Dissertations / Theses: 'African trypanosomiasis. Dynein. Trypanosoma brucei'

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Kinzel, Kathryn Whitney. "Functional analysis of inner-arm dynein knockdowns in Trypanosoma brucei /." Connect to online version, 2008. http://ada.mtholyoke.edu/setr/websrc/pdfs/www/2008/268.pdf.

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Millar, Amanda E. "T-cell responses during Trypanosoma brucei infections." Thesis, University of Glasgow, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363151.

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Hickey, Meghan C. "Exploring an unusual beta-hydroxybutyrate dehydrogenase from Trypanosoma brucei." Click here for download, 2010. http://proquest.umi.com.ps2.villanova.edu/pqdweb?did=2011158651&sid=1&Fmt=7&clientId=3260&RQT=309&VName=PQD.

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Jamnadass, Harmanjeet Ramni. "Identification and characterisation of an extrachromosomal element from a multidrug-resistant isolate of Trypanosoma brucei brucei." Thesis, Brunel University, 1995. http://bura.brunel.ac.uk/handle/2438/4314.

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Drug resistance together with difficulties involved in the development of new trypanocides are a major problem in the present control of African trypanosomiasis. DNA based diagnostics for drug resistance would overcome problems in the identification of drug-resistant populations and contribute to effective control measures. However, this requires a detailed knowledge of the mode of action and the mechanisms by which trypanosomes can overcome the toxic effects of trypanocides. In this study, a search for molecular differences between a multidrug-resistant isolate of Trypanosoma brucei brucei, CP 547, and a reference drug-sensitive population, ILTat 1.4, led to the identification of a 6.6 kbp extrachromosomal element in the multidrug-resistant population. In light of the involvement of extrachromosomal elements in drug resistance in Leishmana spp. and cancer cells, the identification of the 6.6 kbp element warranted its characterisation. Several different approaches sere attempted before a sequence which hybridised to the 6.6 kbp element its eventually isolated. This sequence is represented by a 108 bp repeat sequence which forms long arrays of tandem repeats. Since N/a III is the sole restriction enzyme that cuts within the repeat, it has been referred to as an N/a III repeal The repeat is flanked by a 5 bp spacer sequence. However, a unique 5 bp direct repeat flanking two complete, and one partial copy of the N/a III repeat may signify the transposition of these sequences. Hybridisation with the N/a III repeat revealed the presence of 'higher' hybridising elements which also appear to be predominantly composed of long tandem arrays of the N/a Ill repeal Through exploitation of the p01) merase chain reaction using arbitrary primers (AP-PCR), additional sequences were identified which are associated with some of the 6.6 kbp and 'higher' hybridising elements. The 6.6 kbp element and some of the 'higher' hybridising elements display features of circular DNA molecules. The 6.6 kbp element also displays some level of size and sequence heterogeneity within different populations of the same trypanosome isolate. The copy number of the 6.6 kbp element is also not stable and appears to be directly affected by the application of selective drug pressure, but a direct association between the presence of the element and the expression of multidrug resistance could not be determined. The N/a III repeat family represents a newly identified repetitive family specific to members of the Trypanozoon subgenus. This repeat family, representing about 5% of the parasite genome, is dispersed through all size classes of chromosomes, in addition to its presence on the extrachromosomal elements. Transcriptional studies of the N/a III repeats have revealed that their transcription is developmentally regulated, since heterogeneous transcripts ranging from greater than 10 kb to smaller than 300 bp are present in the actively dividing long slender bloodstream and insect stage procyclic forms of the parasite but not nondividing, stumpy bloodstream forms. Lastly, the N/a III repeat lacks an open reading frame and transcripts do not appear to have a spliced leader sequence at the 5' end. Furthermore, there is almost an equal representation of polyadenylatcd and non-polyadenlyated transcripts.

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Mabbott, Neil A. "Nitric oxide : host-protective or host-destructive during African trypanosomiasis." Thesis, University of Aberdeen, 1995. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU543723.

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The aims of the research presented in this thesis were concerned with investigating the effect of inducible nitric oxide (NO) synthase expression during Trypanosoma brucei infections on both host and parasite. NO was shown to exhibit a potent cytostatic effect on parasite proliferation. Oxyhaemoglobin is a potent scavenger of NO. The cytostatic effects of NO on the trypanosomes were completely prevented through the addition of erythrocytes to the cultures. This implies that in the host blood-stream, NO is unlikely to be involved in the eradication of the parasites. Through the adoptive transfer of suppressor macrophages from T.brucei-infected donor mice to naive recipients, it was demonstrated that NO mediates a suppressive effect on host lymphocyte responses in vivo. Furthermore, suppressor macrophages were shown to have a finite life-span and undergo NO-mediated apoptosis. Evidence also suggested that elevated NO production in the bone marrow of T.brucei -infected mice is likely to play a significant role in the anaemia resulting from T.brucei infection. Experiments demonstrated that a soluble lysate prepared from freeze-thawed blood-stream form T.brucei, activated interferon (IFN)-gamma primed macrophages to express high levels of NO synthase. Experiments also demonstrated that viable blood-stream forms, but not procyclic form trypanosomes, released a soluble factor which in combination with IFN-gamma induced NO synthase. The absolute requirement of IFN-gamma priming for NO synthase activation by T.brucei was studied using macrophages from mutant mice lacking functional IFN-gamma receptors (IFN-gamma R -/- mutant mice). In comparison to macrophages from wild-type mice, cells from IFN-gamma-R-/- mutant mice were unable to produce NO following stimulation in vitro or infection in vivo. Finally, utilising mice with specific immunodeficiencies it was demonstrated that natural killer cells and a/b T-lymphocytes were important sources of IFN-gamma during murine T.brucei infections.

