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Virulent Trypanosoma cruzi, and the non-pathogenic Trypanosoma conorhini and Trypanosoma rangeli are protozoan parasites with divergent lifestyles. T. cruzi and T. rangeli are endemic to Latin America, whereas T. conorhini is tropicopolitan. Reduviid bug vectors spread these parasites to mammalian hosts, within which T. rangeli and T. conorhini replicate extracellularly, while T. cruzi has intracellular stages. Firstly, this work compares the genomes of these parasites to understand their differing phenotypes. Secondly, genome architecture of T. cruzi is examined to address the effect of a complex hybridization history, polycistronic transcription, and genome plasticity on this organism, and study its highly repetitive nature and cryptic genome organization. Whole genome sequencing, assembly and comparison, as well as chromosome-scale genome mapping were employed. This study presents the first comprehensive whole-genome maps of Trypanosoma, and the first T. conorhini strain ever sequenced. Original contributions vii to knowledge include the ~21-25 Mbp assembled genomes of the less virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E, containing ~10,000 to 13,000 genes, and the ~36 Mbp genome assembly of highly virulent T. cruzi CL with ~24,000 genes. The T. cruzi strains exhibited ~74% identity to proteins of T. rangeli or T. conorhini. T. rangeli and T. conorhini displayed greater complex carbohydrate metabolic capabilities, and contained fewer retrotransposons and multigene family copies, e.g. mucins, DGF-1, and MASP, compared to T. cruzi. Although all four genomes appear highly syntenic, T. rangeli and T. conorhini exhibited greater karyotype conservation. T. cruzi genome architecture studies revealed 66 maps varying from 0.13 to 2.4 Mbp. At least 2.6% of the genome comprises highly repetitive repeat regions, and 7.4% exhibits repetitive regions barren of labels. The 66 putative chromosomes identified are likely diploid. However, 20 of these maps contained regions of up to 1.25 Mbp of homology to at least one other map, suggestive of widespread segmental duplication or an ancient hybridization event that resulted in a genome with significant redundancy. Assembled genomes of these parasites closely reflect their phylogenetic relationships and give a greater context for understanding their divergent lifestyles. Genome mapping provides insight on the genomic evolution of these parasites.

The problem of controlling tsetse flies in Africa is an old one. The tsetse fly transmits the trypanosome parasites which cause sleeping sickness in humans and disease in cattle. Because cattle are a favoured food source for tsetse much work has been done looking at the use of insecticide treated cattle as a control strategy for the tsetse fly. Such treatment methods possess many advantages; they are safe and relatively environmentally benign, they can be applied by individual farmers without the need for logistically demanding and costly traditional control programmes and, in addition to tsetse flies the insecticides are effective against a wide range of other harmful cattle parasites. The cost of the insecticide is however a significant constraint to the number of livestock keepers who can afford to employ the technique and as a result many cattle remain untreated. Following the discovery that tsetse had a significant predilection for feeding on the legs and belly of cattle, it was hypothesised that restricting the insecticide to only those areas could offer comparable protection to treating the whole animal. Such an approach would use up to 80% less drug and thus make the treatment per animal much cheaper. In addition, preferentially targeting areas favoured by tsetse, and leaving the rest of the animal untreated, preserves some important ecological balances between cattle and their parasites which traditional treatment methods destabilise. This thesis describes the design, implementation and analysis of a longitudinal study run over 8 months in south east Uganda that sought to compare the effect of applying insecticide to cattle only on the regions favoured by tsetse flies. Cattle were recruited to the study and assigned one of four treatment groups; a whole body application of deltamethrin insecticide pour-on; a restricted application of deltamethrin spray, applied to the front legs, ears and belly; a prophylactic trypanocide injection of isometamidium chloride, and a control group, that received no further treatments. All animals in the study were however cleared using twin doses of a trypanocide diminazene aceturate at the start of the study.

