Bloodstream trypanosomes initiate differentiation to procyclic forms in response to a citrate/ cis-aconitate (CCA) signal. A cell line was previously selected (“defective in differentiation-clone 1”; DiD1) that was unable to differentiate to procyclic forms (Tasker et al. (2000)). Additionally, expression profiling of this line in comparison to the parental line by macroarray hybridisation identified two differentially-expressed transcripts from an 8 gene cluster of highly homologous genes we named PAD genes (Proteins Associated with Differentiation). Members of this family show distinct expression profiles throughout the trypanosome lifecycle at both the mRNA and protein level, and are localised to the cell surface membrane of the cell. At least 1 member of the family (PAD1) shows stumpy form specific RNA and protein expression, representing the first useful molecular marker for this stage, and exhibits biochemical specificity for citrate. Additionally, another member of this family (PAD2) is upregulated in response to low temperature, a condition reported to cause hypersensitivity to CCA. Finally, RNAi mediated ablation of the PAD gene transcripts compromised the capacity of stumpy form trypanosomes to differentiate to the procyclic form in response to CCA. These combined expression, cytological, reverse-genetic and biochemical data make PAD proteins excellent candidates for recognition of the signal to initiate differentiation in response to CCA.

The mitochondrion contains its own genome. Replication of the mitochondrial DNA (mtDNA) is an essential process that, in most organisms, occurs through the cell cycle with no known mechanism to ensure spatial or temporal constrain. Failures to maintain mtDNA copy number affects cellular functions causing several human disorders. However, it is not clear how the cells control the mtDNA copy number. The mtDNA of trypanosomes, known as kinetoplast DNA (kDNA), is a structurally complex network of topologically interlocked DNA molecules (minicircles and maxicircles). The replication mechanism of the kDNA differs greatly with all other eukaryotic systems. Key features of the kDNA replication mechanism include defined regions for main replication events, coordination of a large number of proteins to drive the replication process, and replication once per cell cycle in near synchrony with nuclear S phase. Two main regions known as the kinetoflagellar zone (KFZ) and the antipodal sites are where main kDNA replication events are known to occur (i.e, initiation, DNA synthesis and Okazaki fragment processing). So far, the localization of the proteins involved in kDNA replication is restricted to two main regions: the KFZ and the antipodal sites. Three mechanisms that directly regulate kDNA replication proteins and serve to control kDNA replication have been proposed: (1) Reduction and oxidation status of the universal minicircle sequence binding protein (UMSBP) controls its binding to the origin sequence, (2) Trans-acting factors regulate the stability of mRNA encoding mitochondrial Topoisomerase II during the cell cycle and, (3) Regulation of TbPIF2 helicase protein levels by a HslVU-like protease to control maxicircle copy number. These mechanisms seem to be protein specific and it appears that a combination rather than a single mechanism regulates kDNA replication. In this study we used Trypanosoma brucei to understand how mitochondrial DNA replication is controlled. We investigated the mechanism of how proteins transiently localize to the sites of DNA synthesis during cell cycle stages. Our data provides a comprehensive analysis of the first two examples of T. brucei kDNA replication proteins that have a cell cycle dependent localization (Ch. 2 and 3). The localization of two of the three essential mitochondrial DNA polymerases (TbPOLIC and TbPOLID) is under tight cell cycle control and not regulated by proteolysis. TbPOLIC and TbPOLID localize to the antipodal sites during kDNA S phase, however, at other cell cycle stages TbPOLIC becomes undetectable by immunofluorescent analysis and TbPOLID disperses through the mitochondrial matrix. In agreement with this data, TbPOLIC and TbPOLID replication complexes were not detected using affinity purification presumably because only a fraction of these proteins are participating in replication at a given time (Ch. 4). We propose that spatial and temporal changes in the dynamic localization of essential kDNA replication proteins provide a novel mechanism to control kDNA replication.

