Malaria is endemic in 106 countries worldwide. This disease is caused by a parasite from the genus Plasmodium. Of the five species that infect humans, Plasmodium falciparum is the most virulent, with over three billion people at risk and around 660 000 deaths reported in 2011. Of these deaths, 91% were in the African region, while 86% were children under the age of five. In light of the widespread development of resistance by malaria parasites against the classic antimalarial drugs, such as chloroquine (CQ) and now the established tolerance towards the widely used artemisinins, an immense need exists for identifying and developing new and effective antiplasmodial drugs. In search for such new antimalarial drugs, three chalcone based series of compounds were prepared and investigated during this study. The first series (Chapter 3) comprised 4-aminoquinolinyl-chalcone amides, which were synthesized through amidation of carboxylic acid-functionalised chalcone with aminoquinolines, using 1,1'-carbonyldiimidazole (CDI) as coupling agent. These compounds were screened alongside CQ against the CQ sensitive (3D7) and CQ resistant (W2) strains of P. falciparum. Cytotoxicity was assessed against the WI-38 cell line. The amide, featuring the 1,6-diaminohexane linker, was found the most active of all these new novel compounds tested. It was found to be as potent as CQ against 3D7, while displaying a two-fold higher activity than CQ against the W2 strain, coupled with good selective antimalarial activity (SI = 435) towards the parasitic cells. The second series (Chapter 4) consisted of aminoferrocenyl-chalcone amides, synthesized through condensation of a chalcone with an aminoferrocenyl. These compounds were screened against the 3D7, and antifolate- and CQ resistant (FCR3) strains of P. falciparum and cytotoxicity was determined against the WI-38 line. The most active compound of this series was the amide, containing the 1,2-diaminoethane linker, which showed 130- and 42 times less potency than CQ against the 3D7 and W2 strains, respectively. The third series of antimalarials (Chapter 5) involved dihydroartemisinyl-chalcone esters, synthesized through esterification of chalcones with DHA. These compounds were screened against 3D7 and W2 strains of P. falciparum, while the cytotoxicity was determined against the WI-38 line. Those esters featuring oxygenated aryl rings were three- to four-fold more potent than current clinically used artesunate against both P. falciparum strains. They were also screened in vitro against a panel of three cancer cell lines consisting of TK-10, UACC-62 and MCF-7. Thermogravimetric analysis revealed that the targeted hybrids were all thermally more stable than DHA as a result of the presence of the chalcone moiety in their structures. This could prove beneficial to the high temperature storage conditions that prevail in most malaria endemic countries. This study resulted in a number of compounds with varying antiplasmodial activity ranges. The compounds in series 3 were overall the most active, due to the incorporation of the highly active dihydroartemisinin pharmacophore. The chalcone moiety, especially, demonstrated a large scope for future development, owing to the ease of synthesis and the relatively low costs involved. The most active compounds of the three series could serve as potential lead compounds in the future development of more effective antimalarial drugs. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Malaria endemic areas not only pose a public health threat, but affects 3.3 billion people worldwide. In 2011, estimated malaria related deaths amounted to 660 000 out of 219 million reported cases, with 81% of these and 91% of malaria related mortality occurred in the African region. Those most affected were pregnant women, children under the age of five and immunocompromised individuals. Malaria is the fifth deadliest disease worldwide and accounts for the second highest death rate in Africa, following HIV/Aids. To combat this parasitic infection of antiquity, the ideal malaria pharmacotherapy would be a cost effective and easily obtainable monotherapy. The malaria parasite, however, has an intrinsic ability to develop drug resistance through various mechanisms. Widespread resistance towards antimalarial drugs has rendered traditionally used drugs therapeutically ineffective, hence accentuating the efficacy of the artemisinins as first line treatment option for uncomplicated Plasmodium falciparum (P. falciparum). A devastating reality of the challenging battle against malaria