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The African Trypanosomiases are a suite of diseases affecting humans and animals across sub-Saharan Africa, caused by protozoan trypanosome parasites and transmitted by tsetse flies (Glossina spp.). A number of different trypanosome species cause Animal African Trypanosomiasis (or Nagana) in wildlife and livestock, and have a major impact on animal health, agriculture and the economy. Two subspecies of African trypanosomes infect humans: Trypanosoma brucei gambiense causes a chronic disease in Central and Western Africa and Trypanosoma brucei rhodesiense causes an acute disease in Eastern and Southern Africa. Human African Trypanosomiasis (HAT), or sleeping sickness, is fatal if left untreated. Uganda is the only country to have active but geographically discrete foci of both T. b. gambiense HAT (gHAT) and T. b. rhodesiense HAT (rHAT). Recently, Northwards migration of rHAT has been observed in Uganda, associated with movements of cattle that serve as reservoirs for the zoonotic T. b. rhodesiense parasite. Convergence of the two forms of HAT in districts of Northern Uganda would impact on correct diagnosis and treatment since rHAT and gHAT require different diagnostics and treatment. The work described in this thesis explores the impact of recent cattle-based interventions for the control of zoonotic rHAT, specifically targeted at preventing the Northwards migration of T. b. rhodesiense in cattle in Uganda. Previously, a cross-sectional survey of cattle in 150 villages across 32 rHAT-affected districts of Uganda was undertaken in 2014 using the Polymerase Chain Reaction (PCR); screening determined the prevalence of human-infective T. b. rhodesiense and non-human-infective Trypanosoma brucei brucei parasites. In 2018, cattle from 20 villages in three districts of Northern Uganda were screened to determine change in trypanosome prevalence across these 20 sites. Between 2014 and 2018, overall trypanosome prevalence was observed to have significantly reduced (p < 0.0001) from 59.05% (95% CI 56.02% - 62.02%; 607/1,028) to 15.17% (95% CI 13.07% - 17.54%; 150/989), a trend observed for all individual trypanosome species. The prevalence of Trypanosoma brucei sensu lato decreased from 35.51% (95% CI 32.64% - 38.48%; 365/1,028) to 6.88% (95% CI 5.46% - 8.63%; 68/989), (p < 0.0001), and minimum prevalence of human-infective T. b. rhodesiense reduced from 4.67% (95% CI 3.54% - 6.14%; 48/1,028) to 2.12% (95% CI 1.39% - 3.22%; 21/989), (p = 0.0020). Between 2014 and 2018, the prevalence of Trypanosoma vivax decreased from 15.86% (95% CI 13.75% - 18.22%; 163/1,028) to 4.75% (95% CI 3.59% - 6.26%; 47/989), (p < 0.0001), and the prevalence of Trypanosoma congolense decreased from 31.42% (95% CI 28.66% - 34.32%; 323/1,028) to 5.46% (95% CI 4.21% - 7.06%; 54/989), (p < 0.0001). The scale (and/or direction) of change in prevalence was observed to vary at both district and village level. A human survey for HAT was also undertaken in the same 20 villages in 2018; 5,383 individuals were screened for the presence of Trypanosoma brucei sensu lato DNA using PCR. In total, 10 samples were considered positive for T. brucei sl (0.19%; 95% CI 0.10% - 0.35%) and were examined to determine the sub-species. Association between villages with PCR-positive human samples and the presence of humaninfective T. b. rhodesiense parasites was observed. Human-infective T. b. rhodesiense parasites and non-human-infective T. b. brucei parasites co-exist in the cattle population in Uganda. This thesis explores the dynamics of distribution of human- and non-human-infective T. brucei in cattle before and after treatment; in particular, the impact of interventions on the proportion of T. b. rhodesiense within the T. brucei sl population. On very few occasions does the relative prevalence of T. b. rhodesiense appear to exceed that of T. b. brucei in cattle populations, despite epidemiological theory predicting otherwise. Explanations for these observations are discussed, and progress towards elimination of Human African Trypanosomiasis as a public health problem is considered, taking into account the findings of this thesis.

In Zambia, African trypanosomiasis (AT) is endemic in tsetse-infested Mambwe district. Although cases are common in livestock, they are rarely reported in dogs. However, recent studies have detected trypanosomes in Zambian dogs. As a follow-up, the current research was designed to investigate what communities know about tsetse flies and AT and the role that dogs might play as trypanosome hosts. Data from the knowledge, attitudes, and practices survey and focus group discussions revealed that tsetse flies were present and respondents correctly identified and associated tsetse flies with the transmission of AT. This study also followed a cohort of 162 indigenous dogs for a period of six months. Nearly 20% of dogs were lost to follow-up, with the main causes being ill-health, predation, and euthanasia. Various African trypanosome species were detected by microscopy, serology, and PCR. Nzi traps captured a total of 3895 tsetse flies; molecular analyses found 70% of tsetse flies trypanosome positive. Blood meal analysis of 217 tsetse flies found DNA linked to humans (64.1%), wildlife (31.8%), cattle (2.3%), and dogs (1.8%). In conclusion, the results suggest that dogs acquire and maintain trypanosomes long enough for them to be potential reservoirs in the epidemiology of AT in Mambwe district. Therefore, the policy on treatment, prevention, and control of AT in Zambia needs to include dogs as important hosts.