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Giles, Natalie Lydia. "Exploitation of the protein tubulin for controlling African trypanosomiasis /." Access via Murdoch University Digital Theses Project, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20060315.191003.

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7

Hamadien, Maha. "Parasite signalling and host responses in experimental and human African trypanosomiasis /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-266-3.

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Kushwaha, Manish. "TbISWI and its role in transcriptional control in Trypanosoma brucei." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:36aedf26-7bbc-4f29-9fa5-fc57c9477c23.

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ISWI is a member of a versatile family of ATP-dependent chromatin remodelling complexes involved not only in transcription regulation (initiation, elongation and termination), but also in other cellular functions like maintenance of higher order chromatin structure and DNA replication. TbISWI, a novel ATPase of the ISWI family in Trypanosoma brucei, is involved in the transcriptional repression of silent VSG expression sites (ESs) in both bloodstream form (BF) and procyclic form (PF) life cycle stages of the parasite. Using in silico analysis, I have found that TbISWI is well conserved across the eukaryotic lineage, including those members of the order Kinetoplastida that do not exhibit antigenic variation. Compared to the ISWIs of higher eukaryotes, TbISWI has greater representation of random coils within its structure, an indicator of more structural fluidity and flexibility of interaction with multiple protein partners. Using an eGFP reporter based assay, I have studied the role of TbISWI in transcriptional repression of silent areas of the T. brucei genome. TbISWI was found to be involved in preventing inappropriate transcription of the silent VSG repertoires. TbISWI was also found to downregulate transcription in RNA pol I, but not pol II, transcription units. These results argue for the presence of at least two functionally distinct TbISWI complexes in T. brucei. Using DNA staining and fluorescence in situ hybridisation (FISH), I have investigated the potential effect of TbISWI depletion on cell cycle progression and minichromosome segregation. I did not find any evidence for the role of TbISWI in the maintenance of centromeric heterochromatin in T. brucei.

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Lilley, Alison. "An investigation into the Trypanosoma brucei CDP-DAG synthase and downstream pathways." Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/3615.

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Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host, allowing a plethora of novel drug targets to be discovered and validated. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate produced by the enzyme CDP-DAG synthase (CDS), but nothing was known about CDS in T. brucei. Only one gene encodes CDS in Trypanosoma brucei (Tb927.7.220) and this was shown to encode a functional CDS by overexpression in E. coli and complementation of a yeast CDS null, which was created during this study. Expression and activity of TbCDS was confirmed in T. brucei, and was shown to be essential in both life cycle stages. Disruption of TbCDS altered the lipid profile of T. brucei, confirming a central role for CDP-DAG in phospholipid synthesis. Biochemical and morphological characterisation of mutants in TbCDS expression elucidated at least two separately localised and regulated pools of CDP-DAG and phosphatidylinositol in T. brucei. In bloodstream form these pools are localised to the Golgi and the ER, however in procyclics it is possible that both of these pools are localised to the Golgi, since no phosphatidylinositol synthase protein was detected in the ER of procyclics. Reduction in TbCDS was shown to affect cell cycle regulation and Golgi segregation possibly due to a depletion of phosphorylated phosphatidylinositols (PIPs). These studies also indicate that phosphatidylglycerol may be synthesised by the phosphatidylglycerol-phosphate synthase which may be capable of using phosphatidylserine as a substrate in a headgroup swapping reaction. TbCDS has now been genetically validated as a drug target, and has highlighted novel aspects of lipid biosynthesis in T. brucei. Collectively, these findings highlight the central role played by TbCDS and the new knowledge gained here may lead to the discovery and validation of other novel drug targets against African sleeping sickness.

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Mlozen, Madalitso Martin. "Comparative study of the effect of silver nanoparticles on the hexokinase activity from human and Trypanosoma brucei." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1017910.

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Oluwafemi, A. J., O. Okanla, P. Camps, D. Muñoz-Torrero, Z. B. Mackey, P. K. Chiang, Scott Seville, and Colin W. Wright. "Evaluation of cryptolepine and huperzine derivatives as lead compounds towards new agents for the treatment of human African Trypanosomiasis." Natural Products Inc, 2009. http://hdl.handle.net/10454/4534.

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no
The alkaloid cryptolepine (1) and eight synthetic analogues (2-8) were assessed for in vitro activities against Trypanosoma brucei. Four of the analogues were found to be highly potent with IC50 values of less than 3 nM and three of these were assessed against T. brucei brucei infection in rats. The most effective compound was 2,7-dibromocryptolepine (7); a single oral dose of 20 mg/Kg suppressed parasitaemia and increased the mean survival time to 13.6 days compared with 8.4 days for untreated controls. In addition, four huperzine derivatives (9-12) were shown to have in vitro antitrypanosomal activities with IC50 values from 303-377 nM.

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Laxman, Sunil. "cAMP signaling and regulation by phosphodiesterases in trypanosomes /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/6280.

