Malaria is known to be endemic throughout Zambia and continues to be a major public health problem. Eighty three percent (83 %) of Zambian population is reported to be living in malaria high risk areas and the remaining 17 percent is reported living in malaria low risk areas. Zambia has a 16 percent malaria national prevalence. Malaria peak transmission periods are usually during the rainy season ? from November to April and the main transmitting vectors are anopheles species; funestus, gambiae and arabiensis. Plasmodium falciparum is the main transmitting parasite accounting for about 98 percent of all malaria infections in the country. Other species of plasmodium that can be found in Zambia are ovale and malariae. The main intervention measures used in the prevention and control of malaria in Zambia are; Indoor Residual spray (IRS), mass distribution of insecticide treated mosquito nets (ITNs) and Intermittent Prevention Treatment (IPT). The main aim of this thesis is to analyze the efficiency and effectiveness of the the current intervention measures used to prevent and control malaria in Zambia. I approached this issue from time the ?roll back malaria ? project was intiated in 1998 up to 2011. With help of the changes introduced to the health sector under the sector wide approach reforms in 1995, the public health approach in the fight against malaria was even much widened. The second aim of my thesis is to offer acceptable alternative interventions that can used in the prevention and control of malaria. In this qualitative type of research, I chose to use secondary analyzation of data as my research method. I obtained much of the information from studying health literature, journals, laws and other online publications, which I found to be relevant to the topic at hand. I also consulted with the Zambian ministry of health through provincial health offices as well as via district health offices. A series of three open research questions was used as a guide to obtaining the much needed data. However, the results revealed that the current intervention measures used in the prevention and control of malaria are efficient and effective. In 2009, Zambia recorded a 66 percent reduction in deaths due to malaria. This success recorded clearly surpasses the target set by the ?roll back malaria? 2006 which was aimed at reducing mortality due to malaria by 50 percent by year 2010. However, parasitemia results still show great variation in prevalence between urban areas and rural. This inequality is also evident in the access to health care as well. There is much need to scale up on intervention measures if a Zambia without malaria is to be achieved. This work can as used as a public health tool in the prevention of malaria in Zambia and also as a road map to future research concerning malaria and public health.

MSc (Biochemistry) / Department of Biochemistry / Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1. / NRF

MENVSC (Geography) / Department of Geography and Geo-Information Sciences / Semi-arid regions of Africa are prone to epidemics of malaria. Epidemic malaria occurs along the geographical margins of endemic regions, when the equilibrium between the human, parasite and mosquito vector populations are occasionally disturbed by changes in one or more meteorological factors and a sharp but temporary increase in disease incidence results. Monthly rainfall and temperature data from the South African Weather Service and malaria incidence data from Department of Health were used to determine the influence of meteorological variables on malaria transmission in Limpopo from 1998-2014. Meteorological influences on malaria transmission were analyzed using time series analysis techniques. Climate suitability for malaria transmission was determined using MARA distribution model. There are three distinct modes of rainfall variability over Limpopo which can be associated with land falling tropical cyclones, cloud bands and intensity of the Botswana upper high. ENSO and ENSO-Modoki explains about 58% of this variability. Malaria epidemics were identified using a standardized index, where cases greater than two standard deviations from the mean are identified as epidemics. Significant positive correlations between meteorological variables and monthly malaria incidence is observed at least one month lag time, except for rainfall which shows positive correlation at three months lag time. Malaria transmission appears to be strongly influenced by minimum temperature and relative humidity (R = 0.52, p<0.001). A SARIMA (2, 1, 2) (1, 0, 0)12 model fitted with only malaria cases has prediction performance of about 53%. Warm SSTs of the SWIO and Benguela Niño region west of Angola are the dominant predictors of malaria epidemics in Limpopo in the absence of La Niña. Warm SSTs over the equatorial Atlantic and Benguela Niño region results in the relaxation of the St. Helena high thus shifting the rainy weather to south-east Africa. La Niña have been linked with increased malaria cases in south-east Africa. During El Niño when rain bearing systems have migrated east of Madagascar ridging of the St. Helena high may produce conducive conditions for malaria transmission. Anomalously warmer and moist winters preceding the malaria transmission season are likely to allow for high mosquito survival and the availability of the breeding sites thus high population in the beginning of the transmission season hence resulting in increased epidemics. / NRF

