Trypanosoma brucei is a protozoan parasite that is the causative agent of sleeping sickness in sub-Saharan Africa. T. brucei has a complex life cycle involving passage between a mammalian host and the tsetse fly. The parasite evades the mammalian immune system via expression of Variant Surface Glycoprotein (VSG) on the cell surface. VSG genes are expressed at telomeric expression sites and at these sites are a number of Expression Site Associated Genes (ESAGs). One unusual ESAG, ESAG9, is developmentally regulated: RNA for these genes accumulates during the transition from slender to stumpy cells in the mammalian bloodstream and cellassociated protein is only detected transiently in stumpy and differentiating cells. Transgenic cell lines were generated which ectopically express one or more members of the ESAG9 gene family. Biochemical and cytological analyses using these cell lines indicated that some members of this family are glycosylated and GPI-anchored, and also that one gene, ESAG9-K69, is secreted. ESAG9-K69 is also secreted by wild-type stumpy parasites. In vivo experiments with tsetse flies did not conclusively show whether ESAG9 proteins play a role in the establishment of a tsetse fly mid-gut infection by transgenic trypanosomes. However, In vivo and ex vivo experiments using the mouse model of trypanosomiasis indicated that expression of ESAG9 proteins may alter parasitaemia in the mouse and results in a significant decrease in the proportion of CD4+ T cells in the mouse spleen.

La maladie du sommeil ou Trypanosomose Humaine Africaine (THA) est une parasitose vectorielle due à un protozoaire flagellé sanguicole du genre Trypanosoma et d'espèce brucei. Deux sous-espèces de ce parasite sont pathogènes pour l'Homme : T. b. gambiense et T. b. rhodesiense ; transmis par les mouches Tsé-Tsé présentes en Afrique subsaharienne. Cette maladie évolue classiquement en deux stades : le stade hémolyphatique qui est marqué par la présence du parasite dans le sang et la lymphe et le stade nerveux caractérisé par la présence du trypanosome dans le Système Nerveux Centrale. En l’absence de traitement cette maladie est mortelle. Actuellement les traitements accessibles à la population sont stades-dépendants. Pour contrôler un jour cette pathologie, la recherche et l’amélioration des outils de diagnostic de la maladie et le diagnostic de stade sont essentielles. C’est dans ce but que nous avons exploité une biobanque d’échantillons composée de patients infectés par T. b. gambiense et d’individus non-infectés pour : 1) Évaluer l’efficacité de biomarqueurs de stade déjà existants -Néoptérine et CXCL-13- et nous avons évalué leur potentiel sur les échantillons recueillis lors du suivi des patients post-traitements. 2) Rechercher de nouveaux biomarqueurs protéiques par spectrométrie de masse LCMS/MS. Notre étude a permis d’identifier, grâce à l’établissement d’un nouveau catalogue protéomique un grand nombre de biomarqueurs potentiels dans le liquide céphalo-rachidien, l’urine et la salive de patients. Certaines de ces protéines pourraient améliorer la prise en charge et le suivi des patients à l’avenir. / Sleeping sickness, or Human African Trypanosomiasis (HAT), is a parasitic disease caused by a flagellar protozoan of the genus Trypanosoma and brucei species. Two subspecies of this parasite are pathogenic for humans: T. b. gambiense and T. b. rhodesiense; transmitted by Tsé-Tse flies present in sub-Saharan Africa. This disease classically evolves in two stages: the hemolymphatic stage which is define by the presence of the parasite in the blood and lymph and the nervous stage characterized by the presence of trypanosome in the central nervous system. Without treatment, this disease is lethal. Currently the available treatments for patients are stage-dependent. In order to control this pathology one day, research and improvement of tools for the diagnosis of the disease and the staging is fundamental. In this context, we have exploited a samples biobank composed of T. b. gambiense-infected patients and uninfected controles to: 1) evaluate the efficacy of existing stage biomarkers -Neopterin and CXCL-13- and we assessed their potential on the samples collected during post-treatment followup of patients. 2) determine new protein biomarkers using LC-MS/MS mass spectrometry. Our study identified a large number of potential biomarkers in cerebrospinal fluid, urine and saliva through the establishment of a new proteomic catalogue. Taking into account some of these proteins may improve patient management and follow-up in the future.