Une nouvelle voie d’immunomodulation, l’induction de l’arginase par les trypanosomes chez leurs hôtes, a été identifiée et caractérisée. Pour éviter la réponse cytotoxique de l’activation « classique » M1 des macrophages et bénéficier de leur activation « alternative » M2, les parasites induisent l’arginase, qui produit la L-ornithine, indispensable à leur développement. Cette voie d’immunomodulation mise en évidence chez la souris infestée par son parasite naturel, Trypanosoma musculi, est également présente dans d’autres trypanosomoses, en particulier la trypanosomose humaine africaine (THA). Une augmentation de l’arginase, retrouvée dans le sérum de patients trypanosomés, se normalise après un traitement efficace. T. brucei gambiense, parasite de l’homme, induit l’arginase au niveau des macrophages murins et des leucocytes humains. T. lewisi, parasite du rat, induit également l’arginase. Au cours de leur longue coévolution avec leurs hôtes, les trypanosomes extracellulaires ont sélectionné un procédé favorisant leur croissance, l’induction de l’arginase, par des facteurs d’excrétion/sécrétion. Nous avons produit un anticorps monoclonal dirigé contre ce facteur inducteur. Il bloque l’induction de l’arginase par T. musculi in vitro et in vivo. Chez la souris infectée, son injection diminue considérablement la parasitémie. Il a permis l’identification du facteur inducteur, une kinésine orpheline. Cet anticorps, inhibant l’induction de l’arginase par différents trypanosomes, reconnaîtrait une région conservée de la kinésine induisant l’arginase. Cette kinésine se lie à des récepteurs de la membrane des macrophages. In vitro, l’addition de mannose à des co-cultures macrophages-parasites bloque l’induction de l’arginase et la multiplication des parasites. Chez la souris infestée par T. musculi, l’injection de mannose diminue la parasitémie, qui est également réduite chez les souris Mrc1-/-, KO pour le récepteur mannose. L’utilisation de molécules ciblant la voie inductrice de l’arginase et/ou ce récepteur peut représenter une nouvelle approche thérapeutique dans les trypanosomoses. / Arginase induction, a mechanism of immunomodulation elaborated by trypanosomes has been identified. To avoid cytotoxic classical M1 macrophage activation, trypanosomes induce alternative M2 macrophage activation, which leads to L-ornithine production, essential for parasite growth. This immunomodulation pathway has been evidenced in a natural murine trypanosomiasis provoked by Trypanosoma musculi. This mechanism is also evidenced in human African trypanosomiasis (HAT). An increase in serum arginase is measured in HAT patients. A return to normal values is obtained after an efficacious treatment. Trypanosoma brucei gambiense, the causative agent of HAT, induces arginase in mouse macrophages and human leucocytes. T. lewisi, a rat parasite, also induces macrophage arginase.During host-parasite co-evolution, extracellular trypanosomes have selected a growth promoting mechanism, macrophage arginase induction by excreted secreted factor (ESF). We have produced a monoclonal antibody which inhibits trypanosome-induced arginase. This antibody blocks in vitro and in vivo T. musculi-induced arginase. Its injection into infected mice provokes a decrease in parasite load. This monoclonal antibody has allowed the identification of an orphan kinesin as the arginase inducing factor. The arginase inducing region of kinesin seems conserved among extracellular trypanosomes. Kinesin binds to macrophage membrane receptors. In vitro, addition of mannose to macrophage-parasite cocultures blocks arginase induction and parasite multiplication. Mannose injection decreases parasite load in infected mice. Compared to WT mice, parasite load is highly reduced in infected Mrc1 -/- KO mice. In trypanosomiasis, molecules targeting arginase pathway and/or mannose receptor, highly conserved in evolution, might represent new therapeutic approaches.