is the confirmed prolonged parasitic clearance times of the artemisinins, despite adequate drug exposure, which emphasises the urgent need for identifying and developing new, effective and safe therapies. During this study, 9-aminoacridines and artemisinin-acridine hybrids were successfully synthesised through nucleophillic substitution and their chemical structures confirmed by means of nuclear magnetic resonance spectroscopy (NMR), high resolution mass spectroscopy (HRMS) and infrared spectroscopy (IR). The hybrid compounds were synthesised through microwave assisted radiation, by covalently linking the artemisinin- and amino-functionalised acridine pharmacophores by means of a liable aminoethyl ether chain. The target compounds were screened in vitro for antimalarial activity against both the chloroquine sensitive (NF54) and chloroquine resistant (Dd2) strains of P. falciparum. Their cytotoxicities were assessed against various mammalian cells of different origins, viz. the Chinese hamster ovarian cells (CHO) from animal origin, and from human origin, hepatocellular- (HepG2), neuroblastoma- (SH-SY5Y) and cervical cancer (HeLa) cells. The synthesised hybrids exhibited antimalarial activity against both Plasmodium strains. Compound 7, featuring an ethylenediamine moiety in the linker, was the most active hybrid, with 50% inhibitory concentration (IC50) values of 2.6 nM and 35.3 nM against the NF54 and Dd2 strains, respectively. It had gametocytocidal activity against the NF54 strain, comparable to dihydroartemisinin (DHA) and artesunate (AS) and it is significantly more potent than chloroquine (CQ), whilst possessing a resistance index value of 14, indicative of a significant loss of activity against the CQ resistant strain. Contrary, the promising hybrid 10, containing a 2-methylpiperazine linker, had gametocytocidal activity, comparable to CQ and was found to be six-fold more potent than CQ against the Dd2 strain, with a resistance index (RI) value of 2, whilst it further showed highly selective action towards the parasitic cells. Compound 10 was also found to possess anticancer activity against the HeLa cell line, comparable to DHA and AS, but fivefold higher than that of CQ, with the same levels of hepatotoxicity and neurotoxicity. The artemisinin-acridine hybrids displayed superior antimalarial activity, compared to the derived 9-aminoacridines against both the Plasmodium strains. They, however, did not have the ability to overcome resistance, reduce the toxicity of acridine, nor induce synergistic activity. The hybrids, indeed displayed promising anticancer activity against HeLa cells. It is anticipated that these compounds may stand as drug candidates for further investigation in the search for new anti-cervical cancer drugs, rather than as antimalarials. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Malaria is endemic in 106 countries worldwide. This disease is caused by a parasite from the genus Plasmodium. Of the five species that infect humans, Plasmodium falciparum is the most virulent, with over three billion people at risk and around 660 000 deaths reported in 2011. Of these deaths, 91% were in the African region, while 86% were children under the age of five. In light of the widespread development of resistance by malaria parasites against the classic antimalarial drugs, such as chloroquine (CQ) and now the established tolerance towards the widely used artemisinins, an immense need exists for identifying and developing new and effective antiplasmodial drugs. In search for such new antimalarial drugs, three chalcone based series of compounds were prepared and investigated during this study. The first series (Chapter 3) comprised 4-aminoquinolinyl-chalcone amides, which were synthesized through amidation of carboxylic acid-functionalised chalcone with aminoquinolines, using 1,1'-carbonyldiimidazole (CDI) as coupling agent. These compounds were screened alongside CQ against the CQ sensitive (3D7) and CQ resistant (W2) strains of P. falciparum. Cytotoxicity was assessed against the WI-38 cell line. The amide, featuring the 1,6-diaminohexane linker, was found the most active of all these new novel compounds tested. It was found to be as potent as CQ against 3D7, while displaying a two-fold higher activity than CQ against the W2 strain, coupled with good selective antimalarial activity (SI = 435) towards the parasitic cells. The second series (Chapter 4) consisted of aminoferrocenyl-chalcone amides, synthesized through condensation of a chalcone with an aminoferrocenyl. These compounds were