As doenças tropicais têm um grande impacto sobre a saúde em países de baixa renda, estando relacionadas com condições de pobreza e desigualdade. A tripanossomíase africana é uma infecção parasitaria negligenciada incluída na agenda da Organização Mundial de Saúde. Esta enfermidade é causada pelo Trypanosoma brucei gambiense e Trypanosoma brucei rhodesiense, sendo transmitida pela mosca tsé-tsé (Glossina sp.) e geralmente fatal se não tratada. Os fármacos usados no seu tratamento são ineficazes, difíceis de administrar e causam severas reações adversas. Portanto, existe a necessidade do desenvolvimento de alternativas quimioterápicas eficazes e seguras. Assim, a enzima fosfoglicerato mutase (PGAM) surge como um importante alvo molecular. Esta enzima esta envolvida no metabolismo de glicose, sendo necessária para a viabilidade do parasito. Somado a isso, ela difere da enzima dos hospedeiros permitindo a identificação de inibidores específicos. Não obstante, esforços têm sido realizados para identificar inibidores da PGAM, bem como para elucidar sua estrutura e mecanismo de reação. Nosso propósito é obter o modelo de alta resolução desta macromolécula sem ligantes e conseqüentemente analisar a mudança de conformação que esta sofre ao se ligar ao seu substrato natural. A PGAM de Trypanosoma brucei obtida na expressão e purificação mostrou-se cataliticamente ativa nos ensaios cinéticos. Por experimentos de cromatografia de exclusão molecular observamos que a amostra purificada se comportava na forma de monômero. Dados de difração de raios-X foram coletados para cristais da macromolécula obtidos na ausência de ligantes. A estrutura cristalográfica foi resolvida a 2.3 Å, apresentando um dímero na unidade assimétrica. Ambas as moléculas do dímero estavam na forma livre e apresentava grande diferença conformacional se comparadas com as PGAMs de estruturas conhecidas que estão ligadas ao substrato ou produto natural. Por espalhamento de raios-X a baixos ângulos confirmamos que a enzima é monomérica em condições que mimetizam a fisiológica. A mudança conformacional induzida pelo ligante não afeta a topologia dos dois domínios da PGAM. Entretanto, há mudanças nos ângulos torcionais da cadeia principal dos laços que conectam os domínios da proteína. Além disso, o metal cobalto parece estar envolvido na estabilização da estrutura terciária da PGAM na conformação livre. Finalmente, este novo modelo estrutural pode contribuir para o esforço internacional de desenvolver fármacos tripanocidas / Tropical diseases represent a major burden on population health in low-incoming countries, being related to poverty and social disadvantage. African trypanosomiasis is a neglected parasitic infection on the agenda of World Health Organization. This disorder is caused by Trypanosoma brucei gambiensis and Trypanosoma brucei rhodesiensis, transmitted by the tsetse fly (Glossina sp.), and usually fatal if untreaded. The drugs used in the treatment are ineffective, difficult to administer, and cause severe adverse reactions. Therefore, there is a need to develop effective and safe chemotherapies. Thus, the enzyme phosphoglycerate mutase (PGAM) emerges as an important molecular target. This enzyme is involved in glucose metabolism, and is necessary for viability of the parasite. Moreover, it differs from the host enzyme allowing the identification of specific inhibitors. Nevertheless, efforts have been made in identifying PGAM inhibitors and to elucidate their structure and mechanism of reaction. Our purpose is to obtain the high resolution model of the macromolecule free from ligands and consequently to analyze the change in conformation that undergoes upon binding to its natural substrate. Trypanosoma brucei PGAM obtained in the expression and purification was shown to be catalytically active in the kinetics assays. In the size exclusion chromatography we observed that the purified sample behaves as a monomer. X-ray diffraction data were collected for crystals of the macromolecules obtained in the absence of ligands. The crystal structure was solved to 2.3 Å, showing a dimmer in the asymmetric unit. Both molecules of the dimmer were in free form, and had a large conformational difference compared with those of know PGAM structures that are connected to the natural substrate or product. Small angle X-ray scattering confirm that the enzyme is monomeric under conditions that mimic the physiological. Ligand-induced conformational change does not affect the topology of the two domains of the PGAM. However, there are changes in torsional angles of the main chain of the loops that connect the protein domains. Additionally, the metal cobalt seems to be involved in stabilizing the tertiary structure of PGAM in the free conformation. Finally, this new structural model may contribute to the international effort to develop trypanocidal drugs.

