Sex ratio theory is a focus in evolutionary biology that explores how natural selection shapes investment in males and females. It has provided some of the best quantitative evidence of evolution and could find utility in public health efforts through its application to malaria parasites. These parasites have distinct male and female forms that are produced following massive asexual replication, and they mate within the blood-feeding insects that transmit them between vertebrate hosts. A very similar population structure is assumed by local mate competition (LMC), a model from sex ratio theory that predicts female-biased sex ratios dependent on the degree of selfing within a mating patch. In this dissertation, I test a series of predictions from LMC for the lizard malaria parasite Plasmodium mexicanum. These include: (i) sex ratios have heritable variation that is not constrained by other life history traits; (ii) single-genotype infections have female-biased sex ratios that are determined by male fecundity; (iii) multiple-genotype infections have less biased sex ratios than single genotype infections; (iv) if males are limiting, sex ratios may be less biased when there are fewer parasites present (an extension of LMC called fertility insurance); and (v) less biased sex ratios may also be favored if increased female production yields diminishing returns on transmission to a new vertebrate host. To test these predictions, I combined the study of natural and experimental infections, microscopy (parasite density and sex ratio), molecular genetics (infection genetic diversity), and mathematical modeling (of how transmission patterns might affect sex ratio evolution). Overall, the results were qualitatively consistent with both LMC and my new model predictions. Sex ratios showed evidence of heritable variation that was unlinked to other life history traits measured. Sex ratios in single-genotype infections were female biased and consistent with the male fecundity observed, and were lower than sex ratios in experimental multiple-genotype infections, as predicted. Sex ratios were not less biased with lower sexual cell density, suggesting that males were not limiting. In fact, the opposite trend was sometimes observed: sex ratios were less biased with more sexual cells. This pattern has been observed previously in this and other species, and the only model that currently predicts such a trend is the new transmission model I outline. This dissertation contributes to our understanding of sex ratio evolution for malaria parasites in a number of ways. First, it adds evidence to the idea that the selective forces implicated in LMC are at work in malaria parasites and that malaria parasites are able to detect and respond to relevant cues. Second, it helps account for discrepancies in existing data, which have often reached conflicting conclusions. Third, it offers one of the first detailed studies of malaria parasite male fecundity, an essential piece of the sex ratio puzzle. Finally, it outlines a new theoretical extension of LMC that provides novel predictions and highlights areas of study that may be fruitful for future work on malaria parasites and other organisms.

Filamentous bacteriophages were engineered to express foreign genes with the ultimate purpose of displaying transmission control anti-malarial peptides as in phage display. It was hypothesized that expression of foreign genes would be possible using the phage’s promoters. This hypothesis was tested by assuming that promoters for the phage major coat protein (MCP) gene would also promote the expression of any foreign gene inserted downstream of the MCP gene. As proof of principle, the bacteriophages Pf3, Pf1, and M13 were engineered in this way to successfully synthesize Enhanced Green Fluorescent Protein (EGFP). Type 88 phage display on the EGFP recombinant Pf3 was attempted by fusing a second copy of its MCP gene to the existing EGFP gene. This resulted in a phage display Pf3 replacement vector which was then used to construct a phage for displaying an anti-malarial peptide.