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MacLean, Lorna. "Diverse clinical responses in Trypanosoma brucei rhodesiense human African trypanosomiasis : genetic variation in parasitic virulence or host immuno-genetics?" Thesis, University of Aberdeen, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446604.

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Diverse clinical responses have been reported in geographically distinct Trypanosoma brucei rhodesiense Human African Trypanosomiasis (HAT) foci giving rise to the idea that HAT manifests as a chronic disease in southern East African countries and increases in virulence towards the north. To study the significance of host and parasite genetics on disease severity in HAT I assessed the clinical and cytokine profiles of 275 HAT patients recruited in two northern foci (Uganda) and one southern focus (Malawi) between 1998 and 2003. This data was correlated to patient ethno-linguistic group, host genotype by analysis of immune response gene polymorphisms and trypanosome genotype by analysis of microsatellite and minisatellite loci. T. brucei in northern and southern HAT foci were distinguished by a polymorphism in the serum resistance-associated gene (SRA C-M/K) which was associated with striking differences in disease pathology and cytokine profiles. The SRA C-K polymorphism was associated with elevated TGF-β and mild disease phenotype in Malawi while the SRA C-M polymorphism was associated with dramatically elevated CSF IL-10 and IL-6, severe neuropathy and outcome in Uganda, suggesting trypanosome genotypes, by their varying ability to regulate host immune responses, caused different levels of disease severity in northern and southern foci. Within Uganda patient ethno-linguistic group was associated with marked differences in disease severity; in particular Western and Eastern Nilotic groups displayed increased disease severity and higher mortality rates which were associated with high CSF IFN-γ, IL-6 and IL-10 indicating differences in CNS immune response between ethno-linguistic groups. Furthermore, genotype frequencies at locus IL-10-1082 significantly differed between the Western Nilotic and Bantu patient groups suggesting variation in host immuno-genetics may also play a role in HAT disease severity.

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Worku, Netsanet, August Stich, Arwid Daugschies, Iris Wenzel, Randy Kurz, Rene Thieme, Susanne Kurz, and Gerd Birkenmeier. "Ethyl pyruvate emerges as a safe and fast acting agent against Trypanosoma brucei by targeting pyruvate kinase activity." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-179599.

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Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results: The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion: Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the bloodbrain- barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.

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Brown, Barber Jennifer Crystal. "Synthesis of Fused Heterocyclic Diamidines for the Treatment of Human African Trypanosomiasis and Fluorescence Studies of Selected Diamidines." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/chemistry_diss/38.

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A class of linear diamidines was synthesized for the evaluation as a treatment of Human African Trypanosomiasis. These fused heterocyclic compounds are thiazole[5,4-d]thiazoles and are of interest because the parent compound, 2,5-Bis(4-amidinophenyl)-thiazolo[5,4-d]thiazole HCl salt, which is also called DB 1929, has exhibited a low nanomolar IC50 value against Trypanosoma brucei rhodesiense and has shown selectivity for binding to the human telomere G-quadruplex over that of DNA duplex. A fluoro and a methoxy derivative have been synthesized and are currently undergoing testing for activity and binding affinity. In addition, fluorescence studies of selected diamidines were done to study the effect of structural variation on fluorescence. This data is useful since it can determine what types of moieties are needed to yield a compound that will fluoresce in the higher wavelengths (500 nm and above) of the visible spectrum, which would be advantageous in determining the uptake of the drug in the trypanosome within the endemic areas of Africa with a simple microscope.

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Geiser, Federico. "Molecular mechanisms of drug resistance in human African trypanosomiasis : investigations on the role of the Trypanosoma brucei adenosine transporter 1 /." [S.l.] : [s.n.], 2005. http://www.zb.unibe.ch/download/eldiss/05geiser_f.pdf.

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17

Fijolek, Artur. "Salvage and de novo synthesis of nucleotides in Trypanosoma brucei and mammalian cells." Doctoral thesis, Umeå : Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1850.

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Pereira, João Luís Gomes. "Infeção experimental por Trypanosoma brucei brucei em modelo murino e estudo da eficácia farmacológica do benznidazol." Master's thesis, Universidade de Lisboa. Faculdade de Medicina Veterinária, 2013. http://hdl.handle.net/10400.5/6322.