The protozoan parasite, Trypanosoma brucei causes devastating diseases in both humans and animals in sub-Saharan Africa. They live extracellularly and undergo a complex life cycle involving the mammalian host and the tsetse fly vector. Two developmental forms of the parasite exist in the bloodstream of the mammalian host; the long proliferative slender forms and the cell-cycle arrested stumpy forms. The slender forms differentiate into stumpy forms upon reaching a density threshold through a quorum-sensing like mechanism. During a trypanosome infection, the parasites release peptidases into the bloodstream of their mammalian host which accumulates as parasitaemia increases. These released peptidases hydrolyse their respective host protein substrates resulting in the generation of oligopeptides. At peak parasitaemia, these oligopeptides accumulate and trigger the slender forms to differentiate into non-proliferative stumpy forms. This helps regulate the parasitaemia in the host as well as prepare the parasites for development in the tsetse fly vector. Two peptidases whose activities can provoke the quorum-sensing response have been identified previously in our lab. In this study, proteins secreted/released by the parasites in the bloodstream and early during differentiation to the tsetse fly midgut procyclic forms were analysed by detailed mass spectrometry. This identified twelve peptidases, belonging to different classes of peptidases, enriched at these developmental stages of the parasite. Each peptidase was then validated for its release from parasites using individually epitope-tagged cell lines. Systematic ectopic overexpression and gene knockout using CRISPR/Cas9 of each peptidase gene and their analysis in vivo in mice revealed that two of the peptidases, oligopeptidase B and metallocarboxypeptidase 1, significantly contribute to the generation of the trypanosome's quorum-sensing signal. Further analysis of peptidases enriched in the secretome of parasites differentiating from stumpy forms to procyclic forms in tsetse flies, however, showed that these peptidases may not be involved in establishing infection in the midgut of the flies but may assist proventricular infection. This work analysed for the first time the contribution of a set of released peptidases by T. brucei involved in the important differentiation from proliferative slender forms into the cell-cycle arrested stumpy forms.

A range of fluorinated heterocyclic scaffolds: quinazolines, benzofurans, benzothiophenes, benzothiazoles, indoles, and phenoxazines have been synthesised in this project using a tandem SNArring closing strategy employing perfluoroarenes as starting materials. A range of novel compounds including quinazolines, benzofurans and many more have been characterised by IR spectroscopy, ¹H/¹⁹F/¹³C NMR spectroscopy, elemental analysis, HRMS, and eight X-ray crystal structures have been determined. The novel fluorinated compounds accessed in this work were screened against T b rhod, T cruzi and L. don. Ax am the causative agents of three neglected tropical diseases: sleeping sickness, Chagas disease and leishmaniasis respectively. Of the compounds screened quinazoline 129 presented itself as a lead compound with IC50 values of 0.84 µM and 0.22 µM against T b rhod and T cruzi respectively. Subsequently synthetic reactions involving further SNAr reaction, or functional group modification have been carried out to explore the chemical space of the lead compound to build up SAR data. An improved synthesis of fluorinated quinazolines involving condensation of fluoroaryl nitriles or ketones with amidines has been developed. Microwave irradiation has been shown to be a viable method to obtain this scaffold in a more efficient and greener synthesis. Mechanistic studies have been undertaken to ascertain the mechanism of the deacylation of polyfluoroaryl aldehydes, ketones and esters identified as a competing side reaction during attempts to access benzofurans, indoles and quinazolines under basic reaction conditions. NMR spectroscopic experiments involving deuterium labelling established that in the presence of base deacylation occurs with generation of an aryl anion, a short-lived intermediate which subsequently reacts with moisture forming protonated fluoroarene products. The findings are in agreement with earlier studies on related perfluoroaryl systems. Additionally, a mechanistic study has been carried out on the ring closing reaction of 2-benzamidophenylsulfanyl fluoroarenes which forms fluorinated phenothiazine scaffold to ascertain whether a Smiles rearrangement occurs before ring closure. Experiments to follow the course of the reaction by ¹⁹F NMR spectroscopy were undertaken, and the determination of the X-ray crystal structure of 367 confirmed that Smiles rearrangement had occurred before ring closure.

The haptoglobin-haemoglobin receptor (HpHbR) is expressed by the African try- panosome, T. brucei, whilst in the bloodstream of the mammalian host. This allows ac- quisition of haem, but also results in uptake of trypanolytic factor 1, a mediator of in- nate immunity against non-human African trypanosomes. Here, the structure of HpHbR in complex with its ligand, haptoglobin-haemoglobin (HpHb), is presented, revealing an elongated binding site along the membrane-distal half of the receptor. A ~50° kink allows the simultaneous binding of two receptors to one dimeric HpHb, increasing the efficiency of ligand uptake whilst also increasing binding site exposure within the densely packed cell surface. The possibility of targeting this receptor with antibody-drug conjugates is ex- plored. The characterisation of the unexpected interaction between T. congolense HpHbR and its previously unknown ligand, haemoglobin, is also presented. This receptor is iden- tified as an epimastigote-specific protein expressed whilst the trypanosome occupies the mouthparts of the tsetse fly vector. An evolutionary pathway of the receptor is proposed, describing how the receptor has changed to adapt to a role as a bloodstream form-specific protein in T. brucei. Apolipoprotein L1 (ApoL1) is the pore-forming component of the trypanolytic factors. An expression and purification protocol for ApoL1 is presented here, and the functionality of the protein established. Initial attempts to characterise the pores and structure of ApoL1 are described.