Introduction Le Québec accueille 50 000 immigrants annuellement dont 30 % viennent d’Afrique et 27 % d’Asie. Dans les dix dernières années, 200 à 600 enfants ont été adoptés annuellement, majoritairement de l’Asie. Plusieurs régions d’origine des immigrants sont endémiques pour des parasites. C’est pourquoi l’immigration influence fortement l’épidémiologie de ces maladies. La giardiase, la filariose, la schistosomiase, la strongyloïdose et la malaria sont les plus prévalentes. Il existe peu de données sur les maladies parasitaires chez les immigrants au Québec. Cette étude vise à décrire l’épidémiologie de cette problématique dans la province. Méthode Une étude descriptive transversale a été privilégiée. Les immigrants ayant consulté au centre des maladies tropicales de l’Université McGill et les enfants de la clinique d’adoption de l’hôpital Maisonneuve-Rosemont ont été ciblés. Une grille standardisée a été utilisée pour la collecte des données dans la base de données de GeoSentinel et les dossiers médicaux. Des analyses descriptives et inférentielles fournissent un profil des immigrants atteints et des facteurs associés. Résultats Au moins un parasite se trouve chez 32 % (202/638) des immigrants à l’étude. Les parasitoses les plus prévalentes chez ces cas sont la strongyloïdose (28%), la filariose (21%), la schistosomiase (17%), et la malaria (12%). Le tiers des cas sont des demandeurs d’asile et 43 % (86/202) viennent de l’Afrique subsaharienne. Une proportion élevée d’infection, 50 % (27/56), se trouve chez les Haïtiens. Chez 15 % (73/499) des enfants adoptés, on trouve au moins un parasite; la giardiase et la strongyloïdose sont les plus prévalentes. La majorité (292/499) des enfants à l’étude viennent de la Chine et 69 % (22/32) des enfants Haïtiens sont infectés par des parasites. Discussion et conclusion La littérature appuie nos résultats. La population immigrante est en croissance au Québec ce qui est une des richesses de la province. Par contre, c’est une population qui est vulnérable au niveau de la santé. Environ 1/3 des immigrants et 1/6 des enfants adoptés sont atteints par des parasitoses. Plus de la moitié (57 %) des immigrants atteints sont infectés par des parasites qui ont des conséquences importantes. Des interventions ciblant cette population sont recommandées afin de diminuer ce fardeau.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The high sickle cell gene frequency has been hypothesised to be related to the protective advantage against malaria disease among heterozygous individuals. In this thesis, we study the interaction between the dynamics of malaria and sickle cell gene. The main aim is to investigate the impact of malaria treatment on the frequency of sickle cell gene. For this, we develop a mathematical model that describes the interactions between malaria and sickle cell gene under malaria treatment. The model includes both homozygous for the normal gene (AA) and heterozygous for sickle cell gene (AS) and assumes that AS individuals are not treated since they do not show clinical symptoms. We first analyse the model without malaria treatment, using singular perturbation techniques, basing on the fact that epidemiological and demographical dynamics occur on two different time scales (fast and slow dynamics). Our analysis on the fast time scale shows that high sickle cell gene frequency leads to high endemic levels for longer duration of parasitemia among heterozygous individuals. However, if the duration of parasitemia is reduced then high sickle cell gene frequency is associated with low endemic levels. We also note that on the slow time scale, the invasion ability of sickle cell gene is dependent on the malaria epidemiological parameters. The invasion coefficient given as the difference in the weighted death rates of AA and AS individuals is used as a measure to determine the establishment of sickle cell gene in the population. Results show that, the gene may establish itself if the weighted death rate of AA individuals is greater than that of AS individuals otherwise it fails. We note that, high mortality of AA individuals leads to establishment of sickle cell gene in the population. Then we analysed the model with treatment, our results indicate that the