African trypanosomes are a major plague in sub-Saharan Africa. They cause sleeping sickness in humans and nagana in cattle. These parasites are transmitted between their mammalian hosts by tsetse flies. They are adapting to their different environments through differentiation processes. These processes involve, amongst other things, the expression of different surface coats. These coats are made of procyclin protein at the insect midgut procyclic stage and of variant surface glycoprotein (VSG) at the mammalian bloodstream stage. At a given time, one VSG is expressed from a single VSG gene out of a repertoire of more than 1500 VSG genes present in the trypanosomes genome. The expressed VSG gene is always located at one of fifteen telomeric polycistronic transcription units called expression sites (ES). The VSG coat is changed regularly in a process called antigenic variation allowing trypanosomes to escape the immune response. The exact mechanism controlling the selection of the active ES is not yet known and controversies have been raised concerning the ES transcription control. Although several molecular factors involved in the ES monoallelic-expression have been identified, none of them seems to be a critical regulator.<p><p>Thus during my thesis we decided to explore two aspects of ES expression: (A) deciphering the level at which this expression is controlled and (B) fishing for new protein factors controlling this expression.<p>A) It is not even clear at which level the ES transcription control takes place. In particular, there has been debate on whether it is taking place at the transcription initiation or elongation level. Previous experiments generated contradictory conclusions and gave rise to two different models. The first model suggested that transcription initiation takes place in all ESs simultaneously. The second model suggested that transcription is initiated in only two ESs, one being fully active and a second being pre-active. These two models were equally able to account for the finding of transcripts from different ES within a trypanosome population provided the pre-active ES differs between individual cells. In order to decide if a single or multiple ES promoters can initiate transcription in a given cell, single cell RT-PCR targeting the beginning of the ES was required. Thus single cell RT-PCR was performed and an analysis of the obtained transcripts showed that transcription initiation is taking place on many ES while only one VSG is transcribed. This permitted the unambiguous conclusion that the monoallelic expression of VSG is exerted by controls operating downstream from transcription initiation, suggesting transcription elongation or RNA processing as critical control steps. <p>B) We have characterized a new nuclear protein, Tb alba3, involved in the repression of silent VSGs. Its invalidation lead to chromatin opening in the silent expression sites and to a raise in their expression. As this protein is cytoplasmic and binding procyclin mRNAs at the procyclic stage, it could be a new versatile factor, shuttling between the cytoplasm and the nucleus and involved both in the inverse regulation of major surface antigens at different differentiation stages and the control of antigenic variation.<p><p>These results enhance our understanding of ES transcription control and of ES monoallelic expression. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The bloodstream form of Trypanosoma brucei differentiates into the stumpy form in the mammalian bloodstream, completing differentiation into the procyclic form on uptake by the tsetse fly. The underlying genetic events occurring during this differentiation process in pleomorphic cell lines were investigated through whole-genome microarray studies of key time points during differentiation from stumpy form cells to the procyclic form found in the insect midgut. The microarray was extensively validated and bioinformatic experiments conducted to detect motifs over represented in stumpy form or slender form cells. A positional-dependent motif was identified that was over represented in stumpy form cells, possibly representing a regulatory domain. The transcripts found to be enriched in stumpy form cells included a chloride channel, although RNAi directed against this gene showed no phenotype, suggesting the protein is redundant, as three other homologous proteins exist in the genome and showed similar mRNA profiles on the microarray. Stumpy form cells are G0 arrested and two proteins implicated in G0/G1 regulation in other organisms, Target of Rapamycin (Tor) and Cdh1, were investigated in T. brucei to determine whether these proteins were involved in differentiation. The result of depletion of either protein was rapid cell death in bloodstream form cells, although treatment with the drug rapamycin did not have any effect on the cells in contrast to other eukaryotes where this drug causes G1 arrest. A method for synchronisation of bloodstream form cells was also designed using a supravital dye and flow cytometry to allow investigation into cell cycle-dependent processes. This method was particularly suitable for harvesting populations enriched in G0/G1 stage cells, however differentiation of the isolated G0/G1 and G2/M populations did not show significantly different differentiation kinetics.

