Every year there are approximately 300 million new cases of malaria with 2 million deaths. The majority of deaths occur in African children between the ages of 1 and 4 years and are caused by the parasite Plasmodium falciparum. Approximately R90-million is spent by the South African government each year to control malaria. Peripheral blood monocytes are the first line of defence during infection and they perform many functions, such as phagocytosis, intracellular and extracellular killing by the generation of reactive oxygen intermediates and the production of cytokines. During malaria infection some of these functions are suppressed or elevated by phagocytosis of hemozoin, fever conditions (heat shock) and the presence of anti-malarial drugs in the bloodstream of the patient. Under normal conditions phospholipase A₂ (PLA₂) is down regulated by heat shock protein 70 (HSP70) but in severe malaria PLA₂ is elevated. Two antigenic peptides were selected from the highly conserved human HSP70 and HSC70 proteins. Anti-peptide antibodies raised in chickens were affinity purified and were able to recognize the free peptide in an ELISA and the native proteins in human and canine heat shocked lymphocyte lysates on western blots. Antibodies against HSP70 detected two major proteins at 70 kDa and 33 kDa, which are most likely native HSP70 and a possible breakdown product of HSP70 respectively. The anti-HSC70 antibodies detected two proteins, an as yet unidentified 100 kDa protein and the 70 kDa HSC70. Due to the monocytes being activated during the isolation procedure, HSP70 was expressed at both 37°C and 44°C in this study. Electron-probe X-ray microanalysis enables determination of the elemental composition of any sample under the electron microscope. When the electron beam interacts with a specimen, X-rays are generated and can be used to identify and quantify the elements in the cell. Canine monocytes were analysed using this technique after incubation with therapeutically relevant concentrations of anti-malarial drugs, β-hematin and under fever conditions. The concentrations of the elements in normal canine monocytes were: Na (518.2 mmoles/kg), Mg (199.1 mmoles/kg), P (439.7 mmoles/kg), S (316.3 mmoles/kg), Cl (279.7 mmoles/kg), K (204 mmoles/kg) and Ca (81.3 mmoles/kg). All the drugs (quinine, chloroquine, primaquine, pyrimethamine, artemisinin, tetracycline, doxycycline, dapsone and suramin), phagocytosis of latex beads and β-hematin as well as heat shock, altered the elemental concentrations of canine monocytes in a unique way. Quinine, artemisinin and suramin were the most influential drugs in altering the concentrations of elements in the cells.Suramin substantially increased the concentration of Ca (356%) after 18 h and decreased K concentration (64%) after 18 h. Quinine decreased the concentrations ofNa (47%), Cl (70%), and K (67%). The concentrations of P (52%) and Ca (72%) were increased by quinine after 10 min. Artemisinin induced small increases in Mg (21 %) and K (38%) concentrations within 10 min and large increases in the concentrations of Na (291%) and Cl (389%) after 18 h. Chloroquine induced a large increase in S (212%). Quinine induced major changes after 10 min whereas artemisinin, suramin chloroquine induced huge changes after 18 h. Although artemisinin did increase the concentrations certain elements after 10 min, it was by much smaller amounts than after 18 h. Quinine, suramin and pyrimethamine altered the P/K ratios by the greatest margins whereas artemisinin had no significant effect. The P/K ratio was increased by quinine (348%) after 10 min and suramin (261%) after 18 h. Pyrimethamine decreased the P/K ratio after 18 h by 49%. The findings suggest that further investigations into the alterations in the elemental concentrations of monocytes by anti-malarial drugs, fever and hemozoin may lead to a greater understanding of the influence of these conditions in a patient during a malaria infection and its treatment. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

No description available.