African trypanosomes are protozoan parasites that cause African trypanosomiasis, diseases that affect humans and their livestock. Not only has trypanosomiasis had an overwhelming effect on the development of tropical Africa in the past, but it also constitutes one of the most significant present economic problems of the continent. Trypanosomes alternate between a mammalian host and a tsetse vector using a complex life cycle. In the mammalian host the trypanosomes live as bloodstream forms (BSFs) that are so proficient at antigenic variation, and thus host immune system evasion, that no suitable vaccine candidates have yet been identified. In contrast, the lifecycle stages that exist in the tsetse vector do not undergo antigenic variation. This potentially makes the vector-occupying trypanosomes much better targets for control if strategies can be devised to disrupt their lifecycle in the vector or to interfere with their transmission to mammalian hosts. The primary impediment to developing strategies for disruption of trypanosome life cycles in tsetse is a lack of understanding of the molecular basis of trypanosome-tsetse interactions. Although several major surface molecules have been identified on insect form trypanosomes, these have not been well studied due to a lack of appropriate antibody probes and to the difficulty in obtaining sufficient quantities of the different parasite life cycle stages required for such molecular studies. My thesis research was focused on developing and using monoclonal antibody probes for analysis of expression of major surface molecules of Trypanosoma congolense, a serious pathogen of cattle in Africa. I used this species of trypanosome since in addition to being a socioeconomically important parasite, all four of its major life cycle stages can be grown in vitro in amounts sufficient for immunochemical analysis. I successfully derived and characterized monoclonal antibodies that were useful for detecting the three major surface proteins of T. congolense insect forms: glutamic acid/alanine rich protein (GARP), the T. congolense heptapeptide repeat protein (TcHRP) and congolense epimastogote specific protein (CESP). Selected monoclonal antibody probes were then employed for expression analysis of these molecules throughout the parasite life cycle using in vitro grown trypanosomes and parasites taken directly from infected tsetse. In addition, I determined the peptide epitopes for two of my GARP-specific monoclonal antibodies and in collaboration with Dr. Martin Boulanger and Jeremy Mason was able to localize the epitopes on a high resolution three-dimensional structure obtained by X-ray crystallography. This allowed us to derive a model that describes the orientation of GARP in the trypanosome surface membrane and explains the possible structure-function relationships involved in replacement of the bloodstream form variant surface glycoprotein (VSG) by GARP as trypanosomes differentiate in the tsetse vector after a bloodmeal.

Research was undertaken to discover and study trypanosome proteins that may play important roles in host-parasite or vector-parasite interactions. The methods used mass spectrometry based proteomics ideally suited for analysis of low abundance molecules. First, isobaric tags were used to monitor changes in proteins expression throughout the life cycle of Trypanosoma congolense, an economically important livestock pathogen. This was the first large scale survey of protein expression in trypanosomes. In addition to generating protein expression data for approximately 2000 different parasite proteins, 6 previously undescribed T. congolense proteins were discovered. Several of the proteins with interesting expression trends were selected for molecular characterization and monoclonal antibody derivation. Second, immunoenrichment and mass spectrometry were used to identify the cognate antigen recognized by a T. congolense-specific monoclonal antibody. The antigen, a flagellar calcium binding protein, was expressed as a recombinant protein and used to test its utility as a potential serodiagnostic antigen for diagnosis of T. congolense infections. Third, a “deep-mining” protein discovery mass spectrometric method was used to identify trypanosome proteins present in the plasma of late-stage African sleeping sickness patients. A total of 254 trypanosome proteins were unequivocally identified by tandem mass spectrometry. These findings are unprecedented since never before have such a large number of pathogen proteins been discovered in human blood using a non-biased approach (i.e. without using a targeted assay). The proteins discovered provide insights into host-parasite interactions and are strong candidates as targets for new diagnostic assays. / Graduate