During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists in two different states: the proliferative slender form and the non-proliferative, transmissible, stumpy form. The transition from the slender to stumpy form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. The slender to stumpy transition and the contribution of stumpy formation to within-host dynamics have been difficult to analyse, however, because cell-type specific markers have been restricted to imprecise morphological criteria. PAD1 is a recently identified stumpy-specific protein which acts as a molecular marker for stumpy formation and a functional marker for transmission. Here, the control of stumpy-specific gene expression via the 3’UTR has been analysed, identifying that there are repressive elements in the 3’UTR preventing inappropriate expression during the slender life stage. Further, both pleomorphic and monomorphic transgenic reporter cell lines utilising the PAD1 3’UTR have been created that report on stumpy formation in vitro and these have been used for the analysis of stumpyinducing chemical compounds. Finally, a sensitive and accurate qRT-PCR assay has been developed and optimised that faithfully reports both parasitaemia and stumpy formation throughout host infection. Using a chronic infection rodent model, stumpy levels have been monitored on the basis of conventional morphological and cell cycle assays, as well as by qRT-PCR for PAD1 expression. The results define the temporal order of events that result in the generation of stumpy forms early in a parasite infection and thereafter describe the dynamics of slender and stumpy forms in chronic infections extending over several weeks. This quantitative data has allowed the mathematical modelling of transmission competence in trypanosome infections, suggesting dominance of transmission stages throughout infection.

The unicellular eukaryote Trypanosoma brucei causes African Sleeping sickness and multiplies extracellularly in the bloodstream of the infected host. The parasite evades antibody-mediated lysis by switching its Variant Surface Glycoprotein (VSG) coat. Blocking VSG synthesis results in an abrupt growth inhibition and a precise pre-cytokinesis cell cycle arrest, with an accumulation of cells with two nuclei and two kinetoplasts. Additionally, induction of VSG RNAi triggers a global block in translation, which is not due to a general decrease in transcript levels. The mechanism behind this translation arrest was investigated. It was observed that it correlated with a decrease in polysomes, indicating that translation was blocked at the level of initiation. It was also shown that the VSG RNAi-triggered growth inhibition was reversible, which suggests that this is not a lethal phenotype. The VSG221 RNAi-induced growth arrest could be alleviated if a second different VSG (VSG117), which was not recognised by the VSG221 RNAi, was expressed immediately downstream of the promoter of the active VSG221 Expression site. Further, it was possible to delete the telomeric VSG221 in these VSG double-expressors, leaving the cells completely reliant on the second complementing VSG117 gene. VSG117 expressed from a promoter-adjacent position in the active Expression site was shown to form a functional surface coat that protected the parasites from complement-mediated lysis in vitro. Transiently transfecting cells with anti-VSG221 morpholino oligonucleotides allowed us to specifically block translation of VSG221 mRNA without degrading it. This resulted in a pre-cytokinesis cell cycle arrest similar to that induced by VSG221 RNAi. This indicates that the VSG RNAi-triggered growth inhibition was due to a lack of VSG protein or its synthesis rather than the ablation of the abundant VSG mRNA. In addition, it was shown that blocking VSG synthesis reduced the rate of surface VSG internalisation in cells that were stalled precytokinesis, but had no effect on other endocytic markers. These experiments give us further insight into the importance of the protective VSG coat for pathogenicity in T. brucei.

