF-R expression indicate that inhibitors of the arachidonic acid cascade and modulators of protein kinase-C (PK-C), could influence the binding and internalization of TNF-α and thereby controlling the physiologic future of the cells. The A-MuLV specific V-abl protein, p120, tyrosine phosphorylation was determined by a radio-labelled anti-phosphotyrosine antibody in an antigen capture assay. TNF-α had little effect on p120 phosphotyrosine levels of TNF-α insensitive CL.7 and 3R.1 clones. The, TNF-α sensitive, 6R.1 clone, however, was found to induce its p120 specific phosphotyrosine upon exposure to TNF-α for 8 hr. Thus, TNF-α modulated the tyrosine phosphorylation of p120 only in the TNF-α-sensitive cell line. The mitochondrial toxicity of TNF-α was determined by monitoring the rate of quenching of a cationic spin probe CAT 16. Mitochondrial preparation from CL.7 and 3R.1 clones had higher ability to quench CAT 16 signal with TNF-α incubation time than mitochondria from the 6R.1 cells. This indicates that the differential TNF-α cytotoxicity manifested in A-MuLV transformed clones may, in part, be due to the differential mitochondrial toxicity of this cytokine. The hypothesis that TNF-α cytotoxicity was mediated via an oxidative process was tested on the TNF-α sensitive L929 cells. Using a flow cytometric detection system it was determined that TNF-α produced intracellular hydrogen peroxide in these cells which was sensitive to concentration and incubation time of TNF-α. Superoxide radicals were also generated during TNF-α action on L929 cells, as determined by the use of the spin trap PBN in conjunction with EPR spectroscopic techniques. The PBN-OOH spin adduct spectrum peaked at 9 hr of TNF-α incubation and was inhibitable upto 30 % with 10 µM of desferral-Mn complex (a known SOD mimic). These data indicate that superoxide and hydrogen peroxide are common events in TNF-α dependent cell killing process. The differential TNF-α cytotoxicity was found to depend on differences in the antioxidant status of the target clones. Thus, it was found that Cu/Zn-SOD, Mn-SOD, GSH-Peroxidase and GSH-Reductase enzymes were all induced significantly in the CL.7 clone (p<0.05) upon incubation with 100 units/ml of TNF-α for 18 hrs. TNF-α had little effect on the antioxidant enzymes of both 3R.1 and 6R.1 cells. However, the constitutive levels of most antioxidant enzymes were found to be higher in 3R.1 cells than in the 6R.1 cells. Therefore, the susceptibility of 6R.1 to TNF-α may, in part, be due to a low level of antioxidant enzymes present in this clone. In conclusion we found that the differential cytotoxicity of TNF-a may, in part, due to: (1) differential EGF-R expression, (2) differential mitochondrial cytotoxicity, and (3) differential ability to modulate the tyrosine phosphorylation in untransformed and A-MuLV transformed cells and (4) differential antioxidant status of these cells to handle oxidative stress imposed by TNF-α. / Ph. D.