frequency of sickle cell gene decreases with an increase in the recovery rate of AA individuals. We thus conclude that eradication of malaria disease will lead to a reduction in sickle cell gene frequency. / AFRIKAANSE OPSOMMING: Daar word veronderstel dat die hoë sekelsel geenfrekwensie onder heterosigotiese individue verwant is aan die beskermende voordeel teen malaria siekte. In hierdie verhandeling ondersoek ons die wisselwerking tussen die dinamika van malaria en die sekelsel geen. Die hoofdoel is om die invloed van malaria behandeling op die frekwensie van die sekelsel geen te ondersoek. Hiervoor het ons ‘n wiskundige model ontwikkel, wat die wisselwerking tussen die dinamika van malaria en die sekelsel geen met malaria behandeling, beskryf. Die model sluit beide homosigotiese vir die normale geen (AA) en heterosigotiese vir die sekelsel geen (AS) in, en neem aan dat AS individue nie behandel is nie omdat hulle nie die eerste kliniese simptome getoon het nie. Ons ontleed eers die model sonder malaria behandeling, deur gebruik te maak van enkelvoudige pertubasie tegnieke, wat gegrond is op die feit dat epidemiologiese en demografiese dinamika plaasvind op twee verskillende tydskale (vinnige en stadige dinamika). Ons ontleding op die vinnige tydskaal dui dat hoë sekelsel geenfrekwensie onder heterosigotiese individue lei tot hoë endemiese vlakke vir ‘n langer duur van parasitemie. Nietemin, as die duur van parasitemie afneem, dan word hoë sekelsel geenfrekwensie verbind met lae endemiese vlakke. Ons neem ook waar dat op die stadige skaal die indringingsvermoë van die sekelsel afhanklik is van malaria se epidemiologiese parameters. Die indringingskoëffisiënt wat bereken word as die verskil van die geweegde sterftekoerse van AA en AS individue, word gebruik as ‘n maatstaf om die vestiging van die sekelsel geen in die bevolking te bepaal. Resultate toon dat die geen homself kan vestig as die geweegde sterftekoers van AA individue groter is as di e van die AS individue, andersins misluk dit. Ons let op dat hoë mortaliteit van AA individue lei tot die vestiging van die sekelsel geen in die bevolking. Daarna het ons die model wat behandeling insluit ge-analiseer en ons resultate toon dat die frekwensie van die sekelsel geen afneem met ‘n toename in die herstelkoers van AA individue. Ons kom dus tot die gevolgtrekking dat die uitwissing van malaria siekte sal lei tot die afname in sekelsel geenfrekwensie.

In this thesis we will consider two mathematical models on malaria transmission and patho- genesis. The transmission model is a human-mosquito interaction model that describes the development of malaria in a human population. It accounts for the various phases of the disease in humans and mosquitoes, together with treatment of both sick and partially im- mune humans. The partially immune humans (termed asymptomatic) have recovered from the worst of the symptoms, but can still transmit the disease. We will present a mathematical model consisting of a system of ordinary differential equations that describes the evolution of humans and mosquitoes in a range of malarial states. A new feature, in what turns out to be a key class, is the consideration of reinfected asymptomatic humans. The analysis will include establishment of the basic reproduction number, R0, and asymptotic analysis to draw out the major timescale of events in the process of malaria becoming non-endemic to endemic in a region following introduction of a few infected mosquitoes. We will study the model to ascertain possible time scale in which intervention programmes may yield better results. We will also show through our analysis of the model some evidence of disease control and possible eradication. The model on malaria pathogenesis describes the evolution of the disease in the human host. We model the effect of immune response on the interaction between malaria parasites and erythrocytes with a system of delay differential equations in which there is time lag between the advent of malaria merozoites in the blood and the training of adaptive immune cells. We will study the model to ascertain whether or not a single successful bite of an infected mosquito would result in death in the absence of innate and adaptive immune response. Stability analysis will be carried out on the parasite free state in both the immune and non immune cases. We will also do numerical simulations on the model to track the development of adaptive immunity and use asymptotic methods, assuming a small delay to study the evolution of the disease in a naive individual following the injection of small amount of merozoites into the blood stream. The effect of different levels of innate immune response to the pathogenesis of the disease will be considered in the simulations to elicit a possible immune level that can serve as a guide to producing a vaccine with high efficacy level.