Le paludisme est une cause majeure de morbidité et de mortalité, plus particulièrement en Afrique sub-saharienne. De très nombreuses observations sont en faveur de l'existence de facteurs génétiques contrôlant le devenir de l'infection palustre. Il est très probable que certains variants génétiques de gènes candidats du paludisme affectent la résistance du paludisme à travers leur effet sur la réponse immunitaire acquise. Afin de vérifier cette hypothèse, nous avons étudié, dans une population vivant au Burkina Faso, des variants génétiques de HBB, IL4, IL12B, TNF, LTA, FCGR2A et NCR3 dont l'association avec des phénotypes liés à la résistance au paludisme a été publiée; nous avons évalué leur influence sur les niveaux d'IgG dirigés contre les antigènes de Plasmodium falciparum en utilisant un test d'association basé sur les familles. Ainsi, nous avons détecté, l'effet de l'hémoglobine C, FCGR2A-H131, le TNF-857T, et TNF1304A sur les niveaux des sous-classes d'IgG anti-P. falciparum. Ces résultats constituent une base utile pour des études ultérieures du contrôle génétique de la réponse immunitaire chez des individus vivant dans une zone d'endémie. Un autre projet a porté sur l'étude fonctionnelle du polymorphisme NCR3-412, dont nous avions montré l'association avec les accès palustres simples. Nos résultats basés sur des techniques moléculaires montrent l'effet de ce polymorphisme situé dans le promoteur sur la liaison de protéines nucléaires. / Malaria is the major cause of morbidity and mortality especially in the Sub-Saharan Africa. There is a growing body of evidence for genetic factors controlling the outcome of malaria infection. It is thought that some genetic variants of malaria candidate genes affect malaria resistance through their effect on the acquired immune response. In order to verify this hypothesis, we worked on genetic variants of HBB, IL4, IL12B, TNF, LTA, FCGR2A and NCR3, which have been associated with malaria resistance phenotypes, to determine their influence on levels of anti-P. falciparum IgG in urban population of Burkina Faso. Using family-based association analysis, we detected the effect of Hemoglobin C, FCGR2A-H131, TNF-857T, and TNF1304A on the levels of anti-P. falciparum IgG. This study can pave the way towards further comprehension of genetic control of an individual's immune response against malaria. Another project focused on functional study of polymorphism NCR3-412, which has already been associated to mild malaria. We investigated the functional effect of this polymorphism located in the promoter by using molecular techniques and showed the effect of this polymorphism on the binding of nuclear proteins.

Master of Science / Department of Biochemistry and Molecular Biophysics / Michal Zolkiewski / ClpB is a molecular chaperone that is essential for infectivity and pathogen survival in a host. It belongs to the AAA+ protein family, which cooperates with the DnaK chaperone system to reactivate aggregated proteins. In this study, we purified and then studied the biochemical properties of the apicoplast targeted ClpB isoform from the malaria parasite Plasmodium falciparum: PfClpB1. Plasmodium falciparum is the parasite responsible for the most severe form of malaria. In contrast to the parasitophorous vacuole targeted PfClpB2 from Plasmodium falciparum which contains all characteristic AAA+ sequence motifs, PfClpB1 also includes a 52-residue long non-conserved insert in the middle domain. The ATPase activity study shows that PfClpB1 hydrolyzes ATP in presence of Poly-lysine and α-casein. Similar to most AAA+ ATPases, addition of ATP induces hexamer formation in PfClpB1. Lastly, PfClpB1 reactivates aggregated firefly luciferase. However, PfClpB1 is unable to efficiently reactivated luciferase in the presence of the E. coli DnaK chaperone system or human Hsp70 and Hsp40 (Hdj1). This can be explained by the extra middle domain sequence of PfClpB1. Our data may suggest that PfClpB1 activity is essential for Plasmodium falciparum survival by preserving the activity of apicoplast proteins.

Background: Despite progress made in the last decades, malaria persists as a pressing health issue in sub-Saharan Africa. Pregnant women are particularly vulnerable to infection and serious health outcomes for themselves and their unborn child. Risk can be mitigated through appropriate use of control measures such as insecticide-treated bed nets. Although social networks can influence uptake of preventive strategies, the role of social influence on bed net ownership has not been explored. During an evaluation of a bed net distribution programme, the influence of non-health care advisors on ownership and use of bed nets by pregnant women in Kumasi, Ghana was examined. Methods: Data were collected through in-person interviews with 300 pregnant women seeking antenatal care in an urban hospital in Kumasi, Ghana. Participants were asked about their bed net ownership, bed net use, and information about three personal contacts that they go to for pregnancy advice. Information about these advisors was combined into an influence score. Logistic regression models were used to determine the association between the score and bed net ownership. Those who owned a bed net were further assessed to determine if interpersonal influence was associated with self-reported sleeping under the bed net the previous night. Results: Of the 294 women in the analysis, 229 (78%) reported owning bed nets. Of these bed net owners, 139 (61%) reported using a bed net the previous night. A dose response relationship was observed between the interpersonal influence score and bed net ownership and use. Compared to the lowest influence score, those with the highest influence score (> 1 SD above the mean) were marginally more likely to own a bed net [OR = 2.37, 95% CI (0.87, 6.39)] and much more likely to use their bed net [5.38, 95% CI (1.89, 15.25)] after adjusting for other factors. Conclusions: Interpersonal influence appears to have modest impact on ownership and use of bed nets by pregnant women in an urban area of Ghana. Further investigations would need to be conducted to determine if the relationship is causal or if individuals who associate are simply more likely to have similar practices.

Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood stage malaria and several clinical trials using mostly protein-in-adjuvant vaccines have shown limited success. This thesis describes the development of recombinant adenoviral (AdHu5) and poxviral (MVA) vectors encoding AMA1 from Plasmodium chabaudi murine parasites. In this murine malaria model, AdHu5 and MVA encoding AMA1 when used in a heterologous prime boost regime showed excellent immunogenicity, both humoral and cellular. The vaccination regime was protective against blood stage challenge and both antibodies and CD4+ T cells found to be important for vaccine induced blood stage protection. In parallel to this novel P. falciparum vaccines encoding AMA1 were also developed and administered in a similar prime boost regime to mice and rabbits. The vaccination regime induced cellular immune response and high titre antibodies against AMA1 and these antibodies showed growth inhibitory activity against the homologous parasite strain. In an effort to overcome the issue of antigenic polymorphism and to circumvent pre-existing immunity to human adenovirus, biallelic simian and human adenoviral vectors and MVA encoding AMA1 vaccines were also developed and administered to mice and macaques. These vectors also induced high titre antibodies and the serum from macaques was found to have growth inhibitory activity. These vaccine candidates are now being taken forward to Phase I/II clinical trials in Oxford. This work also described the attempt to improve MVA as a antibody inducing vector to allow better antibody mediated immunity to blood stage malaria.

Seulement 1 à 3% des cas de paludisme dégénèrent en NP. Cependant, les séquelles à long termes touchent 3 à 10% des adultes et 25% des enfants et ces personnes présentent des déficits cognitifs notamment au niveau de l'apprentissage. Au niveau de l'armée française, ce sont 15 000 militaires qui sont exposés chaque année dans les zones impaludées, avec au moins 350 accès palustres mais surtout 1 à 2 morts chaque année.Dans le but de lutter contre P. falciparum mais aussi diminuer les séquelles dans le cas du NP deux molécules ont été étudiées. D'une part, l'atorvastatine (AVA) et d'autre part le bleu de méthylène (BM). Les données, précédemment publiées, ont montré l'efficacité de l'AVA non seulement comme antibactérien, antiviral ou antiparasitique mais aussi comme modulateur de l'immunité et adjuvant potentiel pour les antipaludiques actuels. Le BM est un antipaludique qui jusqu'ici présentait une voie de synthèse avec métaux lourds. C'est une nouvelle voie de synthèse sans métaux lourds et une efficacité démontrée qui nous ont incités à étudier cette molécule.Pour estimer l'efficacité de l'AVA et du BM en association avec les antipaludiques actuels nous avons successivement testé ces molécules dans un modèle in vitro (micro test isotopique simplifié) contre P. falciparum, puis dans un modèle murin de neuropaludisme en utilisant des souris C57BL/6N infectées avec Plasmodium berghei. Un gain d'efficacité a été observé en associant l'AVA ou le BM avec les antipaludiques contre P. falciparum mais aussi une protection par rapport au NP chez les souris traitées par les associations. Le BM protégeant aussi bien contre le paludisme simple que le NP. / Only 1 to 3% of malaria infections turn into CM. Meanwhile, long term neurological sequelae range from 3 to 10 % in adults and 25% of child survivors present long term cognitive impairments. In a military framework, there are 15 000 soldiers localized in endemic malaria areas, with at less 350 infection cases giving clinical malaria syndrome but mainly 2 deaths each year.In the aim to fight against P. falciparum but also to decrease sequelae related to CM, two molecules were studied. In one hand, atorvastatin (AVA) and in the other hand methylene blue (MB). AVA is a synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (3HMG-CoA) reductase used in the treatment of hypercholesterolemia. Previous data, reported in numerous articles support the efficacy of AVA not only as antimicrobial, antiviral or antiparasitic agent but also as immune system modulator and potential adjuvant in vitro for common antimalarial drugs. BM is an antimalarial drug which until now had a synthesis pathway with heavy metals. It is a new synthesis pathway without heavy metals and an efficacy demonstrated which encouraged us to study this molecule.To evaluate AVA and BM efficacies in combination with common antimalarial drugs, we successively tested these molecules in an in vitro model (simplified isotopic microtest) against P. falciparum, then in experimental cerebral malaria using C57BL/6N mice infected with Plasmodium berghei. An increased efficacy was observed when AVA or MB is associated with common antimalarial drugs against P. falciparum but also a protection against CM in mice treated by drugs combinations. MB protects against malaria but also CM.