Submitted by Wesley José dos Santos null (thedeiwz@hotmail.com) on 2016-07-20T20:08:55Z No. of bitstreams: 1 SANTOS WJ - dissertacao 2016.pdf: 3762980 bytes, checksum: 70891a0132a17b5b79842e9ec8acd7bb (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-22T19:05:40Z (GMT) No. of bitstreams: 1 santos_wj_me_bot.pdf: 3762980 bytes, checksum: 70891a0132a17b5b79842e9ec8acd7bb (MD5) / Made available in DSpace on 2016-07-22T19:05:40Z (GMT). No. of bitstreams: 1 santos_wj_me_bot.pdf: 3762980 bytes, checksum: 70891a0132a17b5b79842e9ec8acd7bb (MD5) Previous issue date: 2016-07-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os animais silvestres, tanto os de vida livre como os de cativeiro, podem ser reservatórios e portadores de diversos protozoários, como os integrantes do gênero Trypanosoma. Trypanosoma cruzi é um dos mais conhecidos tripanosomas, agente etiológico da doença de Chagas. Manifesta-se clinicamente com um caráter multifacetado, afetando humanos e várias espécies de animais domésticos e silvestres, como os primatas não humanos, que podem constituir-se em reservatórios do parasito. Em relação aos primatas neotropicais, ocorrem diferentes infecções por tripanosomas que também acometem humanos. No presente estudo, foram coletadas amostras de sangue de 39 primatas não humanos procedentes do Parque Zoológico Municipal de Bauru para pesquisa sorológica anti-T. cruzi e molecular de T. cruzi utilizando-se primers da região de kDNA de T. cruzi (TCZ1 e TCZ2) e da região codificadora da proteína de choque térmico (hsp70). Todos os animais foram negativos ao teste sorológico anti-T. cruzi e molecular para T. cruzi com os primers da região de kDNA. Porém, com a utilização de primers para hsp70, 11 dos 39 (28,2%) animais foram positivos. Os resultados do sequenciamento apresentaram similaridade para protozoários do gênero Trypanosoma. Apesar das dificuldades em se chegar à espécie circulante de tripanosoma entre os primatas, conclui-se que vetores triatomíneos e/ou moscas hematófagas podem estar circulando no ambiente dos animais e que ações de vigilância e busca dos mesmos são altamente recomendadas. / Wild animals, both free-living and the captive ones, can be reservoirs and carriers of various protozoa, such as members of the genus Trypanosoma. Trypanosoma cruzi, the etiologic agent of Chagas disease, is one of the most known trypanosomes. Chagas disease is manifested clinically with a multifaceted character, affecting humans and several species of wild and domestic animals, such as non-human primates, which can be reservoirs of this parasite. Regarding the Neo-tropical primates, different infections occur by trypanosomes that also can affect humans. In this study, 39 blood samples were collected from nonhuman primates from Zoo Park Municipal of Bauru, São Paulo, Brazil, by serological and molecular analysis for T. cruzi infection using specific primers for kDNA region (TCZ1 and TCZ2) and the region encoding the heat shock protein (hsp70). All animals were negative to the serological test for anti-T. cruzi and molecular for T. cruzi with primers for kDNA region. However, with the use of primers for hsp70, 11 of 39 (28.2%) animals were positive. The sequencing results showed similarity to the genus Trypanosoma. Despite the difficulties in stablish the species of trypanosome among these primates, it is concluded that triatomine vectors and/or bloodsucking flies may be present in the animal environment; so, the entomologic vigilance for these insects are highly recommended. / FAPESP: 2014/12186-4

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the in vitro and in vivo trypanocidal activity of free curcumin (F-C) and curcumin-loaded lipid-core nanocapsules (C-LNCs) against Trypanosoma evansi, as well as its antioxidant activity in vivo. In vitro tests were performed in culture medium containing T. evansi, using eight concentrations of F-C (100, 75, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 mg mL-1), and four concentrations of C-LNCs (100, 75, 50, 25 mg mL-1). The counting of alive parasites was performed in a Neubauer chamber after 1, 3, 6, 9 and 12 hours of incubation. In vitro tested showed the death of all trypanosomes treated with F-C one hour post-incubation (PI), except at concentrations below 6.25 mg mL-1. Mobile trypanosomes, treated with C-LNCs, were observed until the third hour PI, except at the highest concentration. For in vivo tests, 54 animals were divided into 8 groups (A, B, C, D, E, F, G and H), as follows: group A (not infected and treated with saline); B (not infected and treated with C-LNCs); C (infected with T. evansi and treated with blank nanocapsules of curcumin); D (infected with T. evansi and treated with DMSO ); E (infected with T. evansi and treated with saline); F ( infected with T. evansi and treated with C-LNCs at a dose of 10 mg.kg); G (infected with T. evansi and treated with F-C at a dose of 10mg/kg) and H (infected with T. evansi and treated with F-C at a dose of 100mg/kg). The results showed that the animals treated with curcumin presented a lower parasitemia compared with untreated animals. Additionally, infected animals showed an increase of nitrite/nitrate and protein peroxidation, while