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

The global One Health movement has become firmly entrenched in both political and scientific discourse pertaining to emerging infectious diseases in the past decade. Since the discovery of the H5N1 strain of Highly Pathogenic Avian Influenza in Hong Kong in 1997, the promotion of more holistic programmes for the control of emerging infectious disease has garnered “unprecedented support” in terms of donor funding and political mobilisation (Scoones 2010). Advocates of One Health argue that intersectoral approaches promoting better communication between the veterinary, medical and environmental disciplines at all levels of governance make not only sound economic sense, they are fundamental to the “new approach” required to address the growing disease threats of the 21st century. However, despite international endorsement of the One Health rhetoric, there is growing pressure to now “turn the rhetoric into reality” (Okello et al 2011). Using a multiple, embedded case study methodology, this thesis seeks to examine questions surrounding the practical implementation of One Health interventions, particularly in developing countries which experience limited resources and competing health priorities. Through examining the livestock and public health policy processes at both local and national levels in Uganda and Nigeria, I attempt to identify whether policy spaces exist for the formal inclusion of One Health approaches in future policy decisions. Furthermore, by scrutinising the current internationally dominant One Health narratives in light of global health governance perspectives and the emerging One Health Global Network, I question whether One Health can be better “packaged” to include endemic diseases and a more focussed sustainable livelihoods approach; arguably inciting greater motivation for developing countries to truly participate. Data from my three empirical chapters are presented in the context of three overriding “One Health propositions” for consideration; by questioning “whose world, whose health”, I aim to delve further into the issues of not whether, but how this “new health paradigm” can be operationalised, and how to address the potential gaps which may ultimately prevent One Health from becoming a truly global phenomenon.

Over the past two decades movement of cattle towards the north of Uganda has enabled the Trypanosoma brucei rhodesiense focus in south-eastern Uganda to spread into previously unaffected districts. This thesis brings together important epidemiological data regarding the impact of mass cattle drug treatment on the point prevalence of several different species of trypanosome in a newly endemic area of human sleeping sickness. Crucially the findings illustrate mass drug treatment is effective in reducing the prevalence of T. b. rhodesiense in cattle, thus minimising the reservoir potential of these animals in the epidemiology of human disease. During 2006 a control programme was launched to halt the northward spread of this zoonotic parasite. This programme, entitled ‘Stamping Out Sleeping Sickness’ (SOS) proposed to reduce the prevalence of Human African Trypanosomiasis (HAT) in the newly affected districts by reducing the prevalence of this parasite in the main animal reservoir of infection – domestic cattle. Cattle were mass treated using trypanocides to clear infections. Previous work demonstrated the prevalence of T. brucei s. l. and T. b. rhodesiense in cattle was higher in the districts of Dokolo and Kaberamaido than in the other SOS intervention districts (Selby 2011). To determine whether animals in these areas were also exposed to pathogenic cattle trypanosomes samples were screened for the presence of T. vivax and T. congolense savannah using PCR. Chapter three of this thesis determined the prevalence of these trypanosomes in cattle in these districts. Before treatment had taken place the prevalence of T. vivax was 2% (4/200, 95% CI 3.57 – 0.12%) in Dokolo and 7.3% (21/310, 95% CI 10.17 - 4.24 %) in Kaberamaido. The prevalence of T. congolense savannah at baseline was 3.5% (7/200, 95% CI 7.08–1.42 %) in Dokolo and 9.1% (21/230, 95% CI13.6–5.7 %) in Kaberamaido. Monitoring was conducted three, nine and 18 months post treatment and both pathogens were detected at all time points. The impact the treatment had on point prevalence varied by trypanosome species and between the two districts. Several clusters of villages in Dokolo and Kaberamaido continued to report cases of HAT after the initial SOS intervention due in part to their proximity to livestock markets (Batchelor et al., 2009). In 2008 re-treatment of these ‘high risk’ areas was undertaken. Monitoring was performed before and six months after treatment. Cattle blood samples were collected at 20 village sites from ten ‘case-positive villages’ (from which human sleeping sickness cases had been reported six months prior to June 2007) and from ten ‘case-negative villages’ (no reported human sleeping sickness cases six months prior to June 2007). These samples were screened for all of the aforementioned trypanosomes using species specific PCR protocols. Chapter five details the results of this screening, and assessed whether re-treatment in Dokolo and Kaberamaido was effective in reducing the prevalence of trypanosomiasis. The re-treatment had a dramatic effect, significantly reducing the point prevalence of overall trypanosomiasis in the 20 villages screened from 38.1% (95% CI = 40.5 – 35.79%) at baseline to 26.9% (95% CI 28.96 – 24.97, p < 0.0001) at six months. Looking at each species separately, point prevalence of three out of four detected species of trypanosome fell significantly, including T. b. rhodesiense, which was reduced to 25% of its baseline prevalence. Finally the two SOS treatment cycles were compared both statistically and spatially with emphasis on trends at village level and the occurrence of mixed infections.

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.