Mon travail de thèse a porté sur l'étude des facteurs influençant le devenir de l'infection palustre. Une étude de liaison génétique basée sur des marqueurs répartis sur l'ensemble du génome nous a permis de mettre en évidence la liaison génétique de la région chromosomique 17p11-p13 avec la parasitémie. Nous avons étudié les variations génétiques des gènes HS3ST3A1 et HS3ST3B1 du chromosome 17p12 humain car ces gènes pourraient influencer l'infection de cellules humaines par le Plasmodium. Nous avons ainsi observé la liaison génétique de certain SNP de ces gènes avec la parasitémie. Les anticorps IgG dirigés contre le parasite P. falciparum sont connus pour jouer un rôle important dans la réponse immunitaire. Certain gènes associés à la résistance au paludisme pourraient également être associés au niveau de production d'IgG. Nous avons montré que certains polymorphismes situés dans les gènes HBB, FcRIIA, et du TNF influencent le niveau de production de différentes sous classes IgG. Ces résultats nous permettent de mieux comprendre le contrôle génétique de la réponse humorale contre la malaria.J'ai aussi caractérisé l'inflammation non infectieuse dans un modèle murin afin de tester l'influence de l'état inflammatoire lors de l'infection par le plasmodium. L'étude transcriptomique de trois organes réalisée sur un modèle de souris injectée avec de l'acide oléique, nous a permis de mettre en évidence une réponse inflammatoire principalement au niveau des poumons et du cerveau. Ces résultats vont nous permettre de tester l'hypothèse qu'une réponse inflammatoire pré-existante favoriserait la survenue de forme sévère du paludisme chez des souris infectées par Plasmodium. / My thesis focused on the study of factors influencing the future of malaria infection. A genetic linkage study based on markers distributed throughout the genome has allowed us to highlight the linkage of 17p11 - p13 chromosome region with parasitaemia. We investigated the genetic variation of genes and HS3ST3A1 HS3ST3B1 human chromosome 17p12 because these genes may influence the infection of human cells by Plasmodium . We observed genetic linkage of some SNPs in these genes with parasitaemia.IgG antibodies against the parasite P. falciparum are known to play an important role in the immune response . Certain genes associated with resistance to malaria could also be associated with the level of IgG production . We have shown that some polymorphisms located in genes HBB , Fc  RIIA , and TNF influence the level of production of different IgG subclasses . These results allow us to better understand the genetic control of humoral response against malaria.I also characterized non- infectious inflammation in a mouse model to test the influence of the inflammatory condition during infection by Plasmodium . The transcriptomic study performed on three organs mouse model injected with oleic acid , has allowed us to demonstrate an inflammatory response mainly in the lungs and brain. These results will allow us to test the hypothesis that pre-existing inflammatory response favor the occurrence of severe malaria in mice infected with Plasmodium.

