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.

Trypanosoma brucei is a digenetic eukaryotic parasite that develops in different tissues of a mammalian host and a tsetse fly. It is responsible for sleeping sickness in sub-saharan Africa. The parasite cycle involves more than nine developmental stages that can be clearly distinguished by their general morphology, their metabolism and the relative positioning of their DNA-containing organelles. During their development, trypanosomes remain exclusively extracellular and encounter changing environments with different physico-chemical properties (nutritional availability, viscosity, temperature, etc.). It has been proposed that trypanosomes use their flagellum as a sensing organelle, in agreement with the established role of structurally-related cilia in metazoa and ciliates. Recognition of environmental triggers is presumed to be at the initiation of differentiation events, leading to the parasite stage that is the best suited to the new environment. These changes are achieved by the modification of gene expression programmes, mostly underlying post-transcriptional control of mRNA transcripts. We first demonstrate that the RNA-binding proteins ALBA3/4 are involved in specific differentiation processes during the parasite development in the fly. They are cytosolic and expressed throughout the parasite cycle with the exception of the stages found in the tsetse fly proventriculus, as shown by both immunofluorescence and live cell analysis upon endogenous tagging with YFP. Knock-down of both proteins in the developmental stage preceding these forms leads to striking modifications: cell elongation, cell cycle arrest and relocalization of the nucleus in a posterior position, all typical of processes acting in parasites found in the proventriculus region. When ALBA3 is over-expressed from an exogenous copy during infection, it interferes with the relocalization of the nucleus in proventricular parasites. This is not observed for ALBA4 over-expression that does not visibly impede differentiation. Both ALBA3/4 proteins react to starvation conditions by accumulating in cytoplasmic stress granules together with DHH1, a recognized RNA-binding protein. ALBA3/4 proteins also partially colocalize with granules formed by polyA+ RNA in these conditions. We propose that ALBA are involved in trypanosome differentiation processes where they control a subset of developmentally regulated transcripts. These processes involving ALBA3/4 are likely to result from the specific activation of sensing pathways. In the second part of the thesis, we identify novel flagellar proteins that could act in sensing mechanisms. Several protein candidates were selected from a proteomic analysis of intact flagella performed in the host laboratory. This work validates their flagellar localization with high success (85% of the proteins examined) and defines multiple different patterns of protein distribution in the flagellum. Two proteins are analyzed during development, one of them showing down-regulation in proventricular stages. The functional analysis of one novel flagellar membrane protein reveals its rapid dynamics within the flagellum but does not yield a visible phenotype in culture. This is coherent with sensory function that might not be needed in stable culture conditions, but could be required in natural conditions during development. In conclusion, this work adds new pieces to the puzzle of identifying molecular switches involved in developmental mRNA control and environmental sensing in trypanosome stages in the tsetse fly. / Trypanosoma brucei ist ein digenetischer, eukaryotischer Parasit, der zwischen Säugetier und Tsetsefliege alterniert, in welchen er unterschiedliche Gewebe besiedelt. Er ist die Ursache für die Schlafkrankheit in Afrika südlich der Sahara. Der Lebenszyklus der Trypanosomen besteht aus mehr als neun Parasitenstadien, die eindeutig anhand ihrer Morphologie, ihres Metabolismus und der Positionierung ihrer DNA Organellen unterschieden werden können. Trypanosomen bleiben ausschließlich extrazellulär und kommen im Laufe ihres Infektionszyklus mit sich verändernden Umwelteinflüssen in Berührung, z. B. Temperaturschwankungen, Variation in vorhandenen Energiequellen, erhöhte Viskosität usw. In Übereinstimmung mit der anerkannten sensorischen Funktion die Cilien in Vielzellern ausüben, wurde für diese Rolle das strukturverwandte Flagellum in Trypanosomen vorgeschlagen. Die Erkennung wechselnder Umweltparameter ist der vermutliche Auslöser für Differenzierungsprozesse, die ein Entwicklungsstadium hervorbringen, welches am besten an die neue Umgebung angepasst