Plasmodium falciparum is a protozoan parasite that causes malaria, a disease that is widely spread in the tropical world. Chloroquine has been very effective against malaria since it was introduced into the field until the emergence of chloroquine resistant malaria. Chloroquine resistant malaria has become widely spread in the endemic area. In addition, cross resistance to other antimalarial drugs that are different in structure and function has been reported, even though some of these drugs had not been previously used in that particular region. The objective of this study was to determine the molecular mechanism of this resistance. Actinomycin D resistant Plasmodium falciparum was selected in vitro from the drug sensitive parental clone, 3D7. Interestingly, we found that the selected strain is resistant to chloroquine, mefloquine, antimalarial drugs, and Rhodamine 123. Comparison between 3D7 parental and 3D7R/act-D2 resistant P. falciparum did not show a difference in the level of expression of pfmdr1 previously implicated in the drug resistance. In addition we found that the level of accumulation of two drugs actinomycin D is reduced in the resistant parasite as compared with the sensitive one. Further studies indicated that the reduction in the drug accumulation was due to the increase in drug efflux. Furthermore, to identify if other P-glycoprotein homologues are involved in the resistance, oligonucleotide primers to conserved sequences in ABC domains have been used. An ABC protein homologous to subunit 4 of the 26S proteasome complex has been cloned. In vitro transcription, translation and immunoprecipitation analysis were done using reticulocytes lysate and polyclonal antibodies generated against peptide sequence in the P. falciparum S4 subunit. Surprisingly a decrease in the expression of this gene was found in the resistant clone, 3D7R/act-D2, compared to its parental cell line as determined by Northern blot analysis. Studies are in progress to determine

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant surface antigens, encoded by the var gene family, play a crucial role in malaria pathogenesis through mediating immunomodulation and host cell adhesion. Var genes can be sub-grouped according to genetic or functional features. This thesis examined var gene transcription of conserved groups of var genes in the context of clinical malaria disease manifestation in African field isolates. Analysis of var gene transcription in 26 P. falciparum field isolates from Malian children revealed that field isolates from children with cerebral malaria show significantly higher transcription of group A var genes than the field isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia). These results suggest that group A var genes are important determinants of parasite virulence and strengthen the growing body of evidence associating group A var expression with severe disease in children. Analysis of var gene transcription in six P. falciparum placental malaria field isolates showed that var2csa was transcribed in all placental malaria field isolates, but not in 10 childhood isolates examined. This finding, also reported in other recent and subsequent studies, suggests that var2csa expression is a critical factor in the onset of clinical malaria disease in pregnant women. Examination of type 3 var gene transcription in laboratory and field isolates established that these var genes were commonly transcribed in blood-stage parasites, and sequence analysis of the transcribed domains confirmed a very high level of conservation across this var gene sub-family. Finally, rosetting is a property of some group A PfEMP1 and is associated with disease severity in African childhood malaria. Certain glycoconjugate compounds can disrupt rosetting, possibly due to the functional similarities of interactions between rosetting PfEMP1 and host rosetting ligands. A non-toxic compound (curdlan sulfate) was found to be effective at disrupting rosettes in all 18 rosetting field isolates examined, showing potential for use in treatment of severe malaria due to rosetting P. falciparum isolates. The findings presented in this thesis expand current knowledge of the role and significance of var genes/PfEMP1 in P. falciparum malaria disease pathogenesis. The work demonstrates the importance of continued research on var genes/PfEMP1 in further understanding this complex parasite, and ultimately in combating this severe disease.

Malaria is caused by the Plasmodium sp. parasite that infects the red blood cells. Of the four types of malaria, the most serious type is transmitted by Plasmodium falciparum species. It can be life threatening. The other types of malaria (P. vivale, P. ovale and P. malariae) are generally less serious and are not life threatening. The existence of malaria as an enemy of humankind certainly predates written history. For thousands of years malaria has been a deadly scourge, and it remains one today. From American president John Adams who nearly succumbed to malaria in Amsterdam while on a diplomatic mission, back down to the timeline to the early Chinese, Indians, Greeks and Romans, malaria has not spared its victims, rich or poor. It wasn’t until the 19th Century that information about the true cause of malaria became known. Yet despite this knowledge, malaria still ravages Sub–Saharan Africa, South–East Asia and Latin America, taking as its victim’s mainly young children and pregnant women. However, without certain discoveries leading to a better