As enzimas desempenham papéis essenciais no metabolismo celular. A ausência ou inativação delas podem trazer sérios prejuízos para o desempenho fisiológico de um organismo. Algumas enzimas são comuns a todos os organismos, outras, por sua vez, podem estar ausentes em determinados grupos biológicos, o que pode representar um excelente ponto de partida para estudos bioquímicos que objetivam o desenvolvimento de fármacos mais efetivos. Nesse sentido, é destacado neste trabalho um grupo de enzimas conhecidas como Old Yellow Enzyme (OYE), que são comumente encontradas em bactérias, leveduras, plantas, protozoários, mas estão ausentes em mamíferos. Conhecer a relação estrutura-função dessas proteínas pode trazer alternativas para tratamentos de doenças nas quais o agente etiológico depende da funcionalidade de uma OYE. Dessa forma, doenças negligenciadas, como as leishmanioses e doença de Chagas tornam-se um alvo interessante para o estudo dessas proteínas. Já foi demonstrado o papel que a OYE de Trypanosoma cruzi (TcOYE) desempenha no metabolismo das prostaglandinas, contudo, os dados sobre o comportamento dessas enzimas frente a vários compostos descritos como substratos e a descrição do papel fisiológico dessas proteínas não é completamente compreendido. Além disso, não há informações estruturais e funcionais sobre a OYE de Leishmania braziliensis (LbOYE) na literatura. Assim, um dos principais objetivos desse trabalho de doutorado foi determinar parâmetros estruturais e funcionais das proteínas LbOYE e TcOYE e analisá-los de forma comparativa. Os resultados apresentados sugerem que a LbOYE foi obtida em sua forma enovelada e que a mesma apresenta uma forma sutilmente mais alongada que sua ortóloga TcOYE. Adicionalmente, os resultados também mostram que a proteína LbOYE apresenta menor estabilidade estrutural e maior flexibilidade comparada à TcOYE. Os ensaios de prospecção de ligantes apresentados nesse trabalho mostraram que as enzimas foram obtidas em sua forma funcional e que o composto menadiona foi o que apresentou menor afinidade de interação dentre os compostos avaliados. Além disso, testes de atividade enzimática em baixa pressão de oxigênio com a proteína TcOYE se mostraram efetivos para determinação de moduladores da atividade funcional da proteína. Este trabalho é pioneiro na caracterização estrutural e funcional da LbOYE. / Enzymes play key roles in cellular metabolism. The absence or inactivation of them can cause serious damage to the physiological performance of an organism. Some enzymes are common to all organisms; others, in turn, may be absent in certain biological groups, which may represent an excellent starting point for biochemical studies aiming at the development of more effective drugs. In this sense, it is highlighted in this work a group of enzymes known as Old Yellow Enzyme (OYE), which are commonly found in bacteria, yeasts, plants, protozoa, but are absent in mammals. Knowing the structure-function relationship of these proteins may provide alternatives for treatments of diseases in which the etiological agent depends on the functionality of an OYE. Thus, neglected diseases such as leishmaniasis and Chagas\' disease become an interesting target for the study of these proteins. However, the role that Trypanosoma cruzi OYE (TcOYE) plays in the metabolism of prostaglandins has been demonstrated, however, data on the behavior of these enzymes against various compounds described as substrates and description of the physiological role of these proteins is not fully understood. In addition, there is no structural and functional information on the OEE of Leishmania braziliensis (LbOYE) in the literature. Thus, one of the main objectives of this doctoral project was to determine structural and functional parameters of LbOYE and TcOYE proteins and to analyze them in a comparative way. The presented results suggest that the LbOYE was obtained in its endovelada form and that it presents a subtly more elongated form than its ortholog TcOYE. In addition, the results also show that the LbOYE protein presents lower structural stability and greater flexibility compared to TcOYE. The ligand prospecting trials presented in this study showed that the enzymes were obtained in their functional form and that the menadione compound had the lowest interaction affinity among the evaluated compounds. In addition, low oxygen pressure enzymatic activity tests with the TcOYE protein proved to be effective for the determination of modulators of the functional activity of the protein. This work is a pioneer in the structural and functional characterization of LbOYE.