This study describes the results of a cross-sectional survey conducted in Mambwe district in the Eastern Province in Zambia. It uses a One Health approach to assess the impact of veterinary, medical, environmental and social determinants on animal and human health and wellbeing. One Health is defined as a holistic and interdisciplinary approach that describes the complexities between people, animals, the environment and their health. Human wellbeing is defined in this thesis as 'a condition in which all members of society are able to determine and meet their needs and have a large range of choices to meet their potential' (Prescott-Allen, 2001). As a first step, eight focus group discussions with the inhabitants followed by key informant interviews with stakeholders in the area were conducted to give a primary impression and narrow down the problems in relation to animal and human health of the area in general. Following this, a randomized selection of 210 households was visited and in each household blood samples were taken from all humans and all animals belonging to five animal species, namely cattle, goats, sheep, pigs and dogs. A third of the households did not keep any of the animal species chosen for sampling, but their inclusion was important for the social analysis. In all of these 210 households a wellbeing questionnaire was administered and, for every human and animal sampled, a health questionnaire. The study area falls within the tsetse-infested region of Zambia. It has a high wildlife density reflecting the proximity of several national parks and is historically endemic for both human and animal African trypanosomiasis (HAT&AAT). Therefore humans and animals were tested for trypanosomiasis using internal transcribed spacer (ITS) polymerase chain reaction (PCR). Since it is important as a differential diagnosis, malaria was tested for by a rapid diagnostic test in the field from human blood. Sera from mature individuals from all animal species except pigs were tested in a field laboratory for brucellosis using the Rose Bengal test. Additionally, cattle and dogs were tested for five genera of tick-borne infections (TBI) including Anaplasma, Ehrlichia, Theileria, Babesia and Rickettsia using reverse line blot (RLB) in the laboratory at the University of Edinburgh (UoE). The blood samples for PCR and RLB analysis at UoE were stored on WhatmanTM FTA cards. A total of 1012 human samples were tested for HAT and none found positive. 1005 (seven people had been tested positive or treated against malaria shortly before the sampling) people tested for malaria showed an overall prevalence of 15% (95% CI 13.2-17.7). None of the 734 Rose Bengal tests showed up positive for brucellosis. The prevalence of AAT in 1275 samples tested was much lower compared to former samplings; in cattle 22% (95% CI 18-27.2), in goats 7% (95% CI 4.5-9.2), in pigs 6% (95% CI 3.2-9.4), in dogs 9% (95% CI 5.2-13.6) and no samples were found positive in sheep. The prevalence of TBIs is much more complex with many multiple infections. A total of 340 cattle and 195 dogs were tested. In cattle the number of samples positive for any microorganism was as follows; 92% (95% CI 88- 94.2). Overall there were fewer positive samples from dogs with 25% of animals infected (95% CI 19.2-31.8). The wellbeing and health questionnaires were designed to help to identify possible risk factors for the above-mentioned diseases and signs, such as fever, diarrhoea and seizures, indicative for several other diseases. The results of these surveys might also help to identify potential reasons for a lower or higher prevalence of trypanosomiasis and malaria found than expected from previous studies. Additionally, information on personal happiness, attitudes towards veterinary and medical services, medical treatments received, education, women's reproductive history, drug abuse, people's perceptions of changes in environment and agriculture, demography, poverty and migration were collected via the questionnaires alongside information on livestock demographics and fertility. One of the main conclusions is that both medical and veterinary health care systems suffer from a number of shortcomings. The distance to appropriate treatment and care facilities is far and the necessary drugs are often unavailable. Also, both the knowledge and technology for diagnosing selected diseases is not in place. This study suggests that neurocysticercosis (NCC) plays an important role in this area due to the high number of seizures reported in people, in whom treatment for epilepsy was unsuccessful. Samples taken from a few pigs indicated the presence of Taenia solium, the causal agent of NCC. Furthermore, many of the TBIs are of zoonotic nature and further investigations must be made to begin to assess the burden of these diseases in humans and animals. Environmental changes such as degradation of the vegetation are likely to have an influence on the prevalence of studied diseases and this aspect is being investigated further in other studies. Due to the nature of a cross-sectional study, only limited conclusions can be drawn on the causal relationships of disease prevalence, but the social analysis conducted in this study confirmed the interactions of selected factors related to health and wealth unique for this study area.