screened against the 3D7, and antifolate- and CQ resistant (FCR3) strains of P. falciparum and cytotoxicity was determined against the WI-38 line. The most active compound of this series was the amide, containing the 1,2-diaminoethane linker, which showed 130- and 42 times less potency than CQ against the 3D7 and W2 strains, respectively. The third series of antimalarials (Chapter 5) involved dihydroartemisinyl-chalcone esters, synthesized through esterification of chalcones with DHA. These compounds were screened against 3D7 and W2 strains of P. falciparum, while the cytotoxicity was determined against the WI-38 line. Those esters featuring oxygenated aryl rings were three- to four-fold more potent than current clinically used artesunate against both P. falciparum strains. They were also screened in vitro against a panel of three cancer cell lines consisting of TK-10, UACC-62 and MCF-7. Thermogravimetric analysis revealed that the targeted hybrids were all thermally more stable than DHA as a result of the presence of the chalcone moiety in their structures. This could prove beneficial to the high temperature storage conditions that prevail in most malaria endemic countries. This study resulted in a number of compounds with varying antiplasmodial activity ranges. The compounds in series 3 were overall the most active, due to the incorporation of the highly active dihydroartemisinin pharmacophore. The chalcone moiety, especially, demonstrated a large scope for future development, owing to the ease of synthesis and the relatively low costs involved. The most active compounds of the three series could serve as potential lead compounds in the future development of more effective antimalarial drugs. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

This thesis investigates antigenic variation and its evolution in Plasmodium falciparum, the cause of the most deadly form of human malaria. Antigenic variation is a strategy for evading immunity by switching between antigenic variants during infection. In P. falciparum, such variable antigens confer different binding phenotypes that may affect parasite survival and have also been linked to pathology. Here, a new statistical method is described for determining the switching patterns that underlie antigenic variation. This method is then applied to experimental data to yield a full description of an antigenic switching network in P. falciparum. In light of the findings, theoretical modelling is used to show how immune selection and binding phenotypes may have contributed to the evolution of antigenic repertoire structure, expression order and virulence. Related models are also used to investigate parasite population diversity, providing possible explanations for observations reported here and elsewhere, with implications for vaccine design. Together, these chapters advance understanding of P. falciparum immune evasion and how it relates to pathology. This work further reinforces the role of host immunity in shaping pathogen population diversity at multiple levels.

Malaria is a leading cause of death in some developing countries. The malaria parasite has been around for over a century, and has coevolved with humans. Coming up with an effective vaccine for P. falciparum will save millions of lives and reduce the morbidity and mortality of malaria globally. Understanding the role of exported parasite proteins i.e PfEMP1 a virulence factor and major cause of malarial pathogenesis, has been of great interest to vaccine researchers in the last decade. The focus of this review is to provide a literature review on PfEMP1s, their interaction with the human immune system, and their role in helping P. falciparum parasite to evade the immune system. This review will primarily focus on the intra-erythrocytic stage, which is the stage that results in the symptoms of malaria. A review is necessary to understand the antigenic variation of PfEMP1s, and how PfEMP1s challenge the different arms of the immune response, both the innate and adaptive. This review is unique in touching on the major parts of the immune system's interaction with the PfEMP1 antigen. Furthermore, the review explores the discussion of future research and therapeutic opportunities based on our knowledge of PfEMP1 antigens.