Tsetse flies, Glossina species, are the biological vectors of Trypanosoma species which cause animal African trypanosomiases (AAT) in livestock (especially cattle) in sub-Saharan Africa. This disease is often fatal without treatment and negatively impacts on rural, agricultural and economic development. On the Jos Plateau, north central Nigeria, AAT was historically of little significance due to the presumed absence of tsetse and Fulani pastoralists were encouraged to settle there. But over the last 30 years, the disease has become widespread and highly prevalent in the area. This has been attributed to the expansion of tsetse on the plateau, frequent migrations of cattle to areas with higher tsetse densities and the presence of other biting flies which serve as mechanical vectors. In the current study, the presence and abundance of tsetse was determined in selected villages using biconical tsetse trap surveys. The low number of flies trapped suggests that tsetse expansion has been very limited within the plateau but the fact that trypanosome DNA was present in over half of these flies implicates them in AAT transmission. The migration of a herd of cattle was also tracked and during the period, blood samples were collected from the cattle and examined for trypanosomes using molecular techniques. Despite prophylactic treatment and deltamethrin sprays, results showed that a significant proportion of the animals (52%) had become infected with T. vivax over the migration period. Tsetse flies (G. palpalis) were also slightly more abundant in some of parts of the migration area. Potential mechanical vectors (Stomoxys spp. and Tabanidae) were trapped and results obtained from the examination of their mouthparts for trypanosomes indicate their involvement in transmission. However, it is difficult to make any definite conclusions about their overall contribution which is thought to be minimal and more studies are needed to clarify their significance. It is concluded that trypanosomiasis risk from tsetse on the Jos Plateau is currently low and seasonal migration appears to be the main driver of AAT transmission by exposing cattle to more tsetse for longer periods. Other biting flies may play a limited role which remains undetermined. Continued monitoring of cattle and tsetse across the plateau over the next few years is important and the careful use of trypanocides and insecticide treated cattle is recommended as an appropriate control strategy.

This research aims to utilise ligand-based target prediction to (i) understand the mechanism of action of African natural products (ANPs), (ii) help identify patterns of phylogenetic use in African traditional medicine and (iii) elucidate the mechanism of action of phenotypically active small molecules and natural products with anti-trypanosomal activity. In Chapter 2 the objective was to utilise ligand-based target prediction to understand the mechanism of action of natural products (NPs) from African medicinal plants used against cancer. The Random Forest classifier used in this work compares the similarity of the input compounds from the natural product dataset with compound-target combinations in the training set. The more similar they are in structure, the more likely they are to modulate the same target. Natural products from plants used against cancer in Africa were predicted to modulate targets and pathways directly associated with the disease, thus understanding their mechanism of action e.g. "flap endonuclease 1" and "Mcl-1". The "Keap1-Nrf2 Pathway" and "apoptosis modulation by HSP70", two pathways previously linked to cancer (which are not currently targeted by marketed drugs, but have been of increasing interest in recent years) were predicted to be modulated by ANPs. In Chapter 3, we aimed to identify phylogenetic patterns in medicinal plant use and the role this plays in predicting medicinal activity. We combined chemical, predicted target and phylogenetic information of the natural products to identify patterns of use for plant families containing plant species used against cancer in African, Malay and Indian (Ayurveda) traditional medicine. Plant families that are close phylogenetically were found to produce similar natural products that act on similar targets regardless of their origin. Additionally, phylogenetic patterns were identified for African traditional plant families with medicinal species used against cancer, malaria and human African trypanosomiasis (HAT). We identified plant families that have more medicinal species than would statistically be expected by chance and rationalised this by linking their activity to their unique phyto-chemistry e.g. the napthyl-isoquinoline alkaloids, uniquely produced by Acistrocladaceae and Dioncophyllaceae, are responsible for anti-malarial and anti-trypanosome activity. In Chapter 4, information from target prediction and experimentally validated targets was combined with orthologue data to predict targets of phenotypically active small molecules and natural products screened against Trypanosoma brucei. The predicted targets were prioritised based on their essentiality for the survival of the T. brucei parasite. We predicted orthologues of targets that are essential for the survival of the trypanosome e.g. glycogen synthase kinase 3 (GSK3) and rhodesain. We also identified the biological processes predicted to be perturbed by the compounds e.g. "glycolysis", "cell cycle", "regulation of symbiosis, encompassing mutualism through parasitism" and "modulation of development of symbiont involved in interaction with host". In conclusion, in silico target prediction can be used to predict protein targets of natural products to understand their molecular mechanism of action. Phylogenetic information and phytochemical information of medicinal plants can be integrated to identify plant families with more medicinal species than would be expected by chance.