the infected animals, that received treatment based on curcumin, showed a reduction in these parameters. Infected groups treated with curcumin exhibited a reduction in ALT and creatinine levels when compared with the positive control group. Therefore, it was possible to conclude that F-C and C-LNCs showed trypanocidal activity in vitro; however, the curcumin in its free form appeared to be more effective. A control of parasitemia was observed in in vivo tests for the treated groups, besides a possible protective effect of curcumin on liver and kidney functions, which also would be related to the antioxidant phytochemical action proved in our study. / O objetivo deste estudo foi avaliar a atividade tripanocida da curcumina livre (C-L) e curcumina nanoencapsulada (C-N) contra o Trypanosoma evansi in vitro e in vivo, e sua atividade antioxidante in vivo. Os testes in vitro foram realizados em meio de cultura contendo T. evansi, utilizando-se oito concentrações de C-L (100, 75, 50, 25, 12,5, 6,25, 3,12, 1,56 e 0,78 mg.kg - 1) e quatro concentrações de C-N (100, 75, 50, 25 mg.kg/L- 1). Em 1, 3, 6, 9 e 12 horas após a incubação, a contagem de parasitas vivos foi realizada em câmara de Neubauer. Através dos testes in vitro, foi possível observar a morte de todos os tripanossomas tratados com C-L um hora pós-incubação (PI), exceto em concentrações inferiores a 6,25 mg.kg - 1. Tripanossomas móveis tratados com C-N foram observados até a terceira hora PI, exceto na concentração mais elevada. Para realização dos ensaios in vivo, foram utilizados 54 animais divididos em 8 grupos (A, B, C, D, E, F, G e H), sendo esses: grupo A (não infectados e tratados com solução salina), B (não infectados e tratados com C-N), C (infectados com T. evansi e tratados com nanocápsulas brancas-sem curcumina), D (infectados com T. evansi e tratados com DMSO), E (infectados com T. evansi e tratados com solução salina), F (infectados com T. evansi e tratados com C-N a uma dose de 10mg/kg), G (infectados com T. evansi e tratados com C-L a uma dose de 10 mg.kg) e H (infectados com T. evansi e tratados com C-L a uma dose de 100 mg.kg). Este estudo pode verificar que os animais tratados com curcumina mostraram uma menor parasitemia em relação aos animais não tratados. Animais infectados apresentaram um aumento de nitritos / nitratos e de peroxidação proteica, logo os animais infectados que receberam tratamento a base de curcumina apresentaram uma redução destes parâmetros. Os grupos infectados tratados com curcumina exibiram uma redução nos níveis de ALT e de creatinina em relação ao grupo de controle positivo. Conclui-se que C-L e C-N, apresentam atividade tripanocida in vitro, porém a curcumina em sua forma livre apresenta-se mais efetiva. Nos testes in vivo, foi observado controle da parasitemia nos grupos tratados e uma possível ação hepatoprotetora e nefroprotetora da curcumina, que pode estar relacionada também com a ação antioxidante do fitoquímico, comprovada em nosso estudo.

The objective of this study was to use molecular biological reagents (primers targeting different genomic loci) and methods (PCR and RFLP) to detect and characterize trypanosomes in cattle, wild animals and tsetse flies in KwaZulu-Natal, thus contributing to improved understanding of the genetic diversity of trypanosome species infecting cattle at the game/livestock interface in the KwaZulu-Natal Province of South Africa. Primers based on sequences of different loci in the trypanosome genome were used in conducting polymerase chain reactions (PCRs) on samples collected from cattle at 14 diptanks and one commercial farm around the Hluhluwe-Umfolozi Game Reserve, wild animals within Hluhluwe-Umfolozi Game Reserve and tsetse flies from the commercial farm, Hluhluwe-Umfolozi Game Reserve and two other Game Reserves. Trypanosome isolates were grown under laboratory conditions in cattle, rodents and culture medium for molecular characterizations. Overall, a total of 673 cattle, 266 tsetse flies, 141 buffaloes and 6 rhinoceros samples were analyzed. The following observations were made: two species of trypanosomes are present in KZN; T.congolense and T. vivax. The two species were found as single and as mixed infections in cattle. There are two genotypic groups of T. congolense in KZN; the Savannah- and the Kilifi-type. The two genotypic groups were found as mixed infections in cattle and in tsetse flies. Lastly, there are at least five “genomic variants” of Savannah-type T. congolense in KZN. The infection rate in cattle ranged from 5.2-91%; in tsetse flies, 11-97.5% and in wild animals it was 4.3%. Mixed infection of T. congolense and T. vivax were only observed in samples from one diptank. Mixed infections of Savannah- and Kilifi-type T. congolense were observed in samples from Boomerang commercial farm, and in tsetse flies. Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