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Dichloro-diphenyl-trichloroethane (DDT) was extensively used in agriculture pest control and is still used for indoor residual spraying to control malaria. The lipophylicity of DDT and its breakdown product dichloro-diphenyl-dichloroethylene (DDE) dictates that they associate with membranes, lipids and hydrophobic proteins in the biological environment. Their poor degradable nature causes DDT and DDE to persist for decades in the environment and in individuals who are or were in contact with the pesticide. In many countries the synchronised resistance of the mosquito vector to insecticides and the malaria parasite towards antimalarial drugs led to a drastic rise in malaria cases and to malaria epidemics. This study assesses the influence of low level exposure of DDT and DDE on chloroquine (CQ) resistance of the dire human malaria parasite, Plasmodium falciparum. The in vitro activity of p,p’-DDT and p,p’-DDE towards blood stages of chloroquine sensitive (CQS) P. falciparum D10 and chloroquine resistant (CQR) P. falciparum Dd2 was determined using two complementary in vitro assays (Malstat and SYBR Green 1). The 50% inhibition concentrations (IC50s) of p,p’-DDT and p,p’-DDE were found to be ±14 to 38 μM (5-12 μg/mL) and highly similar towards CQS and CQR P. falciparum strains. This result indicated that the proteins involved in CQ resistance have no effect on the activity of the insecticide DDT and it breakdown product DDE. In order to assess the influence of DDT and DDE on CQ activity, in vitro fixed ratio drug combination assays were performed, as well as isobologram analysis. We found that CQ works in synergy with p,p’-DDT and p,p’-DDE against CQS P. falciparum D10. However, both p,p’-DDT and p,p’-DDE were antagonistic toward CQ activity in CQR P. falciparum Dd2. This indicated that p,p’-DDT and p,p’-DDE do have an effect on CQ resistance or on the action of CQ via a target other than hemozoin polymerization. The observation of reciprocal synergism of p,p’-DDT and p,p’-DDE with CQ against CQS D10 and antagonism against CQR Dd2 strain is highly significant and strongly indicates selection of CQ resistant strains in the presence of p,p’-DDT and p,p’-DDE. People who have low levels of circulating DDE and/or DDT could be at a high risk of contracting CQR malaria. However, medium term (nine days) DDE exposure of CQS P. falciparum D10 did not induce resistance, as no significant change in activity of CQ, p,p’-DDT and p,p’-DDE towards blood stages the CQS strain was observed. This exposure was, however, shorter than expected for a malaria infection and would be addressed in future studies. From our results on the interaction of CQ with p,p’-DDT and p,p’-DDE, it was important to assess the residual DDT and DDE variable and how much of residual p,p’-DDT and/or p,p’- DDE would enter into or remain in the different compartments (the RPMI media, erythrocytes and infected erythrocytes) over time. In combination with liquid-liquid extraction, we developed a sensitive GC-MS analyses method and a novel HPLC-UV analysis method for measuring DDT and DDE levels in malaria culturing blood and media. Whilst the HPLC-UV method was relatively cheaper, faster, and effective in determining high DDT and DDE concentrations, the optimised GC-MS method proved to be effective in detecting levels as low as 78 pg/mL (ppt) DDE and 7.8 ng/mL (ppb) DDT in biological media. Using both the HPLC and GC-MS methods we observed that malaria parasites influence distribution of the compounds between the erythrocytic and media fractions. P. falciparum D10 infection at ±10% parasitemia lead to must faster equilibration (less than 8 hours) between compartments. Equimolar distribution of p,p’-DDE was observed, but the parasites lead to trapping of the largest fraction of p,p’-DDT in the erythrocyte compartment. These results indicate that a substantial amount would reach the intra-erythrocytic parasite and could influence the parasite directly, possibly leading to either synergistic or antagonistic drug interactions. This study is the first to illustrate the “good and bad” of the insecticide DDT in terms of CQ resistance and sensitivity toward the human malaria parasite P. falciparum. These results will hopefully have an important influence on how future policies on malaria control and treatment particularly in endemic areas will be addressed and could also have an impact on the anti-malarial drug discovery approach. / AFRIKAANSE OPSOMMING: Dichlorodifenieltrichloroetaan (DDT) is op groot skaal in landbouplaagbeheer gebruik en word nog steeds gebruik vir binnenshuise oppervlakbespuiting om malaria te beheer. Die lipofilisiteit van DDT en sy afbraakproduk dichlorodifenieldichloroetileen (DDE) dikteer dat hulle met membrane, lipiede en hidrofobiese proteïene in die biologiese omgewing assosieer. Stadige