understanding of malaria, new groundbreaking work wouldn’t be possible. Artemisinin and its derivatives are developing into a very important new class of antimalarial and their usage is becoming more common in the fight against malaria. The most commonly used and applied of these derivatives are artesunate, artemether, arteether and dihydroartemisinin. The discovery of artemisinin as the pharmacological active ingredient in an age old Chinese herb, Artemisia annua, was a major breakthrough in malaria chemotherapy. Discovery of qinghaosu in the 1970s sparked a new age for chemotherapy of malaria, and greatly inspired further research on organic peroxides. This generated widespread interest and led to the design and synthesis of organic peroxides into a highly active area of organic chemistry. The artemisinin derivatives act quickly and are eliminated quickly. Their rapid onset makes them especially effective against severe malaria. Their rapid disappearance may be a key reason why artemisinin resistance has been so slow to develop, and may be the reason why recrudences are so common when these drugs are used in monotherapy. Since their isolation, artemisinins have had a substantial impact on the treatment of malaria. Although very potent, the use of artemisinins as prophylactic antimalarials is not recommended. The aim of this study was to synthesise ester derivatives of artemisinin, determine certain physicochemical properties such as aqueous solubility and partition coefficient, and to evaluate their antimalarial activity in comparison to dihydroartemisinin and chloroquine. In this study eight esters of dihydroartemisinin (DHA) were synthesised by substitution at C– 10. The structures of the prepared derivatives were confirmed by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The new artemisinin esters were tested in vitro against the chloroquine sensitive strain of Plasmodium falciparum (D10). All the compounds tested showed activity against the D10 strain. All of the esters showed potency significantly better than chloroquine, except the octyl and decyl esters which were less active. The reason for the low activity could be ascribed to the fact that these two esters are both water immiscible oils, leading to solubility problems. The ethyl, butyl, phenyl and p–nitrophenyl esters all had similar IC50 values making their activity similar. The lowest IC50 value was displayed by the butyl ester with a value of 3.2 x 10– 3 uM. The poorest activity was recorded by the two oils, the octyl and decyl esters, with IC50 values of 38 x 10–3 uM and 90.2 x 10–3 uM respectively. All other compounds showed less antimalarial potency against the D10 strain compared with the other reference drug dihydroartemisinin, except the butyl ester. The butyl ester 12 displayed activity comparable to that of DHA (IC50; 3.2 x 10–3 uM versus 3.8 x 10–3 uM), and is thus worthwhile being further investigated in terms of pharmacokinetics in order to determine its half–life. Statistically it is impossible to make structure–activity relationship (SAR) deductions from the data received as the number of compounds in the series is too small. The butyl (12) (IC50 = 3.2 uM), 4–nitrobenzyl (16) (IC50 =15 uM), 2–(acetyloxy) acetyl (17) (IC50 = 8.6 uM), and 2–phenylacetyl (18) (IC50 = 12.4 uM) esters showed on a 0.05 level statistically significantly better activity against the chloroquine sensitive D10 strain of Plasmodium falciparum than chloroquine itself while the decyl ester (14) (IC50 = 90.2 uM) was statistically significantly less potent. The activity of the octyl (13) (IC50 = 38.0 uM) and benzyl (15) (IC50 = 25.7 uM) esters did not differ from that of chloroquine. In comparison to dihydroartemisinin the propyl (11) (IC50 = 24.1 uM), octyl (13) (IC50 = 38.0 uM), decyl (14) (IC50 = 90.0 uM), and benzyl (15) (IC50 = 25.7 uM) esters proved to be statistically significantly less potent than DHA while the activity of the butyl (12) (IC50 = 3.2 uM), 4– nitrobenzyl (16) (IC50 =15.3 uM), 2–(acetyloxy) acetyl (17) (IC50 = 8.6 uM), and 2–phenylacetyl (18) (IC50 = 12.4 uM) esters did not differ from that of DHA. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Malaria is caused by the Plasmodium sp. parasite that infects the red blood cells. Of the four types of malaria, the most serious type is transmitted by Plasmodium falciparum species. It can be life threatening. The other types of malaria (P. vivale, P. ovale and P. malariae) are generally less serious and are not life threatening. The existence of malaria as an enemy of humankind certainly predates written history. For thousands of years malaria has been a deadly scourge, and it remains one today. From American president John Adams who nearly succumbed to malaria in Amsterdam while on a diplomatic mission, back down to the timeline to the early Chinese, Indians, Greeks and Romans, malaria has not spared its victims, rich or poor. It wasn’t until the 19th Century that information about the true cause of malaria became known. Yet despite this knowledge, malaria still ravages Sub–Saharan Africa, South–East Asia and Latin America, taking as its victim’s mainly young children and pregnant women. However, without certain discoveries leading to a better understanding of malaria, new groundbreaking work wouldn’t be possible. Artemisinin and its derivatives are developing into a very important new class of antimalarial and their usage is becoming more common in the fight against malaria. The most commonly used and applied of these derivatives