The objective of this thesis was to study whether heterocyclic dicationic compounds that are minor groove binders have the ability to inhibit the digestive properties of type II restriction enzymes which bind to the major groove of the DNA. If these compounds do possess the ability to inhibit restriction enzymes, then what factors influence their ability to inhibit the restriction enzymes? The methods used to study the interactions of DNA, compounds, and enzymes are gel electrophoresis, DNA thermal melting, and circular dichroism. The results from this project reveal that the minor grove binding compounds are able to inhibition type II restriction enzymes. The inhibition is heavily influenced by compound structure and the DNA binding sequence of the enzyme.

DNA minor groove binding compounds have had limited therapeutic uses, in part due to problems with sequence specificity. A two-site model has been developed to enhance specificity, in which compounds bind to two short AT sites separated by one or two GC base pairs. Using thermal melting, heterocyclic dications with this capability were tested with various oligonucleotides for binding affinity and specificity. Compounds of interest were further probed using circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling. Several compounds were found to “jump” a GC base pair, binding to AT sites in the minor groove of DNA with a two-site recognition mode. One compound was also found to recognize a single intervening GC base pair. Compounds with terminal, non-polar amidine extensions were found to have increased DNA binding compared to analogs with terminal amidines. This unique, two-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.

Mitochondrial gene expression in trypanosomatid pathogens requires extensive post transcriptional modification called RNA editing. This unique molecular mechanism, catalyzed by a multiprotein complex (the editosome), generates translatable transcripts for essential components of parasite respiratory complex. How editosome proteins are assembled and perform RNA editing is not fully understood. Moreover, previous studies have shown that editosome proteins are essential for parasite survival, which makes editosome as a suitable target for drug discovery. Currently, researchers use radio-labeled based assays to monitor RNA editing process. However, these assays are not suitable for high throughput screening of editosome inhibitors, have low detection limits, and cannot monitor RNA editing in real time. / Therefore, to develop a sensitive high throughput RNA editing assay, we have designed a sensitive hammerhead ribozyme-based fluorescence assay. Ribozyme structure was remodeled by adding or removing uridylate in its conserved catalytic core to make an inactive ribozyme. In the presence of the editosome, inactive ribozyme is edited to an active ribozyme. Consequently, hammerhead ribozyme activity can be measured by cleaving its fluorescently labeled substrate. We have shown that higher sensitivity is achieved using fluorescent based assay than conventional radio-labeled assay. Moreover, we can use this assay for rapid identification and characterization of the editosome inhibitors against RNA editing activities in trypanosomatids.

Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb was purified from bloodstream forms of T. b. brucei by a combination of three phase partitioning (between ammonium sulfate and tertiary butanol), and chromatography on quaternary amine or pepstatin A-Sepharose resins. Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has cathepsin L-like specificity for synthetic substrates and readily degrades various proteins. In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no trypanopain-like activity was detectable in the blood of infected hosts, so it appears that trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host proteins. Antibodies against a peptide corresponding to a region of the trypanopain active site were produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting antibodies were produced and these effects varied with the substrate tested. Thus, the in vivo effects of anti-trypanopain antibodies will only become clearly understood once the physiological substrates of trypanopain have been identified. Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely intracellular target of these inhibitors, indicating that the enzyme is essential for parasite viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus, trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.