The protozoan parasites Trypanosoma brucei brucei, Trypanosoma congolense and Trypanosoma vivax cause Animal African Trypanosomiasis, a disease responsible for costly livestock pathology and economic losses in Africa. Each of these African trypanosomes are vector-borne and transmitted by the blood-feeding tsetse fly. Additional blood-feeding vectors can spread T. vivax, extending its range into South America. T. b. brucei infection of the mammalian host progresses in waves, with periodic clearance of antigenic variants. Accumulation of slender parasites in the blood is accompanied by accumulation of the density-sensing factor, SIF (stumpy induction factor). SIF drives differentiation from the proliferative slender form to the growth arrested stumpy form at the peak of parasitaemia. This differentiation step aids host survival, and the stumpy form is pre-adapted for continuation of development in the tsetse fly, ensuring transmissibility. Despite facing challenges comparable to T. b. brucei during their life cycle, T. congolense and T. vivax are not found to have morphologically distinguishable slender and stumpy forms. The growth control mechanisms used by these important veterinary pathogens have been investigated in this thesis. Particular focus has been placed on the conservation of quorum sensing pathways within the African Trypanosomes. The potential for cross-species communication has implications for co-infections. T. congolense was found to undergo growth arrest at peak parasitaemia, and transcriptomic changes occurring between ascending and peak parasitaemia were identified and comparisons made to T. b. brucei slender and stumpy transcriptomes. In an examination of the conservation of the SIF-responsive pathway, expression of a T. congolense orthologue was found to rescue stumpy formation in an otherwise SIF-resistant null mutant for the corresponding T. b. brucei gene. The capacity for cross-talk between density-sensing signals in different trypanosome species was tested using conditioned medium from T. congolense bloodstream form cultures. This could activate the expression of a stumpy specific reporter protein in T. b. brucei. A cell line deficient in a SIF-responsive gene showed resistance to the conditioned medium with a delay in reporter expression. These results highlight the unanticipated capacity for different trypanosome species to exhibit intra and inter specific cell-cell communication in the mammalian bloodstream, with possible consequences for their virulence, transmission and evolution.

Background: Despite the acknowledged importance of economic assessments for public health interventions at the human-animal-ecosystem interface, there are currently limited economic methodologies for doing so. In this thesis studies were undertaken to ascertain the economic impact of interventions to control trypanosomiasis and taeniasis/cysticercosis in south-east Uganda and northern Lao PDR respectively. Also, in Uganda studies were done to find out if demand of draft cattle would be an important economic driver for spreading trypanosomiasis due to inter-district trade. Method: In Uganda, a one year recall cross-sectional baseline survey and an 18 month longitudinal survey of 660 households was conducted; to determine the benefits and changes due to restricted application of deltamethrin insecticide to only the legs, belly and ears of cattle. During the 18 month study, the households participating in the study were divided into six regimes depending on the type of intervention done in their cattle and these were; diminazine injection only, deworming only, no treatment and those had 25%, 50% and 75% of the total village cattle sprayed. Thus, the first three regimes were those households that had their cattle not sprayed with insecticide at all as opposed to the last three. Additionally, cattle trade data was collected for network and value chain analysis in all markets in Tororo and Namutamba districts from 199 cattle traders. In northern Lao PDR, stochastic modelling was done to determine the burden of neurocysticercosis associated epilepsy and soil transmitted helminthes. A cross-sectional study was carried out in 49 households, focusing on the prevalence of cysticercosis and soil transmitted helminths before and after a twelve month intervention to control a hyperendemic focus of Taenia solium. The village data was then extrapolated to the wider northern Lao PDR population. Results: The Uganda study indicated that the restricted application of deltamethrin in cattle induced change of USD 31 per head of adult bovine per year; this was the change in income that directly occurred due to restricted spraying of cattle with deltamethrin. During the intervention period, the annual difference in income between those households that had their cattle sprayed using restricted application protocol and those that did not was USD 123; and this was significant (t= 7.18, p= < 0.001). Analysis of variance using households that had their cattle receive no treatment as control showed that restricted application of deltamethrin significantly increased household income compared to diminazine aceturate injection and deworming of cattle only. The incremental benefit cost ratio of spraying 0% to 25% of the cattle was found to be the highest (16:1) compared to spraying 25% to 50% (3:1) and 50% to 75% (1:1) of the cattle. Cattle trade network and value chain analysis revealed that the key cattle markets