afbraak veroorsaak dat DDT en DDE vir dekades in die omgewing agterbly, asook in individue wat in kontak is, of was met die insekdoder. In baie lande het gesinkroniseerde weerstand van die muskietvektor teenoor insekdoders en die malariaparasiet teenoor antimalariamiddels gelei tot 'n drastiese styging in malariagevalle en tot malariaepidemies. In hierdie studie word die invloed van lae vlak blootstelling van DDT en DDE op chlorokien (CQ) weerstand van die mens malariaparasiet, Plasmodium falciparum, geëvalueer. Die in vitro aktiwiteit van p,p'-DDT en p,p'-DDE teenoor die bloedstadia van chlorokiensensitiewe (CQS) P. falciparum D10 en chlorokien-weerstandbiedende (CQW) P. falciparum Dd2 is bepaal deur gebruik te maak van twee komplementêre in vitro toetse (Malstat en SYBR Groen toetse). Die 50% inhibisie konsentrasies (IC50s) van p,p'-DDT en p,p'-DDE is bepaal as ±14 to 38 μM (5-12 μg/mL) en was hoogs vergelykbaar tussen CQS en CQW P. falciparum stamme. Hierdie resultaat het aangedui dat die proteïene betrokke by CQ weerstand geen effek op die aktiwiteit van die insekdoder DDT en die afbraakproduk DDE het nie. Om die invloed van DDT en DDE op CQ aktiwiteit te evalueer, is die aktiwiteit van kombinasies van die verbindings in vaste verhoudings getoets, tesame met isobologram ontleding. Ons het gevind dat CQ sinergisties saam met p, p'-DDT en p, p'-DDE teen CQS P. falciparum D10 werk. Daarteenoor het beide p, p'-DDT en p, p'-DDE antagonistiese werking getoon teenoor CQ aktiwiteit met CQW P. falciparum Dd2 as teiken. Dit het aangedui dat p,p'-DDT en p, p'-DDE wel 'n invloed op CQ weerstand het of ‘n aktiwiteit van CQ, anders as hemozoin polimerisasie, beïnvloed. Die waarneming van resiproke sinergisme en antagonisme van p, p'-DDT en p, p'-DDE in kombinasie met CQ teenoor die CQS D10 en CQW DD2 stamme respektiewelik, is hoogs betekenisvol en dui op seleksie van CQweerstandige stamme in die teenwoordigheid van p, p'- DDT en p, p'-DDE. Mense wat lae vlakke van sirkulerende DDE/DDT het, het dus 'n hoër risiko om CQW malaria te kry. Verder is gevind dat medium termyn (nege dae) DDE blootstelling van CQS P. falciparum D10 nie weerstand nie veroorsaak nie, want geen beduidende verandering in die aktiwiteit van CQ, p,p'-DDT en p,p'-DDE teenoor die bloed stadiums van die CQS stam is waargeneem nie. Hierdie blootstelling is egter korter as in 'n malaria-infeksie en sal verder bestudeer word in toekomstige studies. Vanuit die interaksie resultate van CQ met p, p'-DDT en p, p'-DDE was dit belangrik om die residuele DDT en DDE veranderlike te evalueer, asook die distribusie van p,p'-DDT en p,p'- DDE tussen die verskillende kompartemente (die kultuurmedium, eritrosiete en geïnfekteerde rooibloedselle) oor verloop van tyd. In kombinasie met vloeistof-vloeistof ekstraksie, het ons 'n sensitiewe GC-MS en nuwe HPLC-UV analisemetode ontwikkel vir die meet van DDT en DDE-vlakke in bloed (normale en geïnfekteerde eritrosiete) en die kultuurmedium. Terwyl die HPLC-UV metode relatief goedkoper, vinniger en effektief in die bepaling van hoë DDT en DDE-konsentrasies is, was die geoptimaliseerde GC-MS metode doeltreffend in die opsporing van vlakke so laag as 78 pg/mL (dpt) DDE en 7.8 ng/mL (dpb) DDT in biologiese media. Met behulp van beide die HPLC-UV en GC-MS metodes is waargeneem dat die malariaparasiet die ekwilibrasie van die verbindings tussen die eritrosiet- en media kompartemente beïnvloed. P. falciparum D10 infeksie met ± 10% parasitemia lei tot vinniger ekwilibrasie (minder as 8 uur) tussen die kompartemente. Ekwimolêre verspreiding van p,p'- DDE is waargeneem, maar die parasiete het die grooste fraksie van p,p'-DDT in die eritrosiet kompartement vasgevang. Hierdie resultate wys dat 'n aansienlike fraksie die intraeritrositiese parasiet kan bereik en sodoende die parasiet direk kan beïnvloed en moontlik kan lei tot sinergistiese of antagonistiese middel interaksies. Hierdie studie is die eerste om die "goed en sleg" van die insekdoder DDT in terme van CQ weerstand en sensitiwiteit teenoor die menslike malariaparasiet P. falciparum te illustreer. Hierdie resultate sal hopelik 'n belangrike invloed hê op die toekomstige beleid oor die beheer van malaria en behandeling, veral in endemiese gebiede, en mag ook 'n impak hê op die antimalariamiddel navorsing.