Orientador: Luiz Carlos Marques / Banca: Rosangela Zacarias Machado / Banca: Fabiano Antonio Cadioli / Banca: Percílio Brasil dos Passos / Banca: Thais Helena Constantino Patelli / Resumo: Trypanosoma evansi é patogênico para a maioria dos animais, acometendo bovinos, bubalinos, caprinos, ovinos, suínos, cães, quatis, capivaras, camelos e outras espécies animais em áreas tropicais e subtropicais do globo terrestre sendo, no Brasil, a doença endêmica no pantanal mato-grossense. O presente estudo teve como fito principal estudar a evolução clínica, as alterações hematológicas, bioquímicas sérica, imunológicas e anatomopatológicas de bovinos infectados experimentalmente com T. evansi. Para tal, foram utilizados oito bovinos, clinicamente sadios e sorologicamente negativos para T. evansi. Três foram mantidos como testemunhos e cinco inoculados com T. evansi. Exames físicos, parasitológicos, hematimétricos e bioquímicos séricos (proteínograma, índice ictérico e glicose) e do líquido cefalorraquidiano foram realizados. Nos exames físicos realizados nos bovinos até 525° DAI não foi notada nenhuma anormalidade clínica com relação à temperatura retal, batimentos cardíacos, frequência respiratória, movimentos ruminais, aspectos de membranas mucosas (nasal, conjuntival, oral, vaginal e/ou prepucial) e dos linfonodos externos (mandibulares, maxilares, parotídeos, cervicais superficiais, sublíacos e mamários). A presença de tripomastigotas foi demonstrada através da prova biológica nos bovinos 01, 06 e 08 no15° DAI, bovinos 06 e 07 no 30° DAI, bovinos 01 e 06 no 45° DAI, bovino 06 no 60° DAI, bovino 01 no 75° DAI. As contagens de hemácias, os teores de hemoglobina e os volumes globulares dos bovinos, experimentalmente infectados, variaram dentro dos limites de normalidade para a espécie bovina. O VGM, HGM e CHGM, apresentam alterações pontuais. / Abstract: Trypanosoma evansi are pathogenic to most of animals, affecting cattle, buffaloes, goats, sheep, pigs, dogs, coatis, capybaras, camels and other animals in tropical and subtropical areas of the globe, and, in Brazil, it causes an endemic disease in the Pantanal Mato Grosso. This study primarily aimed to study the clinical, hematological, biochemical, immunological and pathological alterations in cattle experimentally infected with T. evansi. For this purpose, we used eight animals, clinically healthy and serologically negative for T. evansi. Three animas were kept as evidence and five were inoculated with T. evansi. Physical, parasitological, hematological and serum biochemical (proteins, icteric index and glucose) and cerebrospinal fluid examination were performed. In the physical examination conducted in cattle up to 525th DAI were not observated any clinical abnormality in concerning rectal temperature, heart rate, respiratory rate, ruminal movements, aspects of the mucous membranes (nasal, conjunctival, oral, vaginal and / or specimen) and external nodes (mandibular, maxillary, parotid, superficial cervical, breast and sublíacos). The presence of trypomastigotes was demonstrated by bioassay in cattle 01, 06 and 08 no 15th DAI, cattle 06 and 07 at 30° DAI, cattle 01 and 06 on the 45 th DAI, cattle 06 in 60 th DAI, cattle 01 in 75 th DAI. Red blood cells counts, hemoglobin content and volume cell of experimentally infected cattle were within normal limits for the bovine species. The MCV, MHC and MCHC, showed specific changes. Physical examination of the cerebrospinal fluid did not show alterations in appearance and coloration. Morever, using the Giensa-stained blood smears, buffy coat technique (BCT) and mouse inoculation procedure were negative for T. evansi tripomastigote. Serum protein concentrations, identified 26 proteins with molecular weights ranging from 20 to 245 KD. / Doutor

Orientador: Simone Baldini Lucheis / Resumo: A família Trypanosomatidae é composta por protozoários flagelados da ordem Kinetoplastidae. Os protozoários do gênero Trypanosoma, causadores das tripanossomíases e gênero Leishmania, causadores das leishmanioses, são os parasitos de maior interesse médico e veterinário As tripanossomíases são causadas por diferentes espécies, dentre elas: Trypanosoma cruzi, Trypanosoma. theileri, Trypanosoma equiperdum, Trypanosoma evansi e Trypanosoma vivax, sendo os três últimos de maior importância para os animais de produção, causando prejuízos econômicos para o setor agropecuário. Com o objetivo de avaliar a ocorrência de tripanosomatídeos no municipío de Bauru-SP, região endêmica para leishmaniose, foram avaliados 200 animais, sendo 100 bovinos (Bos taurus) e 100 equídeos (Eqqus spp.), procedentes de áreas urbanas e periurbanas do município. Para entender a distribuição de fatores de riscos na análise espacial, avaliou-se fatores epidemiológicos das leishmanioses, relacionados a seu diagnóstico nos animais de produção avaliados e também em cães e em humanos em diferentes bairros do município de Bauru, visando sua compreensão e integrá-las a estratégias de controle da doença. O diagnóstico dos animais foi baseado em técnicas parasitológicas: esfregaço sanguíneo e hemocultura; sorológicas: Reação de Imunofluorescência Indireta (RIFI) e Enzyme Linked Immunosorbent Assay (ELISA) e moleculares: Reação em Cadeia da Polimerase (PCR) e seqüenciamento. Não foram encontradas formas sugestivas de... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Poverty and disease are bound together in rural communities of sub-Saharan Africa (SSA) exacerbated by weak social services and conflict. The infectious disease burden in SSA combines the neglected tropical diseases (NTDs) and the 'big three' (malaria, HIV/AIDS and tuberculosis), so-called because they attract more global attention and hence funding. NTDs include human African trypanosomiasis (HAT or sleeping sickness), first noticed by the outside world during the slave trade era and later in the 2-th century by widespread epidemics of disease across the tsetse fly belt. HAT describes two diseases: i) Gambian HAT caused by Trypanosoma brucei gambiense is characteristically chronic with an infectious period lasting up to three years and ii) Rhodesian HAT caused by T.b. rhodesiense is an acute disease, killing its victim within weeks of infection. The two diseases are frequently considered together as both are transmitted by tsetse flies, the parasites are morphologically indistinguishable and the associated diseases are both fatal if left untreated. However, the two diseases are clinical, epidemiologically and geographical distinct, each requiring different control strategies. Under field conditions, where microscopy is the basic diagnostic tool, differentiation is simply by geographical location of the patient; the Great Rift Valley separates the Gambian disease present in West and Central Africa, from East and southern Africa's Rhodesian disease. Control strategies are also distinct; while the Belgian and French colonial strategies to control the disease were patient-centred, the British colonial powers in East Africa were motivated by the effect of tsetse borne diseases on animal health. Towards the end of the colonial ear, both types of disease were heading for elimination but during the immediate post-colonial era in the 1960s, political instability compromised the rigid HAT control programs that had been put in place. For zoonotic Rhodesian sleeping sickness, complex tsetse control programmes proved difficult to maintain and to justify economically; for Gambian sleeping sickness the generalised breakdown of medical services allowed the disease to return, sometimes to devastating levels. The millennium development goals (MDGs) set out in 2000, highlighted specific challenges and opportunities for national and global development. HAT impacts national health goals of national development plans and MDGs and impedes rural development of SSA. NTDs were not addressed directly by MDGs but the World Health Organization (WHO) has reaffirmed its commitment not only to control of HAT but also to eliminate it as a public health problem by 2020. Currently there are 25 countries reporting HAT to WHO, and while the overall prevalence of HAT across Africa continues to fall, epidemics have been recorded, particularly from central Africa, South Sudan and Uganda. Uganda is uniquely, the only country affected by both T.b. gambiense and T.b. rhodesiense and until the present study, there was no evidence to suggest that the two parasite species co-existed in Uganda. The development of a new control paradigm for T.b. rhodesiese in South East Uganda has lowered the incidence of human infections and, more importantly, halted the northerly spread of this parasite. However, recurring epidemics in several established and new disease foci in central Uganda highlight the difficulties involved in eliminating this disease. The present study assesses past and present HAT control strategies centred on Dokolo, Kaberamaido and Soroti Districts located at the centre of Uganda. These districts are highly endemic for T.b. rodesiense, they represent the region of concern for overlap with T.b. gambiense foci in central Uganda, and are the current focus of the Stamp out Sleeping sickness control initiative. The point prevalence of T. brucei s.1 in cattle reservoir from villages with (out) reported human disease located at specific distances to Otuboi, Chagwere and Ochero cattle markets, was evaluated before and six months after trypanocidal treatment, to assess the transferrable impact of zoonotic T.b. rhodesiense to the human population. Overall, the proportion of T. brucei s.1 