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Dissertação de Mestrado Integrado em Medicina Veterinária
A Tripanossomose Africana (TA) é uma doença parasitária provocada por várias espécies de Trypanosoma, transmitidas por dípteros do género Glossina, vulgo moscas tsé-tsé. Esta doença afeta humanos e animais, tomando nos humanos o nome de Doença do Sono, e nos animais o nome de Nagana. O diagnóstico pode ser realizado por meio de técnicas de visualização parasitária, técnicas serológicas e técnicas moleculares. A terapêutica depende da fase da doença, da espécie do parasita e da espécie do hospedeiro, tendo em atenção a elevada toxicidade dos fármacos. Este facto aliado à inexistência de uma vacina eficaz surge como justificativa deste trabalho, o estudo de alternativas terapêuticas para a TA. Os objetivos deste trabalho foram a determinação da eficácia farmacológica do Benznidazol (BNZ), um antichagásico da família dos nitroimidazóis, bem como a determinação de uma dose infetante por via oral. Para este trabalho foram utilizados 25 murganhos (Mus musculus) BALB-C e 37 murganhos CD-1, e parasitas da espécie Trypanosoma brucei brucei estirpe GVR35 distribuídos por três ensaios. Em dois ensaios de eficácia farmacológica os animais foram infetados por via IP com 500 parasitas por animal. Foi então administrado 10mg/kg BNZ SID PO, durante 5 dias no primeiro ensaio e 11 dias no segundo. Foram medidas parasitémias, pesos, e taxas de sobrevivência. Na segunda experiência foram medidos ainda títulos de IgG total, IgM total e subclasses de IgG anti-T.b.brucei, parâmetros hematológicos e concentração das citocinas IL-4, IFN-γ, NO e TGF-β1. Num terceiro ensaio pretendeu-se determinar a dose infetante por via oral, e consistiu na administração de 500 parasitas por animal, em suspensão de PBS-Glucose a um grupo de animais e a administração de 2x105 parasitas por animal a outro grupo. A análise estatística foi realizada recorrendo aos testes Wilcoxon rank-sum, Correlação de Spearman, Análise de regressão linear, Mantel-Cox log-rank test e Two-way ANOVA. Os resultados dos ensaios revelaram que não foi possível estabelecer infeção por via oral até uma dose de 2x105 parasitas/animal em veículo aquoso. Foi também possível determinar que o BNZ foi ineficaz nos protocolos estudados não controlando significativamente a parasitémia nem aumentando a sobrevivência. Relativamente a achados hematológicos o tratamento falhou em controlar a anemia, evidenciando-se uma tendência significativa para a macrocitose no grupo tratado. Os animais tratados apresentavam maiores títulos de subclasses de IgG, especialmente de IgG2a e IgG3, assim como uma maior libertação de IFN-γ, com significância confirmada por teste estatístico (Two-way ANOVA). É possível concluir que embora o BNZ não seja um bom candidato para a terapêutica de TA, é um bom imunomodulador, estimulando uma resposta Th1. É também possível concluir que com uma dose inferior a 2x105 parasitas/animal em veículo aquoso não se desenvolve infeção por via oral.
ABSTRACT - TRYPANOSOMA BRUCEI BRUCEI MURINE EXPERIMENTAL MURINE INFECTION AND STUDIES ON PHARMACOLOCICAL EFFECTIVENESS OF BENZNIDAZOLE - African Trypanosomiasis (AT) is a parasitic disease caused by several species of Trypanosoma, transmitted by diptera of the Glossina genus, also known as the tsetse flies. This disease affects humans and animals, in humans takes the name of Sleeping Sickness, and in animals takes the name of Nagana. Diagnosis can be performed by parasite visualization, serology and molecular techniques. The treatment depends on the stage of the disease, the species of parasite and host species, knowing that all the drugs for AT are very toxic. With this knowledge, and due to the lack of an effective vaccine, the justification of this work is to find new therapeutic approaches for AT. The objectives of this study were to determine the pharmacological effectiveness of Benznidazole (BNZ), a nitroimidazole antichagasic drug, and ascertaining an infective dose for oral infection, that may be important in carnivores. For this purpose, 25 (Mus musculus) BALB-C and 34 CD-1 mice, and Trypanosoma brucei brucei strain GVR35 parasites were used in this study divided by three experiments. In two of these experiments the pharmacological effectiveness was tested. The animals were treated with 10mg/kg of BNZ, once a day for 5 days for the first experiment and 11 days for the last. Parasitemias, weight gain and survival rates were measured. In the final experiment, anti-T.b.brucei antibody titers, hematological parameters and concentration of cytokines (IL-4, IFN-γ, NO and TGF-β1) were also measured. In the remaining experiment, which tested an infective dose per os, two groups of mice were exposed, using a feeding probe, to a dose of 500 parasites per animal and 200 000 (2x105) parasites per animal, suspended in Glucosed PBS, respectively. Statistical analysis was performed using the Wilcoxon rank-sum test, Mantel-Cox log-rank test, Two-way ANOVA, Spearman’s correlation and Linear regression analysis. The results of these experiments revealed that it was not possible to establish oral infection with dose of up to 2x105 parasites per animal in an aqueous medium. It was also possible to determine that BNZ was ineffective in the protocols studied, due to a lack of control of parasitemia or a significant increase of host survival. As for hematological values the anemia was not controlled, showing a significant trend for macrocytosis in the treated group. Treated animals had higher titers of IgG subclasses, especially IgG2a and IgG3, as well as increased release of IFN-γ, with significance confirmed by statistical testing (Two-way ANOVA). It was concluded that although BNZ is not a good candidate for therapy of AT, it is a good immunostimulator, enhancing a Th1 response. It is also possible to conclude that a dose 2x105 parasites per animal in an aqueous medium does not establish oral infection.

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Burger, Adélle. "Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1011618.

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One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.

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Bonnet, Julien. "Exploitation d'une biobanque de patients atteints de Trypanosomose Humaine Africaine à Trypanosoma brucei gambiense : recherche et validation de biomarqueurs." Thesis, Limoges, 2017. http://www.theses.fr/2017LIMO0117/document.

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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

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Felu, Cécile. "Characterisation of the mechanism of human serum resistance in T.b.gambiense." Doctoral thesis, Universite Libre de Bruxelles, 2006. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210844.