In October 2006 an intervention was initiated to arrest the northerly advance through Uganda of the zoonotic parasite Trypanosoma brucei rhodesiense. This is a protozoal infection that is vectored by the tsetse fly. It is the aim of this thesis to review the impact of this large scale treatment programme in terms of animal health and human disease. The Stamp Out Sleeping Sickness (SOS) campaign was designed to target the cattle reservoir of T. b. rhodesiense in these newly affected areas by block treating >180,000 head of cattle. This was achieved in collaboration with final year vet students from the University of Makerere, Uganda. Farmers were also encouraged to spray their animals with deltamethrin in order to suppress the tsetse population. In order to monitor the impact of this intervention a base line survey was carried out. Evaluation of the logistics and implementation of the SOS campaign was assessed through interviews with personnel involved. Analysis by PCR revealed the prevalence of T. brucei s.l. as 15.57% (T. b. rhodesiense as 0.81%) within the cattle reservoir prior to SOS treatment. Follow up sampling was carried out at 23 locations at three, nine and 18 months. The prevalence of T. brucei s.l. was reduced post treatment, but in the absence of sustained vector control infections amongst the animals returned by nine months and subsequently exceeded the base line findings (P=<0.0001). It was observed that across most of the SOS area, T. b. rhodesiense did not re-establish following treatment. However, a significant cluster was identified where cases of both human and animal disease were continually reported. This cluster was noted to include the area immediately surrounding the Otuboi cattle market. This link between cattle movement and the spread of T. b. rhodesiense is an established one and is addressed by Ugandan governmental policy which states that ‘cattle traded at market must be treated with trypanocidal drugs prior to movement’. The findings presented here suggest that this policy may not be strictly enforced. The risk of spread is compounded at the northern districts of Uganda restock their domestic livestock following years of civil conflict. The majority of animals are traded in a northward direction – transporting infected animals from the endemic south. The scale of this trade is assessed through questionnaires, analysis of trade records and animal screening. Specific consideration is given to the implications of this cattle trade and impact this may have on the sustainability of the SOS campaign.

Acute and chronic sleeping sickness are fatal neglected tropical diseases caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense respectively (members of the sub-genus Trypanozoon). Accurate diagnostics are needed to guide treatment since the symptoms of disease are non-specific and the drugs that are used for treatment are too toxic to be administered to unconfirmed cases. Tests need to be simple enough to confirm clinical diagnosis of sleeping sickness in poorly-resourced, peripheral health centres and for use as epidemiological tools to detect T. b. rhodesiense in the zoonotic reservoirs of infection. This study focuses upon LAMP (loop-mediated isothermal amplification) as a novel diagnostic for sleeping sickness that may serve to bridge the gap between the need for sensitive, specific molecular diagnostics on the one hand and ‘field-friendly’ diagnostics on the other. Here, two previously published LAMP assays for Trypanozoons were compared to classic PCR based methods for the diagnosis of Trypanozoon infection status in 428 cattle blood samples. The results did not support the use of LAMP as an improved system for surveillance of T. b. rhodesiense in the zoonotic cattle reservoir. T. b. rhodesiense and T. b. gambiense subspecies specific LAMP assays were evaluated against traditional reference subspecies specific PCR tests, using DNA purified from 86 cryopreserved trypanosome isolates. Novel LAMP assays for these subspecies were also designed and evaluated. Both the published and novel assays for T. b. rhodesiense (targeting different regions of the SRA gene) were sensitive, specific and reliable when applied to purified DNAs, but were less consistent on field samples. The novel T. b. gambiense LAMP (targeting TgsGP) was sensitive and specific but this was not the case for the published LAMP assay (targeting the 5.8S rRNA gene). However reliability may be less than optimal for LAMP TgsGP. Finally, simple endpoint readout methods for LAMP were evaluated. The colour change reagent hydroxynaphthol blue was identified as the best currently available method taking cost, ease of use and reliability into consideration. In 2009 the number of reported sleeping sickness cases fell below 10,000 for the first time in 50 years. Improved LAMP diagnostics could facilitate the diagnosis of sleeping sickness and support the continued fight against this neglected, but deadly disease.

Human African trypanosomiasis (sleeping sickness) is a devastating disease which is endemic in parts of sub-Saharan Africa. It is caused by the protozoan parasite T. brucei, which are transmitted by the bite of infected tsetse flies. Although the disease is fatal if left untreated, there is a lack of safe, effective and affordable drugs available; therefore new drugs are urgently needed. The aim of the work presented in this thesis is to develop novel trypanocidal compounds. It is divided into two parts to reflect the two distinct strategies employed to achieve this aim. The first part focuses on the inhibition of glycophosphoinositol (GPI) anchor synthesis by inhibiting the Zn2+-dependent enzyme, GlcNAc-PI de-N-acetylase. Trypanosomes have a variable surface glycoprotein (VSG) coat, which allows them to evade the human immune system. The GPI anchor attaches the VSG to the cell membrane; therefore inhibiting GPI synthesis should expose the parasite to the immune system. Initially, large substrate analogues were synthesized. These showed weak inhibition of the enzyme. Zinc-binding fragments were screened, and small molecule inhibitors based on salicylhydroxamic acid were then synthesized. These compounds showed modest inhibition, but the excellent ligand efficiency of salicylhydroxamic acid indicates this may be a promising starting point for further inhibitors. The second part details the P2 strategy. The P2 transporter is a nucleoside transporter unique to T. brucei, which concentrates adenosine. The transporter also binds and selectively concentrates compounds that contain benzamidine and diaminotriazine P2 motifs, which can enhance the potency and selectivity of these compounds. The sleeping sickness drugs melarsoprol and pentamidine contain P2 motifs. Compounds comprising a P2 targeting motif, a linker and a trypanocidal moiety were synthesized. Initially, a diaminotriazine P2 motif was attached to a trypanocidal tetrahydroquinoline (THQ) protein farnesyl transferase (PFT) inhibitor, with limited success. The P2 strategy was also applied to a non-selective, trypanocidal, quinol moiety. The quinol moiety was attached to diaminotriazine and benzamidine P2 motifs, and an increase in selectivity for T. brucei over MRC5 cells was observed.

