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.

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.

This thesis deconstructs the social, ecological, and colonial elements of the 1900-1910 Human African Trypanosomiasis (African Sleeping Sickness) epidemic which affected British Uganda and Belgian Congo. This paper investigates the epidemic’s medical history, and the subsequent social control policies which sought to govern the actions of the indigenous population. In addition, this paper argues that the failure to understand and respect the region’s ecological conditions and local knowledge led to disease outbreaks in epidemic proportions. Retroactive policies sought to inflict western medical practices on a non-western population, which resulted in conflict and unrest in the region. In the Belgian Congo, colonial authorities created a police state in which violence and stringent control measures were used to manage the local population. In Uganda, forced depopulation in infected regions destabilized local economies. This thesis compares and contrasts the methods used in these regions, and investigates the effects of Germ Theory on Sleeping Sickness policy and social perceptions during the colonial period in Africa.

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.

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.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds have the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds ave the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biological Sciences. / Includes bibliographical references (leaves 51-53).

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Biological Sciences. / Includes bibliographical references (leaves 46-50).

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