in cattle dropped significantly from 22% at baseline to 9% six months after trypanocide treatment (P < 0.05, Chi-square + 17.92, 95% C.I. + 1.71 to 4.49). All villages located in sub-counties that received at least 80% treatment coverage had a drop in T. brucei s.1 prevalence from 30.4% (95%, C.I + 22.8 to 38.0) before treatment was done, to 12.9% (95%, C.I. + 7.4 to 18.4) six months after treatment. More specifically, impact on human infective T.b. rhodesiense was also halved. In fact only three cattle were detected with the parasite six months after treatment compared with six from those sampled as baseline. This study also utilises documented cases between 2009 and 2012 to assess the current HAT reporting system for monitoring and evaluating transmission dynamics of the disease. Using a questionnaire, capacity and preparedness of healthcare professionals to respond to disease epidemics was assessed. The point prevalence of sleeping sickness in the three districts in 2009 was determined by screening volunteers. Microscopic examinations detected trypanosomes in four volunteers (4/5311 or 0.075 %) while PCR detected significantly more infections (24, p < 0.001). Multiplex PCR showed that ten of the Trypanozoon infections were T.b. rhodesiense while nested PCR identified four infections as T.b. gamiense, indicating that the distribution of the two forms of sleeping sickness overlaps in Uganda. Second phase investigations followed up the PCR positive cases; these people were screened again, together with members of their homestead and the inhabitants of three neighbouring homes. Besides microscopy and PCR, study subjects were examined clinically for sleeping sickness and completed a questionnaire to assess community recognition of the disease. This extended screen revealed no new cases underlining the importance of stringent early screening that PCR techniques can provide. At local healthcare centres, 54% of reported sleeping sickness cases were diagnosed only at the late stage, indicating a weakness in early diagnosis and hence early reporting. Interviews with local health workers also revealed weaknesses in recognition of clinical signs and a gap in diagnostic capacity. While records at treating hospitals remain a useful indicator for targeting active foci of infection, improvement in capacity to diagnose HAT at an early stage should contribute both to rural health and disease control strategies and also towards WHO's 2020 target of elimination of HAT.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

Over a billion individuals worldwide suffer from neglected diseases. This equates to approximately one-sixth of the human population. These infections are often endemic in remote tropical regions of impoverished populations where vectors can flourish and infected individuals cannot be effectively treated due to a lack of hospitals, medical equipment, drugs, and trained personnel. The few drugs that have been approved for the treatments of such illnesses are not widely used because they are riddled with inadequate implications of cost, safety, drug availability, administration, and resistance. Hence, there exists an eminent need for the design and development of improved new therapeutics. Influential world-renowned scientists in the Consortium for Parasitic Drug Development (CPDD) have preformed extensive biological testing for compounds active against parasites that cause neglected diseases. These data were acquired through several collaborations and found applicable to computational studies that examine quantitative structure-activity relationships through the development of predictive models and explore structural relationships through docking. Both of these in silico tools can contribute to an understanding of compound structural importance for specific targets. The compilation of manuscripts presented in this dissertation focus on three neglected diseases: trypanosomiasis, Chagas disease, and leishmaniasis. These diseases are caused by kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., respectively. Statistically significant predictive devices were developed for the inhibition of the: (1) T. brucei P2 nucleoside transporter, (2) T. cruzi parasite at two temperatures, and (3) two species of Leishmania. From these studies compound structural importance was assessed for the targeting of each parasitic system. Since these three parasites are all from the Order Kinetoplastida and the kinetoplast DNA has been determined a viable target, compound interactions with DNA were explored to gain insight into binding modes of known and novel compounds.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2010. Title from PDF t.p. Includes bibliographical references.

The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.