ist. Dies wird durch eine Modifizierung der Genexpression erreicht, die in Trypanosomen fast ausschließlich auf posttranskriptioneller Ebene erfolgt. Diese Arbeit zeigt, dass die RNA bindenden Proteine ALBA3 und ALBA4 an der Differenzierung von Trypanosomen in der Tsetsefliege beteiligt sind. Immunfluoreszenzanalyse und Lebendvideomikroskopie von Zellen, die eine an YFP gekoppelte Variante der Proteine enthalten, haben gezeigt, dass sich ALBA3/4 im Zytosol befinden und dass sie in jedem Parasitenstadium exprimiert sind, mit Ausnahme derer, die im Proventrikel der Tsetsefliege zu finden sind. Das Herunterregulieren der Proteine in vorangehenden Stadien, führt zu markanten Veränderungen, die mit denjenigen, die in Parasiten im Proventrikel zu finden sind, vergleichbar sind: z. B. Verlängerung der Zelle, Zellzyklusarrest und Lokalisierung des Zellkerns in eine posteriore Position. Im Gegenteil dazu findet die Umpositionierung des Zellkerns nicht statt, wenn ALBA3 während der Entwicklung des Parasiten in der Tsetsefliege überexprimiert wird. Ein vergleichbarer Effekt wird mit ALBA4 Überexpression nicht erreicht, welches die Entwicklung nicht negativ zu beeinflussen scheint. Wenn Trypanosomen Hungerstress ausgesetzt sind, reichern sich beide ALBA Proteine zusammen mit DHH1, einem anerkannten RNA bindenden Protein, in zytoplasmatischen Aggregaten an, die nur teilweise mit denjenigen kolokalisieren, die durch polyA+ RNA in diesen Bedingungen verursacht werden. Diese Arbeit zeigt, dass ALBA Proteine eine wichtige Rolle in der Entwicklung von Trypanosomen spielen und legt nahe, dass sie an der entwicklungsbedingten Kontrolle eines Teils der mRNA Expression beteiligt sind. Der zweite Teil dieser Arbeit handelt von der Identifizierung neuer flagellarer Proteine, die eine sensorische Funktion haben könnten. Hierfür wurden mehrere Proteinkandidaten aus einer durchgeführten Proteomanalyse intakter Flagellen gewählt. Die vorliegende Arbeit bestätigt die flagellare Lokalisierung der Proteine mit großem Erfolg (85% der untersuchten Proteine) und zeigt, dass sie unterschiedliche Verteilungsmuster vorweisen. Zwei der Proteine werden während der Infektion des Parasiten in der Tsetsefliege untersucht, was aufdeckt, dass eines davon in den Stadien im Proventrikel herunterreguliert ist. Die Funktionsstudie eines neu identifizierten flagellaren Membranproteins weist seine schnelle Dynamik im Flagellum auf, führt jedoch zu keinem sichtbaren Phänotyp in Laborbedingungen. Diese Beobachtung passt zu der Annahme, dass Proteine mit sensorischer Funktion in stabilen Laborverhältnissen nicht essentiell sind aber eine wichtige Rolle während der Entwicklung des Parasiten in natürlichen Bedingungen spielen. Zusammenfassend fügt diese Arbeit Teile zum Puzzle der Identifizierung molekularer Schalter, die in Trypanosomenstadien in der Tsetsefliege an der mRNA Kontrolle und der Erkennung der Umwelt beteiligt sind.

Trypanosoma brucei is an obligate parasite and causative agent of severe diseases affecting humans and livestock. The protist lives extracellularly in the bloodstream of the mammalian host, where it is prone to attacks by the host immune system. As a sophisticated means of defence against the immune response, the parasite’s surface is coated in a dense layer of the variant surface glycoprotein (VSG), that reduces identification of invariant epitopes on the cell surface by the immune system to levels that prevent host immunity. The VSG has to form a coat that is both dense and mobile, to shield invariant surface proteins from detection and to allow quick recycling of the protective coat during immune evasion. This coat effectively protects the parasite from the harsh environment that is the mammalian bloodstream and leads to a persistent parasitemia if the infection remains untreated. The available treatment against African Trypanosomiasis involves the use of drugs that are themselves severely toxic and that can lead to the death of the patient. Most of the drugs used as treatment were developed in the early-to-mid 20th century, and while developments continue, they still represent the best medical means to fight the parasite. The discovery of a fluorescent VSG gave rise to speculations about a potential interaction between the VSG coat and components of the surrounding medium, that could also lead to a new approach in the treatment of African Trypanosomiasis that involves the VSG coat. The initially observed fluorescence signal was specific for a combination of a VSG called VSG’Y’ and the triphenylmethane (TPM) dye phenol red. Exchanging this TPM to a bromo-derivative led to the observation of another fluorescence effect termed