from which trypanosomiasis is likely to spread into Tororo District are Molo, Namutumba and Soroti. Also, it was found that the risk of spread of human African trypanosomiasis from south-east to north-west Uganda is high due to the increased demand for male cattle for draft work. In northern Lao PDR, 5,094 (95% CI: 25.6-28,940) DALYs were estimated to be imposed annually due to Taenia solium associated epilepsy, with 446.4 (95% CI: 2.2- 2,536) DALY imposed per 100,000 person-years. Due to the high benefits to pig production, the net monetary cost per DALY averted for simultaneously controlling T. solium, soil transmitted helminthes and classical swine fever was only USD 14, which fell to USD 11 if the separable cost method were applied. If the intervention did not target pigs, then the cost per DALY averted was USD 44; well below the current standard for ’very cost effective ‘of the 1 year’s per capita GDP. Conclusion: This study provided empirical evidence for evaluating the impact of quantifying the benefits of controlling zoonotic diseases in the livestock sector (Uganda case study) and in both livestock and human health populations (Lao PDR case study); this economic assessment approach can be used for planning future integrated health interventions. The results of this study support the policy of preventing the spread of infection by spraying at least 25% of the cattle using RAP, as well as injecting all cattle in key livestock markets in south east Uganda with diminazine aceturate to prevent HAT. In northern Lao PDR, simultaneous control of T. solium, soil transmitted helminths and classical swine fever is the most cost-effective approach. There are still difficulties in incorporating human and animal parameters into a single analytical framework; consequently there is a need to adapt the approaches undertaken in this study to the analysis of other zoonotic diseases in different settings to improve on their robustness.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-26Bitstream added on 2014-06-13T19:19:51Z : No. of bitstreams: 1 silva_mta_dr_araiq.pdf: 3269290 bytes, checksum: 226805a8de91ee25f88e83234f61efa0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A família Trypanosomatidae inclui diversos parasitas protozoários responsáveis por diferentes doenças humanas. Várias evidências sugerem importantes diferenças entre a maquinária de tradução e processamento de mRNA (trans-splicing) em Tripanosomatídeos quando comparados com eucariótos superiores. Neste contexto, alguns fatores importantes para o funcionamento da célula eucarióticas são os pequenos complexos constituídos de proteínas e RNA, chamados de ribonucleoproteínas (U snRNPs). Esta partículas possuem papel essencial no processamento de RNA mensageiros e durante a reação de splicing apresenta um core comum composto por proteínas (proteínas Sm) and RNAs estruturais (U snRNAs) e um conjunto de proteínas específicas de cada complexo. Embora bem definidas em mamíferos, snRNPs permanecem pouco caracterizadas em Tripanosomatídeos. Ferramentos de bioinformática identificaram quatro possíveis proteínas específicas do complexo U5 snRNP (U5-15K, U5-40K, U5-102K e U5-116K), e importantes parâmetros foram determinados, como peso molecular estimado, domínios e motivos conservados. Este trabalho demonstrou que U5-15K e 45-102K são altamente conservadas entre o Tripanosomatídeos e os domínios Dim1 and Prp1 foram identificados, respectivamente. Técnicas de purificação de complexos (PTP-tag) revelaram que estas proteínas interagem com o U5 snRNA, sugerindo que participem do complexo U5 snRNP. Análises funcionais demonstraram que U5-15K é essencial para viabilidade celular e que de alguma forma esta asssociada tanta a reação de cis quanto de tras-splcing. Experimentos de imunolocalização de U5-15K and U5-102K corroboram este dados, uma vez que as protínas em questão possuem localização nuclear. / There are several protozoan parasites in Trypanosomatidae family, including different agents responsible for human diseases. Several evidences suggest important differences in the translational system and mRNA processing (trans-splicing) in Trypanosomatids when compared to higher eukaryotes. In this context, some important factors for the functioning of eukaryotic cells are the small complexes of RNA and proteins; these particles of ribonucleoproteins (UsnRNPs) have an essential role in the pre-mRNA processing, mainly during splicing. UsnRNP presents a common protein core associated between itself and with the snRNA, named Sm proteins and specific proteins of each snRNP. Even though they are well defined in mammals, snRNPs are still not well characterized in certain Trypanosomatids. Bioinformatics analysis identified four possible U5 snRNP specific proteins (U5-15K, U5-40K, U5-102K and U5-116K), and important parameters were determinated, as estimated molecular weight, motifs and conserved domains. This work shows that the U5-15K and U5-102K proteins are highly conserved among different Tryponosomatids species and Dim1 and Prp1 domains were identified, respectively. Tandem affinity pull-down assay revealed that these proteins interact with U5snRNA, suggesting its participation in U5snRNP particle, and functional analysis showed that U5-15K is essential for cell viability and it is associated in some way to trans and cis-splicing machinery. Immunolocalization experiments corroborated those data, showed U5-15K and U5-102K in the nucleus of the cell.