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The two human pathogenic sub-species T.b.gambiense and T.b.rhodesiense can be distinguished from the morphologically identical T.b.brucei by their ability to infect humans, enabling them to cause sleeping sickness. This is because they are resistant to lysis by the lytic factor (APOL-I) present in normal human serum (NHS). In T.b.rhodesiense resistance to this lytic factor is due to a truncated VSG gene termed SRA which blocks lysis by interacting with APOL-I in the lysosome. SRA does not exist in T.b.gambiense. The search for a similar truncated VSG gene lead to the identification of a T.b.gambiense specific glycoprotein termed TGSGP. TGSGP transfected alone into the sensitive T.b.brucei is unable to confer resistance to this sub-species. This is either due to incorrect processing of this gene is this sub-species or because TGSGP requires a partner to confer resistance.

In the search for a partner, the genomic locus of TGSGP was cloned and sequenced. We found that TGSGP is linked to a truncated gene homologous to the S.cerevisiae AUT1 gene, a gene implicated in autophagy and more specifically in membrane expansion. Southern blot hybridization and PCR analysis on genomic DNA from several isolates demonstrated that this feature was a specific to T.b.gambiense. In addition, we observed a correlation between the aut1 allele size and the geographical origin of the isolate.

Since in trypanosomes lysis by NHS is due to an uncontrolled expansion of the lysosome, we speculated that the truncation of the aut1 allele could be implication in the resistance to human serum. We characterized the genomic organisation of the AUT1 locus. T.b.brucei possesses two native AUT1 alleles whilst T.b.gambiense possesses a truncated aut1 allele, as well as a native AUT1 allele. We showed that in the T.b.gambiense LiTAR isolate (aut1/AUT1), despite the presence of a wild-type allele this gene is no longer expressed at the mRNA and protein level. Our complimentary results by run-on transcription assay showed that the AUT1 region is transcribed but that the messenger is unstable. LiTAR is a functional knock-out for AUT1, but Northern blot analysis on several T.b.gambiense isolates showed that this is not a generalised T.b.gambiense characteristic.

We explored the role of AUT1 in trypanosomes by invalidation of the AUT1 gene in T.b.brucei and by the over-expression of the AUT1 and aut1 alleles in T.b.brucei. By functional analysis of AUT1 knocked-down cells we showed that AUT1 is not essential in trypanosomes. By recreating in T.b.brucei the T.b.gambiense AUT1/aut1 genotype we were able to show that the expression of the aut1 UTR down-regulated the expression of the wild-type AUT1 allele. We speculated that this may be due to a natural RNAi mechanism. Par northern blot, using probes covering the potential target region of AUT1, we detected a 50nt small RNA specific to T.b.gambiense. In addition, we showed that in a LiTAR strain in which the RNAi pathway was abolished AUT1 expression is restored.

We continued to investigate TGSGP’s role in the resistance to human serum by invalidation of TGSGP in T.b.gambiense and by expressing TGSGP in the NHS-sensitive T.b.brucei. Because T.b.gambiense cannot be cultured in vitro we established a new in vivo transfection technique and as the knock-out of TGSGP is most probably lethal, we created an inducible RNAi T.b.gambiense cell strain. These indispensable tools will be used to test whether invalidation TGSGP is sufficient to confer resistance to NHS. Many strategies were tested in order to correctly expressing TGSGP in T.b.brucei; in none of these transfectants was TGSGP correctly located in the flagellar pocket as is the case in T.b.gambiense and only partial resistance was ever obtained. In order to identify the factors in human serum that could interacts with TGSGP, we subjected NHS to affinity chromatography using TGSGP as bait. We showed that TGSGP interacts with APOA-I, a major component of HDLs.

Doctorat en sciences, Spécialisation biologie moléculaire
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22

Sokolova, Antoaneta Y. "Nitroaromatic pro-drug activation and resistance in the African trypanosome." Thesis, University of Dundee, 2011. https://discovery.dundee.ac.uk/en/studentTheses/52c1537e-4a37-446c-b62c-86df5b95b2ea.

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Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is declining, strengthening of the current control efforts, including introduction of more adequate chemotherapeutic options, is needed to prevent the re-emergence of yet another epidemic. Nitroaromatic compounds, such as nifurtimox (in combination with eflornithine) and fexinidazole (in clinical trials), have been recently introduced for the treatment of the second stage of sleeping sickness. These compounds are believed to act as pro-drugs that require intracellular enzymatic activation for antimicrobial activity. Here, the role of the bacterial-like nitroreductase TbNTR as a nitrodrug activating enzyme is examined through overexpression and knock-out studies in T. brucei. Multiple attempts to purify soluble recombinant TbNTR from E. coli were unsuccessful, because the recombinant protein was found to be membrane associated. In keeping with the role of TbNTR in nitrodrug activation, loss of an NTR gene copy in T. brucei was found to be one, but not the only, mechanism that may lead to nitrodrug resistance. Furthermore, in the bloodstream form of T. brucei, resistance was relatively easy to select for nifurtimox, with no concurrent loss of virulence and at clinically relevant levels. More worryingly, nifurtimox resistance led to a decreased sensitivity of these parasites to other nitroaromatic compounds, including a high level of cross-resistance to fexinidazole. Conversely, generation of fexinidazole resistance resulted in cross-resistance to nifurtimox. Should these findings translate to the field, emerging nitrodrug resistance could reverse all recent advances in the treatment of sleeping sickness, made since the introduction of eflornithine 20 years ago. Therefore, all efforts should be made to ensure nitroaromatic drugs are used only in drug combination therapies against sleeping sickness, in order to protect them from emerging resistance.