Background: Challenges persist in ensuring access to and optimal use of long-lasting, insecticidal bed nets (LLINs). Factors associated with ownership and use may differ depending on the history of malaria and prevention control efforts in a specific region. Understanding how the cultural and social-environmental context of bed net use may differ between high- and low-risk regions is important when identifying solutions to improve uptake and appropriate use. Methods: Community forums and a household, cross-sectional survey were used to collect information on factors related to bed net ownership and use in western Kenya. Sites with disparate levels of transmission were selected, including an endemic lowland area, Miwani, and a highland epidemic-prone area, Kapkangani. Analysis of ownership was stratified by site. A combined site analysis was conducted to examine factors associated with use of all available bed nets. Logistic regression modelling was used to determine factors associated with ownership and use of owned bed nets. Results: Access to bed nets as the leading barrier to their use was identified in community forums and cross-sectional surveys. While disuse of available bed nets was discussed in the forums, it was a relatively rare occurrence in both sites. Factors associated with ownership varied by site. Education, perceived risk of malaria and knowledge of individuals who had died of malaria were associated with higher bed net ownership in the highlands, while in the lowlands individuals reporting it was easy to get a bed net were more likely to own one. A combined site analysis indicated that not using an available bed net was associated with the attitudes that taking malaria drugs is easier than using a bed net and that use of a bed net will not prevent malaria. In addition, individuals with an unused bed net in the household were more likely to indicate that bed nets are difficult to use, that purchased bed nets are better than freely distributed ones, and that bed nets should only be used during the rainy season. Conclusion: Variations in factors associated with ownership should be acknowledged when constructing messaging and distribution campaigns. Despite reports of bed nets being used for other purposes, those in the home were rarely unused in these communities. Disuse seemed to be related to beliefs that can be addressed through education programmes. As mass distributions continue to take place, additional research is needed to determine if factors associated with LLIN ownership and use change with increasing availability of LLIN.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Having caused the deaths of more than 10 million individuals since 2000 with most of them occurring in Africa, malaria remains a serious disease that requires undivided attention. To this end a detailed kinetic model of Plasmodium falciparum glycolysis was constructed, validated and used to determine potential drug targets for the development of novel, effective antimalarial therapies. The kinetic model described the behaviour of the glycolytic enzymes with a set of ordinary differential equations that was solved to obtain the steady state fluxes and concentrations of internal metabolites. The model included a glycerol branch represented in a single fitted equation. This present study set out to detect, characterise, and incorporate into the model the enzymes that constitute the glycerol branch of P. falciparum glycolysis. The kinetic parameters of glycerol 3-phosphate dehydrogenase (G3PDH), the first enzyme in the branch and catalyst of the dihydroxyacetone phosphosate (DHAP) reducing reaction, was determined and added to the detailed kinetic model. The model was subsequently validated by comparing its prediction of steady state fluxes with experimentally measured fluxes. Once it was evident that the predictions of the unfitted model agreed with experimentally measured fluxes, metabolic control analysis was performed on this branched system to ascertain the distribution of control over the steady state flux through the glycerol branch. The control G3PDH exercised over its own flux was less than expected due to the enzyme’s sensitivity to changes in NADH and thus the redox balance of the cell. Attempts were made to detect the enzymes responsible for the conversion of glycerol 3-phosphate (G3P) to glycerol. Very low levels of glycerol kinase activity was observed. Although G3P-dependent release of inorganic phosphate was detected results were inconclusive as to whether a non-specific phosphatase also mediated the conversion. Overall, the expansion of the model to include G3PDH did not affect the steady state metabolite concentrations and flux adversely. / AFRIKAANSE OPSOMMING: Vanaf die jaar 2000 