The mitochondrial genome (kinetoplast or kDNA) of Trypanosoma brucei is highly complex in terms of structure, content and function. It is composed of two types of molecules: 10-50 copies of identical ~23-kb maxicircles and 5,000-10,000 highly heterogeneous 1-kb minicircles. Maxicircles and minicircles form a concatenated network that resembles chainmail. Maxicircles are the equivalent of mitochondrial DNA in other eukaryotes, but 12 out of the 18 protein-coding genes encoded on the maxicircle require post-transcriptional RNA editing by uridylate insertion and removal before a functional mRNA can be generated. The 1-kb minicircles make up the bulk of the kDNA content and facilitate the editing of the maxicircle-encoded mRNAs by encoding short guide RNAs (gRNAs) that are complementary to blocks of edited sequence. It is estimated that there are at least hundred classes of minicircle, each class encoding a different set of gRNAs. At each cycle of cell division the contents of the kDNA genome must be faithfully copied and segregated into the daughter cells. Mathematical modelling of kDNA replication has shown that failure to segregate evenly will eventually result in loss of low copy number minicircle classes from the population. Depending on the type of minicircle that is lost this can result in immediate parasite death or, if the loss occurred in the bloodstream stage, render the cells unable to complete the canonical life-cycle in the tsetse fly vector. In order to investigate minicircle complexity and replication in T. brucei further we i) first established the true complexity of the kDNA genome using a Illumina short read sequencing and a bespoke assembly pipeline, ii) annotated the minicircles to establish the editing capacity of the cells, iii) analysed expression levels of predicted gRNA gene cassettes using small RNA data, and iv) carried out a long term time course to measure how kDNA complexity changes over time and compared this to preliminary model predictions. The structure of this thesis follows these steps. Using these approaches, 365 unique and complete minicircle sequences were assembled and annotated, representing 99% of the minicircle genome of the differentiation competent (i.e. transmission competent) T. brucei strain AnTat90.13. These minicircles encode 593 canonical gRNAs, defined as having a match in the known editing space, and a further 558 non-canonical gRNAs with unknown function. Transcriptome analysis showed that the non-canonical gRNAs, like the canonical set, have 3' U-tails and showed the same length distribution. Canonical and non-canonical sets differ, however, in their sense to anti-sense transcript ratios. In vitro culturing of bloodstream form T. brucei for ~500 generations resulted in loss of ~30 minicircle classes. After incorporating parameters for network size and minicircle diversity determined above, model fitting to longitudinal kDNA complexity data will provide estimations for the fidelity of kDNA segregation. The refined mathematical model for kDNA segregation will permit insight into time constraints for transmissibility during chronic infections due to progressive minicircle loss. It also has the potential to shed light on the selective pressures that may have led to the apparent co-evolution of the concatenated kDNA network structure and parasitism in kinetoplastids.