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23

Moshiri, Houta. "Fluorescence-based reporter substrate for monitoring RNA editing in Trypanosomatid pathogens." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116117.

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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.

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24

Winner, Katherine M. "A fluorescence-based approach to elucidate the subunit arrangement of the essential tRNA deaminase from Trypanosoma brucei." Wittenberg University Honors Theses / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wuhonors1617803573189193.

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25

Chakaingesu, Chikomborero. "Synthesis and structure-activity relationship studies of 1,4-naphthoquinone derivatives as potential anti-trypanosomal agents." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1020959.

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Human African Trypanosomiasis (HAT) is an infectious, vector-borne protozoal disease which is amongst the so-called neglected diseases. In 2000, at a summit of the United Nations, eight Millennium Development Goals (MDGs) were set, to be achieved by 2015. MDG 6 states “to combat HIV/AIDS, malaria & other diseases”. With just under 2 years to go before the end of 2015, HAT is still thriving in developing countries. The drugs currently used for the treatment of HAT are in short supply, have severe side effects and those used to treat late stages of the disease are very difficult to administer. The aforementioned challenges call for research into this neglected disease in order to develop new, safe and easy-to-use medicines. Naphthoquinones are a class of compounds shown to possess anti-parasitic activity, amongst a variety of other biological activities, and therefore this pharmacophore was selected for this study. The purpose of this study was to synthesise derivatives of 2,3-dichloro-1,4- naphthoquinone to be tested for anti-trypanosomal activity and thereafter conduct structureactivity relationship studies. A series of reactions were carried out using thiophenol, phenol and aniline nucleophiles to synthesise thioether (-S-), ether (-O-) and amino (-NH-) derivatives of 2,3-dichloro-1,4-naphthoquinone with various halogen or methyl substituents. Purification of the products was carried out by recrystallisation. Nuclear magnetic resonance (NMR), infra-red (IR) and high pressure liquid chromatography coupled to an electro-spray ionisation mass spectrometer (HPLC-ESI-MS) were the analytical methods used for structural confirmation of the products. There were eighteen 1,4-naphthoquinone derivatives that were successfully synthesised using ethanolic solutions. Unfortunately, attempts to synthesise 1,4-naphthoquinones in reactions involving 2-(trifluoro-methyl)aniline and 2-isopropyl-5-methylphenol were unsuccessful, presumably due to steric hindrance by the bulky ortho-substituents. Although the aims of the synthetic procedures were to obtain both mono- and disubstituted products by nucleophilic displacement of the chlorine atom(s) of 2,3-dichloro-1,4- naphthoquinone, only monosubstituted products were obtained from substitution with aniline and phenol nucleophiles. Thiol nucleophiles, however, selectively yielded disubstituted products only. Synthesised naphthoquinone derivatives were tested against Trypanosoma brucei and calculation of the EC₅₀ values from the obtained dose-response curves was carried out using the four parametric equation. All the 1,4-naphthoquinones showed a degree of potency, except compounds 1b, 3c and 3e, which had little or lack of potency. Structure-activity relationship studies (SARs and QSARs) were carried out to determine which structural features or functional group substituents of the naphthoquinone derivatives contribute or take away from the desired anti-trypanosomal activity. It was found that compounds with the best in vitro anti-trypanosomal potencies in the series of analogous 1,4-naphthoquinone derivatives had EC₅₀ values in the range 2.137 to 2.884 μM. The most potent compound in the series was 2-chloro-3-(4-(trifluoromethyl)phenylamino)-1,4- naphthoquinone 1e; but it was 142-fold less potent than the reference standard of melarsoprol.

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26

Vanhollebeke, Benoît. "The trypanosome lytic factor of human serum, a Trojan horse." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210395.

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The trypanolytic factor of human serum :a trojan horse.

African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites of huge clinical, veterinary and economical importance. They develop in the body fluids of various mammals (including humans) where they face and manipulate many different aspects of the immune system. The extent of this interplay is pivotal to both host and parasite survival, and depending on parasite virulence and host susceptibility, infection duration ranges from some months to several years. At the end, host survival is invariably compromised.

Humans and few other primates provide however a striking exception to this fatal outcome. They are indeed fully protected against most trypanosome infections through the presence in their blood of a so-called trypanosome lytic factor (TLF). The TLF is known to circulate mainly in the form of a high density lipoprotein particle characterized by the simultaneous presence of two primate-specific proteins: haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I).

We have contributed to delineate the respective roles played by Hpr and apoL-I in the lysis process.

ApoL-I was shown to be the exclusive toxin of the TLF. In its absence humans get fully susceptible to any trypanosome infection. The toxin was shown to kill the parasite after endocytosis through the generation of ionic pores in the lysosomal membrane. Those pores dissipate membrane potential and trigger the influx of chloride ions from the cytoplasm into the lysosomal compartment, leading to an eventually fatal uncontrolled osmotic phenomenon.