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.

The tsetse transmitted fatal disease of humans, sleeping sickness, is caused by two morphologically identical subspecies of the parasite T. brucei; T. b. rhodesiense and T. b. gambiense. Current distributions of the two forms of disease are not known to overlap in any area, and Uganda is the only country with transmission of both. The distribution of Rhodesian sleeping sickness in Uganda has expanded in recent years, with five districts newly affected since 1998. This movement has narrowed the gap between Rhodesian and Gambian sleeping sickness endemic areas, heightening concerns over a potential future overlap which would greatly complicate the diagnosis and treatment of the two diseases. An improved understanding of the social, environmental and climatic determinants of the distribution of Rhodesian sleeping sickness is required to allow more effective targeting of control measures and to prevent further spread and possible concurrence with Gambian sleeping sickness. The work presented in this thesis investigates the drivers of the distribution and spread of Rhodesian sleeping sickness in districts of central and eastern Uganda which form part of the recent disease focus extension. The spatial distribution of Rhodesian sleeping sickness was examined in Kaberamaido and Dokolo districts where the disease was first reported in 2004, using three different methodologies. A traditional one-step logistic regression analysis of disease prevalence was compared with a two-step hierarchical logistic regression analysis. The two-step method included the analysis of disease occurrence followed by the analysis of disease prevalence in areas with a high predicted probability of occurrence. These two methods were compared in terms of their predictive accuracy. The incorporation of a stochastic spatial effect to model the residual spatial autocorrelation was carried out using a Bayesian geostatistical approach. The geostatistical analysis was compared with the non-spatial models to assess the importance of spatial autocorrelation, to establish which method had the highest predictive accuracy and to establish which factors were the most significant in terms of the disease’s distribution. Links between Rhodesian sleeping sickness and landcover in Soroti district were also assessed using a matched case-control study design. Temporal trends in these relationships were observed using an annually stratified analysis to allow an exploration of the disease’s dispersion following its introduction to a previously unaffected area. This work expands on previous research that demonstrated the source of infection in this area to be the movement of untreated livestock from endemic areas through a local livestock market. With regards to the comparison of regression frameworks, the two-step regression compared favourably with the traditional one-step regression, but the Bayesian geostatistical analysis outperformed both in terms of predictive accuracy. Each of these regression methods highlighted the importance of distance to the closest livestock market on the distribution of Rhodesian sleeping sickness, indicating that the disease may have been introduced to this area via the movement of untreated cattle from endemic areas, despite the introduction of regulations requiring the treatment of livestock prior to sale. In addition, several other environmental and climatic variables were significantly associated with sleeping sickness occurrence and prevalence within the study area. The temporal stratification of the matched case-control analysis highlights the dispersion of sleeping sickness away from the point of introduction (livestock market) into more suitable areas; areas with higher proportions of seasonally flooding grassland, lower proportions of woodland and dense savannah and lower elevations. These findings relate to the habitat preferences of the predominant vector species in the study area; Glossina fuscipes fuscipes, which prefers riverine vegetation. The findings presented highlight the importance of the livestock reservoir as well as the climatic and environmental preferences of the tsetse fly vector for the introduction of Rhodesian sleeping sickness into previously unaffected areas, the subsequent spread of infection following an introduction and the equilibrium spatial distribution of the disease. By enhancing the knowledge base regarding the spatial determinants of the distribution of Rhodesian sleeping sickness within newly affected areas, future control efforts within Uganda may be better targeted to decrease prevalence and to prevent further spread of the disease.

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

The protozoan pathogen, Trypanosoma brucei, is the causative agent of sleeping sickness in humans and nagana in livestock in sub-Saharan Africa. T. brucei cycles between tsetse fly and mammalian hosts, and it is adapted to survive in diverse host tissues. Variant Surface Glycoprotein (VSG) plays a key role in immune evasion in the mammalian host. The VSG membrane anchor requires two myristates, 14-carbon saturated fatty acids (FAs) that are scarce in the host. T. brucei can synthesize FAs de novo, but also readily takes up exogenous FAs, despite lacking homologs to fatty acid uptake proteins found in other organisms. Previous work in our lab found that Triacsin C, an inhibitor of acyl-CoA synthetases (ACSs), inhibited the uptake of fluorescent FAs and was toxic to T. brucei. I hypothesized that Grassofermata (GF), an inhibitor of FA transport proteins (FATPs), might also affect T. brucei, as FATPs have intrinsic ACS activity. I found that both mammalian bloodstream forms (BFs) and tsetse fly midgut procyclic forms (PFs) of T. brucei internalized fluorescently-labeled FAs (BODIPIY-FAs). Treatment with GF decreased BODIPY-FA uptake as measured by fluorescence microscopy and flow cytometry, though GF was unable to completely inhibit BODIPY-FA uptake. The residual BODIPY-FA uptake observed under GF inhibition was not due to endocytic mechanisms. Treatment with GF also killed both BF and PF T. brucei, and cell death was dependent on GF concentration as well as treatment time. Addition of unlabeled C12:0 and C16:0 FAs to GF-treated cells was unable to “rescue” cell growth. Finally, the GF concentrations required to inhibit BODIPY-FA uptake (EC50s of ~70–100 µM) were much higher than that for Triacsin C inhibition (2–8 µM). Importantly, the EC50s for BODIPY-FA uptake inhibition by GF were higher than that for cell killing by GF (EC5048hr of 4-17 µM). Based on these results, we conclude that GF killing of T. brucei does not seem to be a result of inhibiting FA uptake and/or ACSs. Instead, GF may be affecting other known targets in the cell, such as ADP-ribosylation factor (ARF) and its accessory factors. Future studies will be needed to confirm the mode of action of GF in T. brucei.