La trypanosomose est la maladie parasitaire la plus dévastatrice en Afrique, et affecte à la fois les hommes et le bétail. Vu l’inefficacité des stratégies de contrôle actuelles, une stratégie alternative dite “anti-maladie” a été proposée dans le cadre de la trypanosomose animale. Elle vise à neutraliser les effets de la maladie plutôt qu’à éliminer le parasite. Une telle stratégie nécessite une meilleure compréhension du développement de la pathologie ainsi qu’une caractérisation détaillée des facteurs de virulence impliqués. Dans ce contexte, nous nous sommes intéressés à l’étude de l’interaction hôte/pathogène entre les trypanosomes Africains et l’endothélium de l’hôte mammifère. En comparant quatre espèces différentes de trypanosomes Africains, nous avons montré que leurs capacités d’activation des cellules endothéliales étaient distinctes. Nous avons clairement démontré que T. congolense, T. vivax et T. b. gambiense activent les cellules endothéliales via la voie de NF-ƘB, alors que T. b. brucei est incapable d’activer cette voie. Cette activation a induit une résponse pro-inflammatoire in vitro et in vivo, ce qui souligne l’importance de ce mécanisme dans le développement de la maladie. Pour la première fois, nous avons identifié une activité sialidase chez le parasite de l’homme T. brucei gambiense, et nous avons démontré que les trans-sialidases trypanosomales sont les médiateurs de cette activation endothéliale et de la réponse inflammatoire consécutive, et ceci à la fois chez les trypanosomes africains d’homme et d’animaux. De plus, nous avons montré que l’activation endothéliale implique l’activité lectin-like des trans-sialidases et non pas l’activité catalytique, ainsi que des récepteurs sialylés sur la surface endothéliale. En conclusion, ce travail a apporté des avancées considérables dans la compréhension de la relation hôte/pathogène et a permis de désigner les sialidases comme un facteur de virulence central dans le dialogue intermoléculaire durant les trypanosomoses, en faisant une cible de choix pour le vaccin « anti-maladie ». / Trypanosomiasis remains by far the most devastating parasitic disease in Africa affecting both humans and livestock. The current control strategies being not efficient, an alternative “anti-disease” strategy aiming to neutralize the pathological effects of the parasite rather than to eliminate it, was proposed. Therefore, it is essential to understand the development of pathogenesis and characterize the involved pathogenic factors. In this context, we wanted to elucidate the host-pathogen interaction between the African trypanosomes and the mammalian host endothelium. By comparing four different trypanosomes species, we showed that they displayed distinct capacities for activation of endothelial cells. We clearly demonstrated that T. congolense, T. vivax and T. b. gambiense activate the endothelial cells via the NF-ƘB pathway, but not T. b. brucei. This activation caused a pro-inflammatory response in vitro and in vivo, showing the importance of this mechanism in the development of pathogenesis. For the first time, we identified sialidase activity in the human parasite T. brucei gambiense, and demonstrated that the trypanosomal trans-sialidases are the mediators of this endothelial activation and its consequent inflammatory response, for both human and animal trypanosomes. Additionnally, we showed that endothelial cell activation is mediated by the lectin-like domain of the trans-sialidase rather than the catalytic site, and involves sialylated receptors of the endothelial cell surface. In conclusion, our study brings considerable insights into the host-pathogen relationship and designates sialidases as a central virulence factor in the molecular crosstalk during trypanosomiasis, which makes it a perfect target for the anti-disease strategy.

In 2004, the polyketide natural product, chamuvarinin (72) was isolated by Laurens et al. from the roots of Uvaria chamae, a member of the Annonaceae plant family. This unique tetrahydropyran containing acetogenin displayed potent levels of cytotoxic activity against the KB 3-1 cell line with an ED50 value of 0.8 nM. Upon initial isolation the relative and absolute stereochemical assignment of chamuvarinin (72) was unable to be readily achieved through ¹H and ¹³C NMR analysis. The initial synthetic route described herein has enabled the relative and absolute stereochemical determination of chamuvarinin (72) through the first total synthesis completed in 20 longest linear steps in 1.5% overall yield. A revised synthetic strategy towards chamuvarinin (72) was completed in 17 longest linear steps in 2.2% overall yield. The revised route facilitated the assembly of non-natural chamuvarinin-like analogues and their trypanocidal and cytotoxic activities have been assessed. The synthesis of these analogues has formed the basis of a more focussed study through the design and synthesis of simplified triazole (295), isoxazole (325) and butenolide triazole (305) analogues as potential Trypanosoma brucei (causative agent in African Sleeping sickness) inhibitors.