Die vorliegende Arbeit beschäftigte sich mit der Entwicklung und Synthese von Inhibitoren der Deoxyhypusin-Hydroxylase (DOHH), die einen wichtigen Schritt in der Aktivierung des eukaryotischen Translationsinitiations-Faktors-5A (eIF-5A) katalysiert. Die Hemmung dieses Metalloenzyms durch kleine Moleküle, die mit dem katalytischen Eisenatom im aktiven Zentrum der DOHH einen Chelatkomplex bilden, hat einen antiproliferativen Effekt auf parasitäre Erreger, wie Plasmodien, Trypanosomen und Leishmanien zur Folge. Ausgehend von den antiplasmodial wirksamen Eisenkomplexbildnern und Pyridon-Derivaten Ciclopirox und Mimosin wurden besser wirksame 2,6-Diaryl-4-oxopiperidincarbonsäuremono- und -diester-Derivate abgeleitet, deren 4-Piperidon-Grundgerüst als Leitstruktur für die Entwicklung von antiplasmodialen und antitrypanosomalen Wirkstoffen fungierte. Entsprechend dieser Leitstrukturen gelang im Zuge dieser Arbeit durch verschiedene Modifikationen der Doppel-Mannich-Reaktion die Erstellung einer weitreichenden Bibliothek 52 strukturell diverser 4-Hydroxytetrahydropyridin-3,5-dicarbonsäurediester 1 – 6, darunter auch erstmals Derivate mit t-Butyl-esterfunktionen und 4-Hydroxytetrahydropyridin-3-carbonsäuremonoester 7 – 8. Dabei konnten vor allem Derivate mit der gewünschten nitroaromatischen Substitution in den Positionen 2 und 6 synthetisiert werden. Darüber hinaus wurden vielfältige Strukturabwandlungen dieser Substanzen in Form von verschiedenen 4-Piperidonderivaten ohne Esterfunktionen, deren Oximen sowie von 4-Hydroxychinoloncarbonsäureestern syn-thetisiert. Die hergestellten Derivate wurden In-vitro-Testungen an Plasmodium falciparum, Trypanosoma brucei brucei und Leishmania major unterzogen. Zusätzlich wurde die Zytotoxizität an der Makrophagen-Zelllinie J774.1 ermittelt. / The present work focused on the development and synthesis of inhibitors of deoxyhypusine hydroxylase (DOHH) that catalyzes an important step in the activation cascade of eukaryotic initiation factor 5A (eIF-5A). The inhibition of this metal-containing enzyme by small molecules chelating the essential catalytic iron at the active site of DOHH leads to an antiproliferative effect on parasites such as plasmodia, trypanosoma and leishmania. Derived from the antiplasmodial iron chelators and pyridine derivatives ciclopirox and mimosine more potent 2,6-diaryl-4-oxopiperidiniumcarboxylic acid mono- and -diester derivatives were developed. Its 4-piperidone skeleton served as a lead structure for the development of antiplasmodial and antitrypanosomal agents. Based on these lead structures it was possible to build a broad variety of 52 4-hydroxytetrahydropyridine-3,5-dicarboxylates 1 – 6 (for the first time including derivatives with t-butyl-ester functions) as well as 4-hydroxytetrahydropyridine-3-monocarboxylates 7 – 8 with varying substitution patterns by various modifications of the double mannich reaction. Thereby it was possible to synthesize derivatives with the advantageous nitro-aromatic substitution in position 2 and 6. Additionally structural modifications of these compounds such as 4-piperidones without ester functions, corresponding oximes and 4-hydroxychinolone carboxylates have been synthesized, too. The synthesized substances were tested against Plasmodium falciparum, Trypanosoma brucei brucei and Leishmania major. Furthermore their cytotoxity was determined using the macrophage cell line J774.1.

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Science, 1998. / This study was initiated in order to determine the identities and distribution patterns of the anopheline fauna, more especially the malaria vectors, in regions of Botswana prone to malaria epidemics. Field samples collected from Shakawe, Maun and Kasane over three consecutive years were subjected to morphological, cytogenetic, isoenzyme and PCR analyses. The results established that Anopheles arabiensis and Anopheles funestus were the predominant vector species.