Part I: Target identification studies are one of the most difficult but rewarding challenges in chemical biology. Part I of this thesis describes target identification studies for 5-nitrofuran containing hits. The 5-nitrofurans used in this study were identified in a phenotypic screen for compounds that induced melanocyte cells death in zebrafish. Chapter 1 provides brief overviews on three related areas of the project: 1) the use of zebrafish as a model organism in drug discovery; 2) phenotypic screening using zebrafish and 3) the strategies used in target identification studies. Chapter 2 describes the synthesis of and SAR studies on two series of 5-nitrofuran containing analogues. The design and preparation of biotinylated chemical probes based on the SAR data is also described. These chemical tools are then used in affinity chromatography studies and genetic validation of a potential target (zebrafish Aldh2) of the 5-nitrofuran compounds is reported. Chapter 3 provides a review of the biological and chemical processes that human ALDHs are known to mediate. In addition, small molecules that modulate ALDH2 activity are reviewed. A detailed study of the interaction between 5-nitrofurans and human ALDH2 including in vitro enzymatic assays is described leading to the conclusion that the 5- nitrofurans under study are substrates of human ALDH2. Further mechanism of action investigations using model reactions are also presented. Chapter 4 introduces the use of 5-nitrofuran containing drugs in the clinic and highlights the reported side-effects. Further investigation of the interaction between ALDH2 and 5- nitrofurans in zebrafish and yeast using ALDH2 inhibitors is described. Based on these results, a combination therapy strategy is proposed. Finally, the trypanocidal activity of the newly synthesised 5-nitrofurans is discussed. Experimental details and future work for Part I are presented in Chapters 5 and 6 respectively. Part II: Human sirtuins are associated with various biological functions and diseases, including cancer and neurodegeneration. Previous work from the Westwood Lab has led to the discovery of the tenovins that act as inhibitors of SIRT1 and SIRT2. Part II of the thesis reports the development of potent fixed ring tenovin analogues with high SIRT2 selectivity. Chapter 7 provides a brief review of the biology of human SIRT2 and the reported SIRT2 inhibitors available to date. This is followed by a short summary of the previous work on the tenovins in the Westwood Lab and the design of the fixed ring tenovin analogues. Chapter 8 describes the synthesis of three series of fixed ring tenovin analogues. SAR data is generated based on in vitro enzymatic assays against both SIRT1 and SIRT2 and the prepared analogues showed relatively high potency and selectivity against SIRT2. Further cell-based deacetylation assay are also discussed. All the experimental details are reported in Chapter 9 and Chapter 10 provides with conclusions and proposed future work.

Yes / African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. IMPORTANCE. Most African trypanosomes are lysed by the ApoL1 protein in human serum. Only the subspecies Trypanosoma b. gambiense and T. b. rhodesiense can resist lysis by ApoL1 because they express specific serum resistance proteins. We here report a complex interplay between trypanosomes and an ApoL1 variant characterized by a homozygous missense substitution (N264K) in the domain that we hypothesize interacts with the endolysosomal membranes of trypanosomes. The N264K substitution knocks down the lytic activity of ApoL1 against T. b. gambiense strains lacking the TgsGP defense mechanism and against T. b. rhodesiense if N264K is accompanied by additional substitutions in the SRA-interacting domain. Our data suggest that populations with high frequencies of the homozygous N264K ApoL1 variant may be at increased risk of contracting human African trypanosomiasis. / This work, including the efforts of Stijn Deborggraeve, was funded by Research Foundation Flanders (1501413N). This work, including the efforts of Bart Cuypers, was funded by Research Foundation Flanders (11O1614N). This work, including the efforts of Jean-Claude Dujardin and Etienne Pays, was funded by Interuniversity Attraction Poles Program of Belgian Science Policy (P7/41). This work, including the efforts of Jean-Claude Dujardin, was funded by Flemish Ministry of Sciences (SOFI-B SINGLE). This work, including the efforts of Etienne Pays, was funded by EC | European Research Council (ERC) (APOLs 669007).