het malaria die dood van meer as 10 miljoen mense veroorsaak. Die meeste sterftes het in Afrika voorgekom —’n aanduiding van hoe ernstige siekte dit is en een wat onverdeelde aandag moet geniet. Om hierdie rede is ’n gedetaileerde kinetiese model van glikoliese in Plasmodium falciparum gebou, gevalideer en gebruik om potensiële dwelm teikens te identifiseer vir die ontwikkeling van nuwe, meer effektiewe anti-malaria terapieë. Die kinetiese model beskryf die gedrag van die glikolitiese ensieme in terme van gewone differensiële vergelykings wat opgelos is om die bestendige toestand fluksies en interne metaboliet konsentrasies te bepaal. Die model sluit ’n gliserol-tak in wat deur ’n enkele aangepaste vergelyking verteenwoordig word. Hierdie studie het voorgeneem om die ensieme van die gliserol-tak van P. falciparum glikoliese te identifiseer, karakteriseer en in die model te inkorporeer. Ons het die kinetiese parameters van die eerste ensiem in die gliserol-tak, gliserol 3-fosfaat dehidrogenase (G3PDH), die katalis van die dihidroksiasetoon fosfaat(DHAP) reduserende reaksie, bepaal. Die kinetiese parameters is by die gedetaileerde model gevoeg. Validering het plaasgevind deur die model se voorspellings met eksperimenteel bepaalde waardes te vergelyk. Toe dit duidelik geword het dat die voorspellings van die model met die eksperimenteel bepaalde fluks ooreenstem, is metaboliese kontrole analiese op die vertakte sisteem uitgevoer. Dit is gedoen om vas te stel hoe die bestendige toestand fluks deur die gliserol-tak beheer word. G3PDH het nie volle beheer oor sy eie fluks nie, in teenstelling met ons vergewagtinge. Daar is gepoog om vas te stel watter ensieme verantwoordelik is vir die produksie van gliserol vanuit gliserol 3-fosfaat (G3P). ’n Lae gliserolkinase aktiwiteit is waargeneem. Alhoewel G3P afhanklike vrystelling van anorganise fosfaat waargeneem is, is dit nie duidelik vanuit die resultate of die proses deur ’n nie-spesifieke fosfatase uitgevoer word nie. Die uitbreiding van die model om ’n G3PDH vergelyking in te sluit het nie die bestendige toestand metaboliet konsentrasies en fluks negatief geaffekteer nie.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new antimicrobial drugs especially with the rise in resistance to almost all available drugs by public health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat to public health. One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research found that the six major Trcs were active against Listeria monocytogenes and Plasmodium falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide unit has an important role in activity. We set out to extend the qualitative structure to activity relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either naturally or by chemical synthesis produced and characterised in terms of chemical character and biological activity. The Trcs were significantly more active than RW peptides, although much more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an aromatic amino acid residue for selective activity towards the leucocin A resistant L. monocytogenes B73-MR1. However, structural properties favouring a tighter membrane interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There was also need for an amphipathic balance between hydrophobicity and size/steric parameters for optimal activity. From our QSAR studies we predict as lead peptide for a future library of antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport system or bacterial dehydrogenases. We confirmed, as previously found, that the Trcs have potent antimalarial activity that is sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in activity. From these results a proposed lead peptide for future research is cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages. This indicated that membrane structures containing neutral lipids, as well as chromatin may be targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic towards each other’s activity. This indicated a shared target and we propose the food vacuole as another of the Trc targets in P. falciparum. / AFRIKAANSE OPSOMMING: Antimikrobiese peptiede (AMPe) is tans die mees nagevorsde groep verbindings in die soeke na nuwe antimikrobiese middels, veral weens 'n toenemende weerstandigheid van patogene in die openbare gesondheidsektor teen alle beskikbare middels. Die doel van hierdie studie was om klein, sikliese AMPe in terme van hul aktiwiteit teenoor twee menslike patogene wat 'n bedreiging vir openbare gesondheid is, Listeria monocytogenes, 'n voedsel-oordraagbare patogeen wat listeriose veroorsaak, asook Plasmodium falciparum, die parasiet verantwoordelik vir malaria, te karakteriseer. Een van die klein, sikliese peptiedbiblioteke wat ondersoek is, is