In all eukaryotic organisms, from yeast to mammals, progression through the cell cycle is ultimately regulated by the actions of a group of highly conserved serine/threonine protein kinases named the cyclin dependent kinases (CDKs). CDK activity is controlled by the association of CDK subunits with regulatory protein subunits named cyclins to form active CDK-cyclin complexes. The trypanosomatids represent one of the earliest known divergent branches of the eukaryotic phylogenetic tree. At the start of this project several putative CDK and cyclin homologues had been described in Trypanosoma hrucei, tbCRKl-3 and tbCYC1. During the project tbCRK2 and tbCYC1 were analysed and a new CRK (tbCRK4) isolated. The function of the tbCRK2 protein and the tbCRK2 gene was investigated using biochemical and molecular genetic approaches. Use of anti-CRK2 antisera showed that tbCRK2 is expressed in both proliferative and nonproliferative life cycle stages of T. brucei. Attempts to create null mutants at the tbCRK2 locus repeatedly failed, with the wild-type allele preserved in all transgenic lines generated. Ectopic expression of a cloned copy of tbCRKl did not allow deletion of wild-type alleles, although the cloned copy of tbCRK1 was demonstrated to generate abnormally high cellular levels of tbCRK2. These results may suggest that tbCRK2 is an essential gene in the procyclic form of the parasite; however, control experiments are needed to verify this. The tbCYC1 protein was previously reported to be a mitotic-type cyclin in T. brucei. Biochemical and molecular genetic approaches were taken to investigate tbCYC1 function. A specific anti-CYC 1 antiserum was generated, which demonstrated that in T. brucei strain STIB 247 tbCYC1 is expressed in the short stumpy bloodstream and procyclic forms, but not in the long slender bloodstream form, suggesting a function in differentiation rather than cell cycle control per se. No significant tbCYC1-associated kinase activity was found in procyclic extracts. In addition, no association of tbCYC1 with any of the known T. brucei CRKs or with the yeast protein p13SUCI or the leishmanial protein pl2CKSI was found in procyclic extracts. These data suggests that tbCYC1 does not fulfil the function of a mitotic-type cyclin. Attempts to generate null mutants for the tbCYC1 locus failed, which may provide preliminary evidence that tbCYC1 serves an essential function in the procyclic form. However, further experiments are required to clarify this. A full-length genomic clone of the tbCRK4 gene was isolated and demonstrated to be homologous to the Crithidia fasciculata cfCRK gene. Computer analysis revealed the presence of two large, highly hydrophilic insert dpmains between the conserved kinase catalysis domains VIb and VII, and X and XI. Specific anti-CRK4 antisera were generated and used to demonstrate that in strain STIB 247, tbCRK4 is expressed in the short stumpy bloodstream and procyclic forms, but not in the long bloodstream form. No tbCRK4-associated kinase activity was detected from procyclic extracts, and no interactions with the yeast protein p13SUCI or the leishmanial protein pl2CKSI was found from procyclic extracts. This study has provided evidence that mechanisms of cell cycle control in trypanosomatids may be based upon the functional CDK-cyclin paradigm observed in higher eukaryotes yet have diverged to serve the parasite's unique biology. If sufficient divergence in terms of biochemical function between trypanosome CRKs and cyclins and the CDKs and cyclin of mammals could be demonstrated, they would provide a tempting drug target.