trypanicidal effect which killed the parasite independent of the expressed VSG and suggests a structurally conserved feature between VSGs that could function as a specific drug target against T. b. brucei. The work of this thesis aims to identify the mechanisms that govern the unique VSG’Y’ fluorescence and the trypanocidal effect. Fluorescence experiments and protein mutagenesis of VSG’Y’ as well as crystallographic trials with a range of different VSGs were utilized in the endeavour to identify the binding mechanisms between TPM compounds and VSGs, to find potentially conserved structural features between VSGs and to identify the working mechanisms of VSG fluorescence and the trypanocidal effect. These trials have the potential to lead to the formulation of highly specific drugs that target the parasites VSG coat. During the crystallographic trials of this thesis, the complete structure of a VSG was solved experimentally for the first time. This complete structure is a key component in furthering the understanding of the mechanisms governing VSG coat formation. X-ray scattering techniques, involving x-ray crystallography and small angle x-ray scattering were applied to elucidate the first complete VSG structures, which reveal high flexibility of the protein and supplies insight into the importance of this flexibility in the formation of a densely packed but highly mobile surface coat. / Trypanosoma brucei ist ein eukaryotischer Parasit welcher bei Menschen und Nutztieren schwere Krankheiten auslöst. Der Protist lebt extrazellulär im Blutstrom seines Säugetier-Wirtes, in welchem er unter konstantem Angriff durch das Wirts-Immunsystem steht. Als ausgeklügelte Methode zur Umgehung der Immunantwort besitzt der Parasit einen dichten Oberflächenmantel des variablen Oberflächen-Glycoproteins (VSG), welcher die Identifikation invariabler Oberflächenproteine durch das Immunsystem erschwert und Wirts-Immunität gegen den Parasiten verhindert. Der gebildete VSG-Mantel muss gleichzeitig eine hohe Dichte besitzt, um invariable Oberflächenproteine vor Immundetektion zu beschützen, und eine hohe Mobilität aufweisen, um ein schnelles Recycling des Schutzmantels während Immunantworten zu gewährleisten. Dieser Mantel schützt den Parasiten effektiv vor dem Wirts-Immunsystem und führt bei fehlender Behandlung des Patienten zur persistenten Parasitemie durch Trypanosoma brucei. Die verfügbaren Behandlung gegen die Afrikanische Trypanosomiasis beinhaltet die Benutzung von Medikamenten welche ihrerseits z.T. stark toxisch sind und den Tod des Patienten verursachen können. Ein Großteil der verfügbaren Medikamente wurden zu Beginn des letzten Jahrhunderts entwickelt und stellen trotz anhaltenden Entwicklungen noch immer die beste Lösung im Kampf gegen den Parasiten dar. Die Entdeckung eines fluoreszierenden VSGs deutete auf eine Interaktionen zwischen dem VSG Mantel und Bestandteilen des umgebenden Medium hin, welche die Entwicklung von Medikamenten mit dem VSG Mantel als Drug Target ermöglichen könnte. Das ursprünglich beobachtete Fluoreszenz-Signal war spezifisch für eine Kombination eines VSG namens VSG’Y’ und dem Triphenylmethan (TPM) Phenolrot. Der Austausch von Phenolrot gegen ein Brom-Derivat führte zur Beobachtung eines weiteren Fluoreszenz-Effekts, welcher unabhängig vom exprimierten VSG auftritt und letal für den Parasiten ist. Dieser so genannten Trypanozide Effekt lässt auf konservierte Strukturen schließen, welche von allen VSGs geteilt werden und als hochspezifisches Drug Target gegen T. b. brucei fungieren könnten. Das Ziel der vorliegenden Arbeit war es, die Mechanismen zu identifizieren, welche die einzigartige VSG’Y’-Fluoreszenz und den Trypanoziden Effekt auslösen. Fluoreszenz-Experimente und Protein-Mutagenese von VSG’Y’, sowie röntgenkristallographische Analysen mit mehreren unterschiedlichen VSGs wurden in dem Bestreben durchgeführt, die Bindung zwischen VSGs und TPMs zu charakterisieren, potentiell konservierte Strukturen von VSGs zu finden und die Mechanismen der einzigartigen VSG’Y’-Fluoreszenz und des Trypanoziden Effekts zu identifizieren. Diese Arbeiten haben das Potenzial die Formulierung hochspezifischer Medikamente mit VSGs als Drug Target anzutreiben. Im Rahmen der kristallographischen Analysen wurden die ersten vollständigen VSG Strukturen ermittelt, welche eine hohe Bedeutung für das Verständnis über die Bildung des VSG-Mantels haben. Die VSG Strukturen wurden u.a. per Röntgenkristallographie und Kleinwinkel-Röntgenstreuung aufgeschlüsselt und zeigten dass VSGs ein hohes Maß an Flexibilität besitzen. Diese Flexibilität ist wichtig für die Bildung eines dichten und hochmobilen VSG-Mantels.