die tyrocidines (Trcs) en analoë (sikliese dekapeptiede [siklo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu]). Hierdie peptiede deur die Gram-positiewe bakterie Bacillus aneurinolyticus word wat nie-ribosomaal gesintetiseer as deel van die tirotrisien kompleks word tydens sporulasie. Vorige navorsing het gewys dat die ses hoof Trcs teen Listeria monocytogenes en Plasmodium falciparum aktief is en dat die identiteit van die aromatiese residue in die aromatiese dipeptiedeenheid 'n belangrike rol speel in die Trc-aktiwiteit. Ons het gepoog om die kwalitatiewe struktuur-aktiwiteit-verwantskap (QSAR) studies uit te brei deur meer Trc analoë en klein sintetiese Arg- en Trp-ryke sikliese peptiede (RW-peptiede) te gebruik en sodoende essensiële struktuur-motiewe en voorvereistes vir aktiwiteit vas te stel. Agt natuurlike en drie sintetiese Trc analoë, asook vyftien RW-peptiede is of deur natuurlike of chemiese sintese geproduseer en gekarakteriseer in terme van chemiese karakter en biologiese aktiwiteit. Die Trcs het beduidend meer aktiwiteit as RW-peptiede getoon, maar is ook meer hemolities en dus meer toksies. Die resultate dui op die belang van waterstofbinding met 'n aromatiese aminosuurresidu vir die selektiewe aktiwiteit teenoor die leucocin A weerstandige L. monocytogenes B73-MR1. Strukturele eienskappe wat tot 'n sterker membraan-interaksie lei, verhinder egter die werkingsmeganisme. Ons het vasgestel dat Gln en die hidroksielgroep van Tyr betrokke kan wees in die interaksie met die vermeende teenmiddelteiken in L. monocytogenes. 'n Balans tussen amfipatiese/hidrofobiese en grootte/steriese parameters is ook noodsaaklik vir optimale aktiwiteit. Vanuit ons QSAR studies word die peptied siklo-(VOMe3LfPWfNQY) as die voorloper vir 'n toekomstige peptiedbiblioteek van antilisteriale Trcs voorgestel. Verder is daar gevind dat die antilisteriese aktiwiteit van die Trcs oorwegend lities en sout-verdraagsaam is, terwyl die RW-peptiede nie-lities en Ca2+ sensitief is. Ons het bevestig dat Ca2+ die Trc antilisteriese aktiwiteit verbeter, deur die Trc se antimetaboliese aktiwiteit verhoog, maar terselfdertyd 'n nie-litiese werkingsmeganisme induseer. Vanuit model-membraan studies word voorgestel dat Trc se nie-litiese werkingsmeganisme, soos teweeggebring deur Ca2+, die gevolg kan wees van nadelige lipied vermenging, die teenwoordigheid van 'n kalsium geïnduseerde Trcsensitiewe nie-membraan teiken in 'n vervroegde kalsium geïnduseerde intrasellulêre anaerobiese vorm van Listeria monocytogenes, en/of dat die Trc-Ca2+ komplekse belangrike komponente soos ’n membraan-gebonde elektron transport sisteem of bakteriële dehidrogenases inhibeer. Daar is ook bevestig, soos voorheen gevind, dat die Trcs kragtige, antimalaria aktiwiteit besit wat volgorde-spesifiek en nie-lities is. Die RW-peptiede het swak aktiwiteit getoon, maar ons resultate het weereens bewys dat peptiede wat meer hidrofobies en hemolities is, meer aktief is, veral die RW-peptiede wat die Trp analoog β-(bensoteïen-3-iel)-alanien (Bal) bevat. 'n Nuwe bevinding is dat een van die meer polêre Trc C analoë, genaamd triptosidien C (Tpc C), in teenstelling met Trc C, sterk antimalaria aktiwiteit het, wat 'n aanduiding is van die spesifieke volgorde en die rol van die Trp7 in aktiwiteit. Vanuit hierdie bevindinge word die peptied siklo- (VOLfP(Bal)fNQ(Bal)) as 'n voorloper vir toekomstige navorsing aangedui. Vir ons soeke na die Trc en Tpc C teiken(s), het ons hoë resolusie fluoressensie mikroskopie aangewend. Resultate toon dat Trc tot die ontwrigting van 'n neutrale lipied strukture en chromatien lei en sodoende groei beperk in die laat trofosoïet/vroeë skisont fases. Dit het aangedui dat die membraanstrukture wat neutrale lipiede bevat, sowel as chromatien, deur die Trcs geteiken word. 'n Verdere nuwe bevinding in hierdie studie was dat chloroquine (CQ) weerstandigheid nie net korreleer met weerstandigheid teen Trcs nie, maar dat die Trcs en CQ antagonisties optree teenoor mekaar se aktiwiteite. Dit dui op 'n gemeenskaplike teiken en die kosvakuool as 'n addisionele Trc teiken in P. falciparum word voorgestel.