African trypanosomiasis is caused by Trypanosoma brucei and is a disease of considerable importance causing infection in both humans and livestock. There is a degree of protection from the antibody response produced by the host but it is ineffective and infections are chronic and debilitating. This chronicity is due to antigenic variation by the trypanosomes and to immunosuppression of the host. Antigenic variation is a classic, and well studied, B-cell evasion mechanism in a number of infections and can be observed at a highly sophisticated level during African trypanosome infections. As yet, it has been little studied as a possible T-cell evasion mechanism and this is the subject of my thesis. Initially, a reliable in vitro assay system was devised in order to examine the proliferative T-cell responses of mononuclear splenocyte populations as T-cell proliferation had not been detected during trypanosome infections in previous studies. Responses against trypanosome lysates, paraformaldehyde-fixed and live trypanosomes were examined with paraformaldehyde-fixed trypanosomes being the preferred choice of antigenic stimuli. The optimal conditions for this assay were also determined as far as mitogen concentration, concentration of trypanosome antigen and incubation time were concerned. Using this reliable proliferation assay system I examined a number of trypanosome infection and immunisation regimes. Most of these analyses were conducted using splenocytes taken from mice at first peak of parasitaemia employing parasite lines in each of which 99% of the trypanosomes present express the same variant antigen type (VAT) during the first parasitaemic wave. The mononuclear splenocytes from infected mice produced a high level of proliferation in response to mitogen stimulation but also produced trypanosome antigen-driven proliferation. This antigen-driven proliferative response was mainly against the homologous VATs but there was also a degree of heterologous antigen-driven proliferation in response to some VATs but not against others. Immunosuppression is considered to be a major contributing factor in continuation of an infection by dampening down any effective immune response during African trypanosomiasis. Immunosuppression is noticeable after the first peak of parasitaemia and involves a number of components with 'suppressor' macrophages appealing to play a key role. I therefore investigated the role of nitric oxide (NO), a major product of activated macrophages, in the regulation of T-cell responses during chronic Trypanosoma brucei infection. Using transgenic mice deficient in inducible nitric oxide synthase (iNOS), the proliferative and cytokine responses were examined. Daily parasitaemias were determined, nitrate levels calculated and antibody isotyping examined using infected and uninfected mice homozygous and heterozygous for the iNOS loci. This work highlighted the key role of NO in the regulation of T-cell responses during trypanosome infections: in the absence of iNOS activity there was an upregulation in proliferation, IFNgamma production and IL-2 receptor expression causing improved clearance of trypanosomes from the systemic circulation. In conclusion, this body of work has successfully designed an in vitro assay system to examine T-cell proliferation to trypanosome VATs and, using this assay, I have successfully detected trypanosome antigen-driven T-cell proliferation during acute infections. This proliferation was observed using mononuclear splenocyte populations from infected and immunised mice and was found to be homologous antigen-driven and of T-helper 1 type. There was however heterologous antigen-driven T-cell proliferation against some trypanosome VATs but a lack of proliferation against others. A pivotal role for NO in causing immunosuppression during chronic T. brucei infections was determined and this immunosuppression influenced the cytokine production and T-cell proliferative responses but did not appear to affect the antibody production. These data suggest that VAT-specific T-cell responses are an important component of the immune response to T. brucei.

This thesis concerns the role of astrocytes in the neuropathogenesis of trypanosomiasis caused by Trypanosoma brucei brucei in mice, a model of the human disease caused by T.b. rhodesiense and T.b. gambiense. Chapter 1 of this thesis includes a literature review of the published work on human African trypanosomiasis (HAT) with a bias on the pathology and possible pathogenic mechanisms, particularly as they relate to the central nervous system (CNS). It also reviews the role of astrocytes in the normal and diseased CNS, and their possible role in the genesis of CNS pathological changes during trypanosome infections. The biological activities of cytokines reported to be produced in the CNS of trypanosome-infected animal models are considered. The chronic trypanosomiasis mouse model, used in this study, is described highlighting the similarities of the disease syndromes in this model to HAT. Chapter 2 describes the experimental techniques used in this study. Chapter 3 demonstrates the occurrence of astrocyte activation, as judged by an increase in the intensity of glial fibrillary acidic protein (GFAP) staining and morphological changes, within the CNS of mice chronically infected with T.b. brucei. It was shown that astrocyte activation first occurred 21 days after infection around the ventricles and the choroid plexus, along the choroid fissure, including, the hippocampus and the base of the cerebellum. It was also demonstrated that astrocyte activation preceded the infiltration of the choroid fissure, the perivascular spaces and the meninges, with inflammatory cells. It would appear that this activation involves production of mediators of inflammation which initiate extravasation of inflammatory cells, and possibly induce further astrocyte activation. From these initial sites, astrocyte activation spreads first to the white matter, the corpus callosum and the cerebellar lobes, and then to the grey matter, of both the cerebrum and the cerebellum. The infiltration of inflammatory cells seemed to enhance the spread and the degree of astrocyte activation. It is known that these areas where initial astrocyte activation occurred, have an incomplete blood-brain barrier and, they have been reported as the first sites of trypanosome invasion, at around day 14 of infection. This suggested that the astrocyte activation observed in these areas, was the first CNS response to invasion by trypanosomes. Subcurative therapy with Berenil, seemed to facilitate inflammatory cell infiltration and astrocyte activation and to hasten the progress of the CNS pathological changes. Treatment of relapsed infections with a second subcurative dose of Berenil increased the inflammatory cell infiltration and the astrocyte activation, resulting in a post-treatment reactive encephalitis (PTRE), similar to that observed in human reactive arsenical encephalopathy (RAE). Curative trypanocidal therapy rapidly removed the infiltrating cells followed by a slow regression of astrocyte activation. It was concluded that the CNS pathological changes are the host's response to invasion by the parasite and that astrocytes played an important role in initiating these changes, possibly by producing inflammatory mediators such as cytokines. Chapter 4 demonstrates the response of cultured astrocytes, as judged by cytokine transcript expression, to in vitro stimulation with trypanosomes, whole trypanosome lysate and variable surface glycoprotein (VSG). Using reverse transcription, cDNA amplification by polymerase chain reaction, and gel electrophoresis, it was found that astrocytes responded by expressing transcripts for the cytokines IL-1alpha and -beta, IL-6, TNFalpha, MIP-1 and GM-CSF. Trypanosomes and whole trypanosome lysate were more potent stimuli than VSG in inducing expression of cytokine transcripts. To investigate translation of the gene transcripts into cytokine protein, immunocytochemistry using antimouse cytokine antibodies, was performed on astrocytes stimulated with trypanosome lysate in vitro. Cytokine protein for IL-1alpha, TNFalpha and IL-6, was detectable by 2 hours after exposure to trypanosome lysate. The cytokines shown to be expressed, in this study, are known to act in tandem and synergy to perform important inflammatory functions, including, leukocyte extravasation, migration, proliferation, adhesion-molecule and MHC-antigen expression, and cytokine production by inflammatory cells, microglia and endothelial cells. In addition, the same cytokines activate astrocytes in an autocrine manner, causing proliferation, MHC-antigen and adhesion-molecule expression. It is proposed that astrocytes respond to trypanosome invasion of the CNS by producing cytokines which recruit inflammatory cells into the subarachnoid and perivascular spaces, and the choroid fissure. The recruited inflammatory cells respond to these cytokines by proliferating, differentiating to antibody-producing plasma cells or to actively phagocytic cells, in the case of macrophages, as well as by producing cytokines.