ApoL-I efficient delivery to the parasite relies on Hpr. African trypanosomes indeed fulfil their heme nutritional requirements by receptor-mediated internalization of the complex formed by haptoglobin, an evolutionary conserved acute-phase protein, and hemoglobin, resulting from physiological intravascular hemolysis. This heme uptake by the auxotrophic parasites contributes to both growth rate and resistance against host oxidative burst. In human serum, the trypanosome receptor is unable to discriminate between Hp and the closely related TLF-bound Hpr, explaining TLF efficient endocytosis.

As such, the TLF acts as a Trojan horse, killing the parasite from inside the cell after having deceived its vigilance through the high similarity between heme-delivering haptoglobin and toxin-associated Hpr.

Doctorat en Sciences
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27

Jansen, Emily. "Identification of non-procyclin molecules expressed by Trypanosoma brucei brucei procyclic culture forms." 2005. http://hdl.handle.net/1828/779.

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28

Baiyegunhi, Omolara O. "Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis." Thesis, 2013. http://hdl.handle.net/10413/11043.

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Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a need for identifying new diagnostic antigens for the serological diagnosis of trypanosomiasis, a disease of humans and animals in Africa caused by protozoa belonging to the genus Trypanosoma. Invariant surface glycoproteins (ISGs) are present in most strains of the parasite and have the potential to replace the variable surface glycoproteins as diagnostic antigens. In order to avoid the challenges of in vivo culturing of bloodstream form (BSF) trypanosomes in laboratory animals, ISG65 and ISG75, the two most common ISGs were heterologously expressed in Escherichia coli and Pichia pastoris expression systems. The extracellular domains of ISG65 and ISG75 of both T. b. brucei and T. b. gambiense were amplified by PCR from genomic DNA using appropriate primers to give inserts of 1121 bp and 1342 bp sizes. These were sub-cloned into the pGEX-4T1 and pET28a expression vectors. Chemically competent E. coli BL21 (DE3) were transformed using the resultant plasmids and the transformed E. coli cells were used for heterologous protein expression. The expressed proteins were purified by three phase partitioning (TPP), nickel or glutathione affinity and molecular exclusion chromatography and analysed by reducing SDS-PAGE. The glycosylation status of ISG65 and ISG75 expressed in the M5 strain of P. pastoris, which has an engineered N-glycosylation pathway that produces glycosylated proteins similar to what is obtained in trypanosomes, was determined. The enzymatic action of Endoglycosidase H resulted in a shift in the electrophoretic migration of ISG65 but not ISG75 on SDS-PAGE, confirming N-glycosylation. Anti-ISG65 and anti-ISG75 antibodies were produced in chickens and affinity purified using the respective recombinant proteins immobilised on affinity matrices. The antibodies recognised native ISG65 and ISG75 respectively in western blots of lysates of T. b. brucei parasites cultured in vitro. Similar recognition of the native ISGs by the anti-recombinant ISG antibodies was also obtained using immunofluorescence microscopy of fixed T. b. brucei parasites. The results obtained demonstrate the potential of application of the recombinant ISG65 and ISG75 and their respective antibodies in the diagnosis of African trypanosomiasis.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

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29

Willert, Erin Kathleen. "The Role of S-Adenosylmethionine Decarboxylase on Regulation of Polyamine and Trypanothione Metabolism in Trypanosoma Brucei." 2008. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=386.

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30

Rasooly, Reuven. "P15 trypanosome microtubule associated protein : structure/function analysis and vaccine development for the prevention of African sleeping sickness." 2001. http://hdl.handle.net/10413/4564.

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Trypanosomes are hemoflagellated protozoan parasites causing chagas disease in South America, Leishmaniasis throughout the world, and African sleeping sickness in humans and nagana in animals in Africa. About 55 million people and 25 million cattle have been estimated to be at risk of contracting African sleeping sickness or nagana respectively. Once injected into the blood stream via the bite of a tsetse fly, the parasite evades the host's immune response by repeatedly changing its surface antigens, thus making the development of a vaccine seem impossible. Furthermore, chemotherapy existing today can be toxic, suggesting that novel methods to prevent diseases caused by trypanosomes are essential. All parasites of the Trypanosomatidae family contain unique microtubular structures called the subpellicular microtubules. Microtubules are made of tubulin and of microtubule associated proteins (MAPs). Unlike other microtubules, the subpellicular microtubules are crosslinked to one another and to the plasma membrane. The unique structure of the subpellicular microtubules has been attributed to unique trypanosome subpellicular MAPs which stabilize the microtubule polymers and crosslink them to one another. Three unique types of subpellicular MAPs have been identified: MARP, which is a high molecular mass MAP that stabilizes microtubules, p52 that is a 52kDa MAP which crosslinks microtubules, and pI5, which is a I5kDa protein which bundles microtubules. Because trypanosome MAPs have been shown to be unique to these parasites, these molecules could serve as useful target sites for therapy. In this study pI5 was cloned and sequenced and shown to contain highly organized, nearly identical tandem repeats with a periodicity of 10 amino acids, rich in positively charged and in hydrophobic amino acids. It was shown that pI5 can also bind phospholipids, suggesting that it may not only bundle the microtubule polymer through its positively charged amino acids but may also crosslink the microtubules to the plasma membrane through its hydrophobic regions, thus contributing to the stable structure of the subpellicular microtubules. To test for the efficiency of pI5 as a vaccine candidate, the recombinant pI5 was cloned into an adenovirus, which was used as a vaccine delivery system for pI5. Mice were vaccinated with the native purified pI5, with the expressed recombinant pI5 and with the adenovirus containing the recombinant pI5 gene (Ad-pI5). The results indicated that pI5 protected 100% of the animals vaccinated with the recombinant molecule (8/8), and 87% of the animals vaccinated with the native protein (7/S), while none of the control animals were protected. Animals that were vaccinated with the Ad-pI5 were protected but so were the control animals vaccinated with an adenovirus containing the lacZ gene. We have shown that vaccination with the adenovirus is associated with an elevated CDS+ T cell response which is known to be trypanostatic (S6), suggesting that animals vaccinated with Ad-pIS may have been protected not only by the specific anti-plS response but also by non specific immunity that was induced by the adenovirus itself. The source of the native and recombinant pI5 was from a different strain of T. brucei that was used for challenge. Since the subpellicular microtubules are common to all members of the Trypanosomatidae family, pI5 may ultimately serve as a common target for therapy to all types of diseases caused by trypanosomes.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.