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The work in this dissertation provides structural and kinetic insight into the mechanism of action of quinoline antimalarial drugs which may aid rational drug design. Quinoline antimalarial drug-ferrihaem (Fe(III)PPIX) complexes were investigated. Single crystal Xray diffraction (SCD) structures of the complexes formed between Fe(III)PPIX and the quinoline methanol antimalarials quinine, quinidine and mefloquine have been determined, and are the first observed structures of complexes formed between free Fe(III)PPIX and quinoline antimalarial compounds. Quinine, quinidine and mefloquine are shown to have a three-point binding mode to Fe(III)PPIX, which comprises direct coordination of the drug to the Fe(III) centre through its benzylic alcohol functionality, π-stacking between the drug and porphyrin aromatic systems, and intramolecular hydrogen bond formation between the porphyrin propionate group and the protonated quinuclidine nitrogen atom of the drug in the case of quinine and quinidine, and formation of an intramolecular hydrogen bonding network in the case of mefloquine. Extended X-ray absorption fine structure spectroscopy (EXAFS) was use to elucidate structural information of Fe(III)PPIX-drug complexes in solution, and indicates that coordination persists in solution. The protocol for lipid-mediated formation of β-haematin, where monopalmitic glycerol was used as a model lipid, was successfully modified to incorporate antimalarial drugs into the aqueous layer in order to investigate drug activity under biologically-relevant conditions. Four compounds were chosen, namely chloroquine and amodiaquine, both 4- aminoquinolines and quinine and quinidine. IC50 values for the inhibition of β-haematin formation show good correlation with biological activities determined against a chloroquine-sensitive Plasmodium falciparum strain. The lipid-water interface system was further used to investigate the effects of quinine, quinidine chloroquine and amodiaquine on the kinetics of β-haematin formation. The results led to the development of a kinetic model based on the Avrami equation and the Langmuir isotherm. The data strongly support a mechanism of antimalarial drug action by adsorption to the growing face of haemozoin, with precipitation of Fe(III)PPIX at high drug concentrations accounting for decreased yields. Adsorptions constants (log Kads) determined for each drug show a strong correlation with biological activity. Finally, the first SCD structure of the μ-propionato dimer of Fe(III)PPIX, the structural unit of haemozoin, has been determined as its DMSO solvate. EXAFS suggests that this species is only formed upon nucleation, with the π-π dimer species being favoured in solution. / AFRIKAANSE OPSOMMING: Die werk in die dissertasie verleen struktuur en kinetiese insig in the meganisme van aktiwiteit vir kinolien antimalariamiddels wat kan bydra tot die ontwikkeling van nuwe medisyne. Kinolien antimalariamiddel-ferriheem (Fe(III)PPIX) komplekse was ondersoek. Navorsing is gedoen op die enkelkristal X-straaldiffraksie strukture van die komplekse gevorm tussen Fe(III)PPIX en die kinolien metanol antimalaria middels kinien, kinidien en mefloquine. Die strukture is die eerste komplekse wat waargeneem is tussen vrye Fe(III)PPIX en kinolien antimalariamiddels. Kinien, kinidien en mefloquine het ʼn driepunt bindingsvorm, direkte koördinasie met die Fe(III) deur die bensielalkohol groep, ʼn π- stapel tussen die middel en die porfirien aromatiese sisteem, ʼn intramolekulêre waterstofbinding tussen the porfirienpropionaat funksie en die geprotoneerde kinuklidien stikstofatoom (kinien en kinidien) en ʼn netwerk van intramolekulêre waterstof bindings (mefloquine) insluit. Uitgebreide X-straal absorpsie fyn struktuur spektroskopie (EXAFS) is gebruik om inligting oor Fe(III)PPIX-middel komplekse in oplossing te verkry en het aangedui dat die koördinasie in oplossing voorkom. Deur gebruik te maak van monopalmitiengliserol as die lipid in the lipid-water interfase sisteem, waar antimalariamiddels suksesvol in die buffer geïnkorporeer was, was die middel se aktiwiteit onder biologiese kondisies geondersoek. Vier middels was gekies naamlik, chloroquine en amodiaquine, albei 4-aminokinoliene en kinien en kinidien om die IC50-waarde vir inhibisie van β-hematien vorming te bepaal. Die IC50 waardes het ʼn goeie korrelasie met biologiese aktiwiteite teen die chloroquine-sensitiewe Plasmodium falciparum stam gewys. Die lipid-water interfase-sisteem was ook gebruik om die effek van kinien, kinidien, chloroquine en amodiaquine op die kineties effek op die vorming van β-hematien te ondersoek. Die resultate het gelei to die ontwikkeling van die kinetiese model gebaseer op die Avrami vergelyking en die Langmuir isoterm. Die data ondersteun ʼn meganisme van middel aksie waar die middel teen die groeiende vlak van hemosoïen kristal adsorbeer. Die neerslag van Fe(III)PPIX wat vorm by hoë konsentrasies, het gelei tot laer opbrengste. Die adsorpsiekonstante (log Kads) bepaal vir elke middel, het goeie korrelasie met biologiese aktiwiteit getoon. Enkelkristal X-straaldiffraksie strukture van μ- propionatodimeer van Fe(III)PPIX, die struktuur eenheid van hemosoïen, was bepaal as ʼn DMSO solvaat. EXAFS het aangedui dat die spesie slegs by kernvorming ontstaan en dat die π-π dimeerspesie in oplossing voorkom.