The epidemiological study of pathogens largely depends on three technologies, serology, microscopy and the polymerase chain reaction (PCR). Serological methods are unable to differentiate between current and past infections. Microscopy has historically been the mainstay of epidemiological study. In recent times the use of microscopy has been in decline, as it has been shown to have an inherent lack of sensitivity and specificity and produces many false negative results. PCR is now the method of choice for screening samples for the presence or absence of pathogens. Although PCR is widely regarded as an extremely sensitive technique, the fact that it assays a very small volume of sample is often overlooked. If the target pathogen is not present in the tiny aliquot of sample from an infected host, then a false negative results will occur. In endemic situations were the pathogen is present at low infection intensities, then the potential for false negatives results of this type is high. This intensity related false negative effect can lead to serious underestimation of diagnosed prevalence and incidence with consequent misinterpretation of the resulting data. This phenomenon has been reported in the literature for a range of pathogens and especially for epidemiological study of schistosomiasis. The extensive occurrence of false negatives during study of schistosomiasis samples was such an obstacle to epidemiological study it prompted the world health organisation to repeatedly call for quantitative methods to be employed to combat the problem. The main objectives of this thesis are to rationalise and simplify the methods of diagnosing African trypanosomes in epidemiological studies and to investigate the consequences of, and methods of dealing with infection intensity related false negative results that occur as a result of widespread sub-patent infections in the study population A new PCR assay was developed that was capable of analysing whole blood placed onto treated filter paper. The PCR assay was capable of differentiating between all the important African trypanosome species, producing a unique size of amplicon for each species of trypanosome. Initial results from repeated screening of human and cattle samples known to be parasitologically positive indicated that many false negative results occur. A more extensive analysis of thirty five bovine blood samples randomly chosen from a collection of field samples revealed that false negative results occurred regularly. The prevalence of infection after a single screening was 14.3% whereas the cumulative prevalence after over 100 repeated screenings rose to 85.7%. This showed that a severe underestimation of prevalence occurs from a single screening of the samples. In order to investigate the consequences of, and develop methods of dealing with this problem, computer based simulations were used to model the dynamics of screening samples with sub-patent infections. In order to construct the model the data obtained from repeat screening of the thirty-five bovine blood samples was fitted to a number of mathematical distributions. A negative binomial distribution best described the distribution of trypanosomes across the hosts. Exploration of the phenomenon with the resulting model showed the extensive underestimation of true prevalence that is possible. The simulations also showed that it is possible for populations with very different patterns of infection and true prevalence to all have the same diagnosed prevalence from a single screening per sample. Statistical comparison of these very different populations by diagnosed prevalence alone would conclude there was no significant difference between the populations. It was therefore concluded that the diagnosed prevalence from a single (or even multiple) screenings is an inadequate and potentially misleading measure of both infected hosts and parasite numbers. In order to deal with these problems new methods were evaluated for use in epidemiological studies. A simple method of producing quantitative measures of infection was advocated. The insensitivity of existing screening methods in detecting significant difference between populations was highlighted and a greatly improved methodology was shown. Finally, a method for inferring the true population prevalence from the data obtained from repeat screening of samples was suggested. Although some of these new methodologies have limitations, they represent a great improvement on the use of a single diagnostic test for each host. The work presented in this thesis highlights a serious potential limitation to our understanding of the epidemiology of pathogens that exist at sub-patent levels, and develops some possible methods of overcoming these limitations.

This thesis describes the dynamics of mitochondrial RNA-binding protein 1 and 2 (MRP1/2) complex in different cell lines of Trypanosoma brucei under an optimized immobilized condition. This study reveals the influence of RNA on the complex's dynamics. Furthermore, the function of RNA-binding complex 1 (MRB1) core has been studied via reverse genetic, biochemical and molecular techniques, with its role in RNA editing being proposed.