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In Africa alone, Plasmodium, the causative agent of malaria is estimated to kill a child, under the age of five every thirty seconds140. The ability of the parasite to rapidly attain resistance, has resulted in immunity of the parasite to all, except one group of frontline drugs. The need to develop novel drugs, vaccines and prevention strategies that are accessible and affordable for third world countries is of the utmost importance to prevent needless human suffering and death. The glycolytic pathway is an attractive drug target since it is the principal source of ATP for the parasite. Many of the glycolytic enzymes have been studied and proposed as drug targets, but the importance of these enzymes for the function of the pathway as a whole has not been considered. It is known, from the frameworks of metabolic control analysis, that control of the flux and metabolite concentration can be divided among the individual steps. Differential control analysis of Plasmodium and erythrocyte glycolysis may reveal potential drug targets. These analyses require a detailed kinetic model of Plasmodium glycolysis, and the feasibility of constructing and validating such a model was the aim of this study. In this work we determined the feasibility of constructing and validating a detailed kinetic model for the Plasmodium falciparum glycolytic pathway. Whether the construction and validation of this kinetic model was feasible or not was decided on the basis of the ability to: i) culture and isolate sufficient asexual parasites for enzymatic and steady state assays , ii) obtain kinetic parameters such as Km and Vmax for each glycolytic enzyme, either from literature or experimentally, iii) measure glycolytic fluxes, iv) determine glycolytic intermediate concentrations, v) construct a kinetic model from the kinetic parameters and vi) validate it with steady state glycolytic fluxes and metabolite concentrations Each of the above criteria were successfully addressed. In summary, the kinetic parameters and glycolytic fluxes that were measured experimentally, were used to construct and partially validate a detailed kinetic model, respectively. Further validation of the model by means of steady state metabolite concentrations was shown to be possible with the development of a suitable protocol to measure the glycolytic intermediate concentrations. The model presented in this work may play an important role in drug target identification and improving the current understanding of host-parasite interactions and glycolytic regulation. / AFRIKAANSE OPSOMMING: Plasmodium, die parasiet wat malaria veroorsaak, is in Afrika alleen elke dertig sekondes verantwoordelik vir die afsterwe van ’n kind jonger as vyf jaar. Die parasiet se vermoë om vinnig weerstand op te bou het daartoe gelei dat Plasmodium weerstandbiedend is teen byna alle nuwe teen-malaria middels, behalwe vir ’n enkele toonaangewende groep. Die ontwikkeling van nuwe malaria teen-middels is van uiterste belang om lyding te voorkom. ’n Goeie teiken vir teen-malaria middels is die glikolitiese padweg omdat die metaboliese padweg essensieël is vir die produksie van ATP, die energiebron van die parasiet. Desondanks die feit dat meeste van die glikolitiese ensieme al goed bestudeer en as teiken voorgestel is, is dit steeds onduidelik hoe hierdie ensieme saam funksioneer om die metaboliese weg, as geheel, tot stand te bring. Metaboliese kontrole analise het aangetoon dat die glikolitiese beheer verdeel is tussen die onderskeie glikolitiese ensieme, m.a.w. geen enkele ensiematiese stap het volledige beheer oor die fluksie van die glikolitiese padweg nie. Die afsonderlike analise en vergelyking van Plasmodium - en rooibloedselglikolise met behulp van differensiële metaboliese kontrole analise sal moontlik gebruik kan word om gasheervriendelike teikens vir nuwe middels aan te toon. So ’n analise benodig ’n omvattende kinetiese model van Plasmodium glikolise. Derhalwe was die doel van hierdie studie om vas te stel hoe uitvoerbaar dit is om ’n kinetiese model van Plasmodium glikolise te konstrueer en te valideer. Die uitvoerbaarheid van die konstruksie en validering van die kinetiese model was geasseseer op grond van die vermoë om: i) parasietkulture te kweek en genoegsame parasiete, wat in die aseksuele fase is, te isoleer sodat ensiembepalings en bestendige toestand-bepalings gedoen kan word, ii) kinetiese parameters soos Km - en Vmax-waardes vir elke glikolitiese ensiem, hetsy vanuit literatuur of eksperimentele werk, te verkry, iii) glikolitiese fluksie te meet, iv) glikolitiese intermediaatkonsentrasies te bepaal, v) ’n kinetiese model van die bepaalde kinetiese parameters op te stel en vi) die model te valideer met glikolitiese flukswaardes en metaboliet- konsentrasies wat in die bestendige toestand