are artesunate, artemether, arteether and dihydroartemisinin. The discovery of artemisinin as the pharmacological active ingredient in an age old Chinese herb, Artemisia annua, was a major breakthrough in malaria chemotherapy. Discovery of qinghaosu in the 1970s sparked a new age for chemotherapy of malaria, and greatly inspired further research on organic peroxides. This generated widespread interest and led to the design and synthesis of organic peroxides into a highly active area of organic chemistry. The artemisinin derivatives act quickly and are eliminated quickly. Their rapid onset makes them especially effective against severe malaria. Their rapid disappearance may be a key reason why artemisinin resistance has been so slow to develop, and may be the reason why recrudences are so common when these drugs are used in monotherapy. Since their isolation, artemisinins have had a substantial impact on the treatment of malaria. Although very potent, the use of artemisinins as prophylactic antimalarials is not recommended. The aim of this study was to synthesise ester derivatives of artemisinin, determine certain physicochemical properties such as aqueous solubility and partition coefficient, and to evaluate their antimalarial activity in comparison to dihydroartemisinin and chloroquine. In this study eight esters of dihydroartemisinin (DHA) were synthesised by substitution at C– 10. The structures of the prepared derivatives were confirmed by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The new artemisinin esters were tested in vitro against the chloroquine sensitive strain of Plasmodium falciparum (D10). All the compounds tested showed activity against the D10 strain. All of the esters showed potency significantly better than chloroquine, except the octyl and decyl esters which were less active. The reason for the low activity could be ascribed to the fact that these two esters are both water immiscible oils, leading to solubility problems. The ethyl, butyl, phenyl and p–nitrophenyl esters all had similar IC50 values making their activity similar. The lowest IC50 value was displayed by the butyl ester with a value of 3.2 x 10– 3 uM. The poorest activity was recorded by the two oils, the octyl and decyl esters, with IC50 values of 38 x 10–3 uM and 90.2 x 10–3 uM respectively. All other compounds showed less antimalarial potency against the D10 strain compared with the other reference drug dihydroartemisinin, except the butyl ester. The butyl ester 12 displayed activity comparable to that of DHA (IC50; 3.2 x 10–3 uM versus 3.8 x 10–3 uM), and is thus worthwhile being further investigated in terms of pharmacokinetics in order to determine its half–life. Statistically it is impossible to make structure–activity relationship (SAR) deductions from the data received as the number of compounds in the series is too small. The butyl (12) (IC50 = 3.2 uM), 4–nitrobenzyl (16) (IC50 =15 uM), 2–(acetyloxy) acetyl (17) (IC50 = 8.6 uM), and 2–phenylacetyl (18) (IC50 = 12.4 uM) esters showed on a 0.05 level statistically significantly better activity against the chloroquine sensitive D10 strain of Plasmodium falciparum than chloroquine itself while the decyl ester (14) (IC50 = 90.2 uM) was statistically significantly less potent. The activity of the octyl (13) (IC50 = 38.0 uM) and benzyl (15) (IC50 = 25.7 uM) esters did not differ from that of chloroquine. In comparison to dihydroartemisinin the propyl (11) (IC50 = 24.1 uM), octyl (13) (IC50 = 38.0 uM), decyl (14) (IC50 = 90.0 uM), and benzyl (15) (IC50 = 25.7 uM) esters proved to be statistically significantly less potent than DHA while the activity of the butyl (12) (IC50 = 3.2 uM), 4– nitrobenzyl (16) (IC50 =15.3 uM), 2–(acetyloxy) acetyl (17) (IC50 = 8.6 uM), and 2–phenylacetyl (18) (IC50 = 12.4 uM) esters did not differ from that of DHA. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Plasmodium falciparum is a protozoan parasite and the causative agent of the most lethal form of malaria, a major disease in the tropical world. Chloroquine has been very effective in treatment of this disease, however the emergence of chloroquine-resistant strains in most geographical regions where malaria is endemic has made difficult the control of malaria. In addition, resistance to other antimalarials has been observed in these regions. The objective of this study was to determine the molecular mechanisms of multidrug resistance in P. falciparum. We have selected in vitro a P. falciparum strain resistant to actinomycin D from a parental drug sensitive clone, 3D7. Interestingly, we found that the actinomycin D resistant clone is less sensitive to chloroquine and mefloquine (antimalarial drugs) and rhodamine123. Comparison between parental 3D7 and resistant P. falciparum did not show differences in the copy number or level of expression of pfmdr1 previously implicated in chloroquine or mefloquine resistance. Furthermore, to identify if other P-glycoprotein homologues are involved in resistance, we used oligonucleotide primers to conserved sequences in ABC domains. An ABC protein, a homologue to the subunit 4, of the 26S proteasome complex has been cloned. To determine if this gene was involved in resistance to actinomycin D, a Northern blot was done. Surprisingly it was found a decreased in the expression of this gene in the resistant cell line, 3D7R/actD2, in comparison with its parental cell line, 3D7. Studies are in progress to determine the role of the PFS4 subunit in the resistance phenotype of 3D7R/actD2.