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31

Mnkandla, Sanele Michelle. "Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense." Thesis, 2013. http://hdl.handle.net/10413/11042.

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Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of the many life threatening tropical diseases posing a serious risk to livelihoods in Africa. The disease is restricted to the rural poor across sub–Saharan Africa, where tsetse flies that transmit the disease, are endemic. Sleeping sickness is known to be caused by protozoan parasites of the genus Trypanosoma brucei, with the two sub-species: T. b. gambiense and T. b. rhodesiense, responsible for causing infection in humans. The disease develops in two stages, firstly, the infection is found in the blood and secondly, when the parasites cross the blood-brain barrier entering the nervous system. To date, no vaccines have been developed, however, there is a range of drugs and treatments available which depend on the type of infection (T. b. gambiense or T. b. rhodesiense) as well as disease stage. The trypanosome parasites have a two-host life cycle i.e. in the mammalian host as well as the tsetse fly vector. Throughout the cycle, the parasite undergoes changes, one of them being the acquisition of a variable surface glycoprotein (VSG) coat prior to entry into the human host bloodstream. Once in the host, the infection progresses and through a phenomenon known as antigenic variation, the parasite expresses a different VSG coat periodically, enabling the parasites to constantly evade the host’s immune response, facilitating their survival. The VSG genes coding for the proteins are activated by an intricate process involving the encoding of a gene which is kept silent, until activated in one of several expression sites. Despite the constant switching of VSG surface coats, there are VSG forms that are predominantly expressed in T. b. gambiense namely VSGs LiTat 1.3, LiTat 1.5 and LiTat 1.6 which are used in diagnostic tests, as antigens to detect antibodies in infected sera of HAT patients. The acquisition of these VSG antigens is, however, of high risk to staff handling the parasites, and so the first part of the study was aimed at cloning, recombinantly expressing and purifying the two VSGs known to be recognised by all gambiense HAT patients: LiTat 1.3 and LiTat 1.5, for possible use as alternative antigens in diagnostic tests. The genes encoding both VSGs, LiTat 1.3 and LiTat 1.5, were first amplified from either genomic or complementary DNA (gDNA or cDNA), cloned into a pTZ57R/T-vector and sub-cloned into pGEX or pET expression vectors prior to recombinant expression in E. coli BL21 DE3 and purification by Ni-affinity chromatography. Amplification and subsequent cloning yielded the expected 1.4 kb and 1.5 kb for the LiTat 1.3 and LiTat 1.5 genes respectively. Recombinant expression in E. coli was only successful with the constructs cloned from cDNA, i.e. the pGEX4T-1-cLiTat 1.3 and pET-28a-cLiTat 1.3 clones. Purification of the 63 kDa cLiTat 1.3His protein following solubilising and refolding did not yield pure protein and there were also signs of protein degradation. For comparison, expression was also carried out in P. pastoris and similar to the bacterial system, expression was only successful with the LiTat 1.3-SUMO construct yielding a 62.7 kDa protein. Purification of LiTat 1.3SUMO also surpassed that of cLiTat 1.3His with no degradation. The diagnostic tests based on VSGs LiTat 1.3 and LiTat 1.5 as antigens operate by binding with antibodies in infected sera, to confirm infection. These antibody detection tests have their limitations, hence an alternative would be antigen detection tests, which use antibodies to detect the respective antigens in infected sera. The second part of the study therefore involved antibody production, where chickens were immunised with the native VSGs LiTat 1.3, LiTat 1.5 as well as recombinant RhoTat 1.2 (a VSG expressed in T. evansi). Antibody production was analysed by ELISA and characterised by western blotting, prior to immunolabelling of T. b. brucei Lister 427 parasites. The chicken IgY showed a response to the immunogens, and were able to detect their respective proteins in the western blot. Interestingly, the anti-nLiTat 1.3, anti-nLiTat 1.5 and anti-rRhoTat 1.2 antibodies were able to detect their respective VSGs on the T. b. brucei trypanosome parasites in the immunofluorescence assay, thus demonstrating cross reactivity. As the antibodies showed specificity, they could potentially detect antigens in infected sera of HAT patients in an antigen detection based test.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

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Dissertations / Theses on the topic 'African trypanosomiasis. Dynein. Trypanosoma brucei'

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