verkry is. Elk van die bogenoemde kriteria was met sukses in hierdie studie aangespreek. Ter opsomming, die eksperimenteel bepaalde kinetiese parameters en glikolietiese flukswaardes was gebruik om onderskeidelik ’n gedetaileerde kinetiese model te konstrueer en gedeeltelik te valideer. Daar was getoon dat verdere validering van die model deur middel van bestendige toestand metabolietkonsentrasies moontlik is met die ontwikkeling van ’n geskikte protokol om glikolitiese intermediaatkonsentrasies te meet. Die model, soos opgestel in hierdie studie, kan moontlik ’n belangrike rol speel om teikens vir nuwe malaria teen-middels te identifiseer en om gasheer-parasiet interaksies en glikolitiese regulering beter te verstaan.

<p>The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo / genease&rdquo / activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001 / van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.</p> <p><br /> Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding /intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons.</p> <p><br /> Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.</p>

Intermittent fevers caused by Plasmodium parasites have been known for millennia, and have caused untold human suffering. Today, millions of people are afflicted by malaria each year, and hundreds of thousands die. Historically, the most successful synthetic antimalarial drug was chloroquine, as it was safe, inexpensive, and highly efficacious. However, plasmodial resistance to chloroquine now greatly limits its utility. Previously in our laboratories it has been shown that attachment of a "reversal agent moiety" to the side chain of chloroquine can result in the restoration of activity against chloroquine-resistant strains of P. falciparum malaria. In the first part of the work presented here, a study has been made of the importance of the quinoline ring substitution pattern to the activity of such reversed chloroquines. The compounds presented here include those bearing a substituent in the 2-, 5, 6-, 7-, and/or 8- position, and include those with chloro, bromo, iodo, fluoro, nitro, trifluoromethyl, methyl, and methoxy substituents. For reversed chloroquines, 2-, 5-, and 8- substituents have been found to decrease in vitro antiplasmodial activity against P. falciparum relative to 7-chloro substitution, whereas 6- and 7- substituted compounds with various substituents have in many cases similar activity to that of 7-chloro substituted compounds. Little difference has been observed between 6- and 7- substitution, or between chlorine and a methyl group in position 6. In most cases these effects on activity are directionally similar to those observed for chloroquine analogs without an attached reversal agent, but the magnitude of the effect is generally smaller, suggesting that the activities of reversed chloroquines are less affected by modifications to the quinoline ring system than is true for chloroquine analogs without an attached reversal agent. The second portion of this work presents an asymmetrical bis-quinoline (PL241) that is highly active against P. falciparum malaria, with an IC50 of less than 0.1 nM for all strains tested. Mechanistic studies have been performed in which the substitution patterns of the two quinoline rings of PL241 are modified in ways that indicate that either ring system is equally capable of participating in the antimalarial activity of these compounds. The excellent in vitro antiplasmodial activity of PL241 makes this a compound of great interest for further development as a potential antimalarial drug. In the third part of this work, a survey has been made of antimalarial treatments recommended in the European medical literature from the time of Pliny the Elder (active in the first century A.D.) through the advent of modern malaria chemotherapy in the early twentieth century. In the fifteen primary sources utilized in this study, 251 distinct substances - primarily plants - were identified as having likely been used in the treatment of malaria. Of the 38 substances that were described in three or more sources, at least fifteen have been examined by other workers for antiplasmodial activity; in many cases, they were found to have antiplasmodial activity in vitro or in vivo. However, the majority of the phytotherapies for malaria identified in this project have not yet been tested against Plasmodium species, and may provide valuable leads in the search for new compounds active against drug-resistant malaria.

The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.