Plasmodium falciparum is the causative agent of malaria, one of the world‟s most prevalent infectious diseases. The emergence of strains resistant to current therapeutics creates the urgent need to identify new classes of antimalarials. Here we present and analyse a constraints-based model (iMPMP427) of P. falciparum metabolism. Consisting of 427 genes, 513 reactions, 457 metabolites, and 5 intracellular compartments, iMPMP427 is relatively streamlined and contains an abundance of transport reactions consistent with P. falciparum’s observed reliance on host nutrients. Flux Balance Analysis simulations reveal the model to be predictive in regards to nutrient transport requirements, amino acid efflux characteristics, and glycolytic flux calculation, which are validated by a wealth of experimental data. Furthermore, enzymes deemed to be essential for parasitic growth by iMPMP427 lend support to several previously computationally hypothesized metabolic drug targets, while discrepancies between essential enzymes and experimentally annotated drug targets highlight areas of malarial metabolism that could benefit from further research.

Plasmodium falciparum is the causative agent of malaria, one of the world‟s most prevalent infectious diseases. The emergence of strains resistant to current therapeutics creates the urgent need to identify new classes of antimalarials. Here we present and analyse a constraints-based model (iMPMP427) of P. falciparum metabolism. Consisting of 427 genes, 513 reactions, 457 metabolites, and 5 intracellular compartments, iMPMP427 is relatively streamlined and contains an abundance of transport reactions consistent with P. falciparum’s observed reliance on host nutrients. Flux Balance Analysis simulations reveal the model to be predictive in regards to nutrient transport requirements, amino acid efflux characteristics, and glycolytic flux calculation, which are validated by a wealth of experimental data. Furthermore, enzymes deemed to be essential for parasitic growth by iMPMP427 lend support to several previously computationally hypothesized metabolic drug targets, while discrepancies between essential enzymes and experimentally annotated drug targets highlight areas of malarial metabolism that could benefit from further research.

Thèse de doctorat : Sciences du vivant. Aspects moléculaires et cellulaires de la biologie : Strasbourg 1 : 2008. Thèse de doctorat : Sciences du vivant. Aspects moléculaires et cellulaires de la biologie : Université d'Abomey-calavi : 2008. / Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. p. 227-252.

Thesis (M.S.)--Florida State University, 2004. / Advisor: Dr. Dean Falk, Florida State University, College of Arts and Sciences, Dept. of Anthropology. Title and description from dissertation home page (viewed June 21, 2004). Includes bibliographical references.

Malaria is a global health concern accounting for approximately 219 million cases and an estimated 660 000 deaths in 2010. The most fatal strain of malarial parasite, Plasmodium falciparum is found to contain 3 Adenylate Kinases (PfAK1, PfAK2 and PfGAK). Adenylate Kinases are important enzymes that essentially catalyze and regulate energy metabolism processes. PfAK1 and PfAK2 catalyze the reversible MG2+ reaction ATP + AMP ←→ 2ADP whereas, the PfGAK catalyzes the Mg2+ dependent reaction GTP+AMP ←→ ADP+GDP. Of all malarial strains, only the Plasmodium falciparum Adenylate Kinase 2 (PfAK2) was found to contain a N-myristoylation sequence and subsequently formed a stable heterodimer with Plasmodium falciparum N-myristoyl transferase (PfNMT). The myristoylation of PfAK2 by PfNMT is believed to help transport PfAK2 to the parasitophorous vacuole membrane (PVM) so that the enzyme can perform its essential functions. With these enzymes being key components in the parasite’s survival, the structural study of these enzymes would provide a lot of insight into targeting these proteins for drug design that would effectively kill the parasite without affecting the human host. In this study, PfAK1 was able to be expressed, purified and crystallized with a dataset collected at 4.3Å. PfGAK was expressed and purified. A GTP analogue called GP5A was used to soak the purified PfGAKand the PfGAK bound to GP5A was crystallized and diffracted. Moreover, PfAK2 and PfNMT was successfully expressed and co-purified. The purified PfAK2-PfNMT heterodimer are undergoing crystal screening for possible crystallization conditions. / published_or_final_version / Physiology / Master / Master of Philosophy

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.