There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. / Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studies with P. c. chabaudi, parasites of the recrudescence were shown to be distinct from parasites of the primary parasitaemia, with respect to antigens expressed on the surface of late trophozoite- and schizont-infected erythrocytes. / Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. / Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.

Malaria has long affected the world both socially and economically. Annually, there are 1.5-2.7 million deaths and 300-500 million clinical infections (WHO, 1998). Several antimalarial agents (such as chloroquine, quinine, pyrimethamine, cycloguanil, sulphadoxine and others) have lost their effectiveness against this disease through drug resistance being developed by the malarial parasites (The- Wellcome-Trust, 1999). Although there is no hard-core evidence of drug resistance shown on the new antimalarial compounds (artemisinin and artesunate), induced resistant studies in animal models have demonstrated that the malarial parasites have capabilities to develop resistance to these compounds (Ittarat et al., 2003; Meshnick, 1998; Meshnick, 2002; Walker et al., 2000). Furthermore, a useful vaccine has yet to be developed due to the complicated life cycle of the malarial parasites (The- Wellcome-Trust, 1999). As such, the re-emergence of this deadly infectious disease has caused an urgent awareness to constantly look for novel targets and compounds. In this present study, Plasmodium falciparum (clone 3D7) was cultured in vitro in human red blood cells for extraction of total RNA which was later reverse transcribed into cDNA. The áI-, áII- and â-tubulin genes of the parasite were then successfully amplified and cloned into a bacterial protein expression vector, pGEX- 6P-1. The tubulin genes were then sequenced and analysed by comparison with previously published homologues. It was found that the sequenced gene of áItubulin was different at twelve bases, of which only six of these had resulted in changes in amino acid residues. áII- and â-tubulin genes demonstrated 100% sequence similarity with the published sequences of clone 3D7, but differences were observed between this clone and other strains (strains NF54 & 7G8) of â-tubulin. Nevertheless, the differences were minor in áI- and â-tubulins and there was greater than 99% homology. Subsequently, all three Plasmodium recombinant tubulin proteins were separately expressed and purified. Insoluble aggregates (inclusion bodies) of these recombinant tubulins were also refolded and have been tested positive for their structural characteristics in Western blot analysis. Both soluble and refolded recombinant tubulins of malaria were examined in a drugtubulin interaction study using sulfhydryl reactivity and fluorescence quenching techniques. Known tubulin inhibitors (colchicine, tubulozole-c and vinblastine) and novel synthetic compounds (CCWA-110, 239 and 443) were used as the drug compounds to determine the dynamics and kinetics of the interactions. In addition, mammalian tubulin was also used to determine the potential toxicity effects of these compounds. Similarities were observed with other published reports in the binding of colchicine with the recombinant tubulins, hence confirming proposed binding sites of this compound on the Plasmodium recombinant tubulins. Two synthetic compounds (CCWA-239 and 443) that have previously tested positive against P. falciparum in vitro were found to bind effectively with all three tubulin monomers, while displaying low binding interactions with the mammalian tubulin, thus indicating that these compounds have potential antimalarial activity. Therefore, this study has satisfied and fulfilled all the aims and hypotheses that have previously been stated.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 99-100).