Neglected tropical diseases (NTDs) encompass a group of infectious diseases with a protozoan etiology, high incidence, and prevalence in developing countries. As a result, economic factors constitute one of the main obstacles to their management. Endemic countries have high levels of poverty, deprivation and marginalization which affect patients and limit their access to proper medical care. As a matter of fact, statistics remain un- collected in some affected areas due to non-reporting cases. World Health Organization and other organizations proposed a plan for the eradication and control of the vector, although many of these plans were halted by the COVID-19 pandemic. Despite of the available drugs to treat these pathologies, it exists a lack of effectiveness against several parasite strains. Treatment protocols for diseases such as American trypanosomiasis (Chagas disease), leishmaniasis, and human African trypanosomiasis (HAT) have not achieved the desired results. Un- fortunately, these drugs present limitations such as side effects, toxicity, teratogenicity, renal, and hepatic impairment, as well as high costs that have hindered the control and eradication of these diseases. This review focuses on the analysis of a collection of scientific shreds of evidence with the aim of identifying novel chalcogen- derived molecules with biological activity against Chagas disease, leishmaniasis and HAT. Compounds illustrated in each figure share the distinction of containing at least one chalcogen element. Sulfur (S), selenium (Se), and tellurium (Te) have been grouped and analyzed in accordance with their design strategy, chemical synthesis process and biological activity. After an exhaustive revision of the related literature on S, Se, and Te compounds, 183 compounds presenting excellent biological performance were gathered against the different causative agents of CD, leishmaniasis and HAT.

African trypanosomes, including Trypanosoma brucei and related subspecies, are the causative agents of sleeping sickness in humans and nagana in cattle. These parasites are the source of significant human mortality and limit economic growth throughout sub-Saharan Africa. T. brucei has a complex life cycle with phases in an insect vector and a mammalian host. Although motility through fluids and on tissue surfaces is central to T. brucei development and disease pathogenesis, a complete description of how the parasite moves through fluids and behave on surfaces is not available. Through the use of novel techniques and classical molecular approaches applied towards the study of T. brucei motility in suspension media and on surfaces, we have discovered unappreciated aspects of protozoan parasite biology. Though the use of high-speed imaging, we found that that T. brucei moves through suspension media via the propagation of kinks generated by the parasite's ability to alternate the chirality of its rotation. The observation that T. brucei moves by generating alternating chiralities disproved the 150 year long notion that the parasite moves by spiraling in one direction and demonstrates that high-speed imaging can be a useful tool for uncovering important features of microorganism locomotion. By studying T. brucei on agarose plates, we discovered that T. brucei engages in social behavior when exposed to surfaces: an unprecedented finding in protozoan parasite biology. This social behavior, termed Social Motility (SoMo) is characterized by the formation of multicellular communities that engage in polarized migrations across the agarose surface and cooperate to divert their movements in response to external signals. Investigation of the mechanisms underlying this social behavior revealed a novel role for a unique family of receptor-like proteins that were previously uncharacterized and implicates a role for cAMP signaling in the regulation of SoMo. Lastly, our search for extra cellular factors that regulate this social behavior has led to the discovery of inter-kingdom cross-talk between T. brucei and bacteria. In short, the investigations presented here have uncovered novel aspects of T. brucei behavior that have transformed our understanding of protozoan biology and will serve as the basis for future investigations.

Residents of lowland Buha, in western Tanzania’s Kigoma Region, faced statesponsored, forced resettlement campaigns in both the colonial and postcolonial periods. In the 1930s, British colonial officials compelled those living in the easternmost areas to resettle closer to the roadway as a public health intervention in response to epidemic sleeping sickness. In the 1970s, Tanzanian officials forced everyone in the region to resettle again, this time in African socialist, or ujamaa, villages. This dissertation examines these schemes as part of a long-term history of resettlement in Buha, demonstrating how resettlement was a decades-long, unfolding process for both Ha people and government officials. In particular, I examine the interplay between the moral visions that different government officials and Ha people had for resettled areas and the material constraints in which they operated. Neither group could unilaterally implement their priorities but instead had to work within a series of limitations. For Ha people, this involved managing the forms of interference that government settlement and natural resource policies placed on their choice of domicile and use of resources. For state officials, they not only had to contend with competing Ha priorities, but also with resource limitations, internal divisions within their bureaucracies, and their own ideological commitments to western science and economic development. In the end, resettled communities created in the wake of removal were not the results of the transformative power of state planning, but instead formed at the intersection of Ha and governmental desires to replicate, adjust, or revolutionize Ha lives and livelihoods.

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.