Uridylyl insertion/deletion mRNA editing is essential for mitochondrial gene expression in Trypanosoma brucei and governed by multi-protein complexes called editosomes. The final step in each cycle of this post-transcriptional process is that of re-ligating the edited mRNA fragments. The ~20S RNA editing core complex contains two RNA editing ligases, REL1 and REL2, located, respectively, in a deletion and an insertion subcomplex. While REL1 is clearly essential for RNA editing, REL2 knockdown by RNAi has not resulted in a detectable phenotype. To explain these findings, alternative scenarios have been suggested: (a) REL2 is not functional in vivo; (b) REL1 can function in both insertion and deletion editing, whereas REL2 can only function in insertion editing; (c) REL1 has an additional role in repairing erroneously cleaved mRNAs. To further investigate respective functions of the two RELs this study used three complimentary approaches: (i) genetic complementation with chimeric ligase enzymes, (ii) deep sequencing of RNA editing intermediates after ligase inactivation, and (iii) evolutionary analysis. In vivo expression of two chimeric ligases, providing a REL2 catalytic domain at REL1’s position in the deletion subcomplex and a REL1 catalytic domain at REL2’s position in the insertion subcomplex, did not rescue the growth defect caused by REL1 ablation. Although the results were not fully conclusive they suggest that it is the specific catalytic properties of REL1 rather than its position within the deletion subcomplex that makes it essential. In order to identify in vivo substrates of REL1, specific editing intermediates that accumulated after genetic knockdown of REL1 expression were captured by 5’ linker and deep sequenced using Ion Torrent and Illumina technology. Analyses of such unligated editing intermediates with bespoke bioinformatics tools suggest that REL1 functions in deletion editing as expected, but also in the repair of miscleaved mRNAs, implying a novel role for this ligase. Neither role can be fulfilled by REL2, at least not with sufficient efficiency. Sequencing data also suggest that either REL1 is not involved in ligation of addition editing substrates, or that REL2 in this case can fully compensate for loss of REL1. REL1, REL2 and KREPA3 sequences were subjected to analysis using MEGA5 and the HyPhy package available on the Datamonkey adaptive evolution server. Results indicated that all three editosome genes are under much stronger purifying than diversifying selective forces. In general this selection pressure to conserve protein sequence increased from KREPA3 to REL2 to REL1, suggesting a requirement to maintain catalytic function for both ligases. Taken together, these experiments reveal a novel function for REL1 during RNA editing, providing a rationale for its essentiality. Deductively, the results also suggest REL2, which was previously thought to be non-essential, may still be required by the cell at its position in the addition subcomplex. Evolutionary analysis suggests that the RELs and KREPA3 are under the same evolutionary forces to maintain their respective functions in RNA editing.

<i>Trypanosoma brucei</i> is a unicellular trypanosomatid protozoan parasite and the etiological agent of sleeping sickness in sub-Saharan Africa. The trypanosomatid order also includes the parasites <i>Trypanosoma cruzi </i>(Chagas disease) and <i>Leishmania major</i> (Leishmaniasis). Sleeping sickness is estimated to cause ~10,000 deaths per year and current treatments are expensive, difficult to administer and toxic. Although genomic sequencing of all three parasites has identified the coding sequences of these organisms, much is still unknown about protein function, with 64% of identified genes annotated as “hypothetical”, lacking obvious homology with proteins of known function. To further understand the unusual biology of this family of eukaryotes, this thesis aimed to provide evidence for protein function in <i>Trypanosoma brucei</i> in a high-throughput manner, utilising global proteomic analyses. This work has encompassed two main approaches: The global analysis of protein interactions and the analysis of proteome changes across the cell-cycle. To enable these approaches, I developed protocols for proteome wide analysis of protein complexes in <i>Trypanosoma brucei</i>, combining multiple forms of chromatography on ‘native’ lysates of cells to produce a proteome wide map of core, soluble protein complexes in this organism. I further performed preliminary studies to optimise in vivo formaldehyde crosslinking in <i>T. brucei</i> in order to characterise membrane bound protein complexes. I also developed methodologies to produce large populations of procyclic <i>T. brucei</i> cells highly enriched in different phases of the cell-cycle for proteomic analysis. In conjunction with the optimisation of methods for isobaric tag quantitation on Fusion mass spectrometers, I provide the first characterisation of protein regulation during cell division in <i>T. brucei</i> at an unparalleled proteomic depth. Together, these datasets provide a wealth of information about the interaction and cell cycle regulation of many thousands of proteins in <i>T. brucei</i>, and contributes greatly to the understanding of protein function in trypanosomatid organisms. I highlight the ability of these methods to predict novel protein complexes, predict interactions between “hypothetical” proteins with proteins of known function, and to identify “hypothetical” cell-cycle regulated proteins that are essential for growth of the parasite, that are a potentially interesting source for novel drug targets. Data visualisation tools to browse the data in a user-friendly format will further allow the trypanosmatid research community to mine these datasets to understand function of proteins of interest and continue to extract functional information from these datasets to extend our understanding of trypanosomatid biology.