Trypanosomiases and leishmaniases are amongst the world’s most neglected infectious diseases. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are the primary human pathogens of the trypanosomatidae family and the causative agents of African sleeping sickness, Chagas’ disease and cutaneous leishmaniasis, respectively. The molecular mechanism controlling each parasite’s life cycle and virulence is poorly understood however protein phosphatases are expected to play a critical role. This study presents the biochemical characterisation data of two groups of phosphatases from the trypanosomatids: the LRR-DSP and LM phosphatases. The LRR-DSPs are a group of twelve proteins, characterised by a unique domain architecture: a leucine-rich repeat (LRR) domain together with a dual- specificity phosphatase (DSP) catalytic domain. In this study, the recombinant expression of a representative LRR-DSP orthologue from T. brucei (TbLRR-DSP), proved to be highly problematic in E. coli. Most full-length and catalytic domain constructs were expressed at very low levels or as insoluble proteins. Soluble protein was obtained by denaturation, treatment with detergents, non-denaturing extraction from inclusion bodies and fusion to solubility-enhancing proteins. However, no method yielded protein with catalytic activity or detectable secondary structure. Soluble expression of TbLRR-DSP was achieved using baculovirus-infected insect cells, but the protein co-purified with endogenous chaperones and exhibited no catalytic activity thus implying a lack of correct folding. In the second part of this study, two phosphatases specific to Leishmania major, LM1 and LM2, were characterised and structural studies were initiated. LM2 was shown to readily hydrolyse phospho-tyrosine substrates in vitro, but not phosphoinositides like its homologue, LM1. Both proteins therefore have a differentiated catalytic profile and are likely to have different functions in vivo. Purification protocols for both proteins were established and crystallisation screenings set up. Preliminary hits were obtained for LM2 and a mutagenesis strategy was developed to improve chances of obtaining diffraction quality crystals. Recombinant LM1 samples exhibited heterogeneity and therefore will require additional engineering to improve chances of crystallization. Promising pilot NMR data was also obtained for both phosphatases. In conclusion, this study demonstrates that the recombinant expression of multi- domain trypanosomatid proteins (like the LRR-DSPs) can be highly problematic and may pose a challenge for their biochemical characterisation and functional elucidation. Future work into trypanosomatid phosphatases, however challenging, will improve our understanding of their cell biology and potentially identify therapeutic targets.

Flagellate protozoa from the order Trypanosomatida have developed a range of strategies to survive in their mammalian hosts. A consequence is that the glycolytic pathway has assumed an important role, especially in bloodstream-form Trypanosoma brucei, where it is essential as the sole producer of ATP. The seventh enzyme in the pathway, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shares no common properties with the corresponding enzyme in humans. This enzyme catalyses the conversion of 3PGA to 2PGA, with the requirement for metal ions to assist the catalytic function. In this study, two important biochemical and structural aspects of the enzyme were investigated: i) The in vitro and in vivo requirements for biologically relevant metal ions to support the activity of iPGAM, and ii) The ability of trypanosomatid iPGAM to exist in multiple conformations and oligomeric states in solution. The maximum activity of iPGAM in vitro requires Co2+, but this cannot be the case in vivo where ICP-OES analyses confirmed that Co2+ was essentially undetectable in T. brucei cytosolic fractions. The activity of iPGAM in vivo is therefore one of the lowest among the glycolytic enzymes. By contrast, Mg2+ and Zn2+ were found to be the most abundant metals in both cytosolic fractions and in purified bacterially expressed iPGAM. Our newly-developed multimode-plate reader discontinuous assay further revealed that of the biologically relevant metals, only Mg2+ can support iPGAM activity, but at less than 50% of the level of Co2+. By contrast, Zn2+ strongly inhibits iPGAM. This assay which was developed with minimal metal interference on the coupling enzymes, also showed that in solution, the ratio of the concentrations of 3PGA:2PGA (substrate:product) at equilibrium is not 1:1 as observed in the crystal structure, but is in fact 12:1, which may be due to the tighter binding of 2PGA to the enzyme. A series of biophysical analyses, notably by SEC-MALS showed that iPGAM from Leishmania mexicana, another trypanosomatid protozoan parasite exists in different forms and oligomeric states in solution, either as the closed-form monomer, openiii form monomer, or closed/open-form dimer which can be successfully separated by ion-exchange chromatography. The open-form LmiPGAM is particularly relevant for drug development, as the catalytic site in the closed-form structure is poorly inaccessible. Both virtual and high-throughput screening approaches were used to identify novel potential inhibitors. Out of a collection of 11 compounds tested at 1 mM, two showed substantial inhibition with 49% and 14% remaining activity. Taken together, the findings from this study demonstrated the potential of iPGAM to be a key modulator in controlling glycolytic flux in trypanosomes, and thus further validated it as an important drug target.

The breakdown of glucose to pyruvate, known as glycolysis, is a central biochemical pathway, critically important for energy production and biosynthesis. Phosphofructokinase (PFK), the third enzyme in the pathway, is a crucial regulator of glycolytic flux, being the first committed step of glycolysis and modulating entry into the pentose-phosphate-pathway. Alterations in PFK activity have been implicated in many neurological conditions, including Tarui's disease, epilepsy, Alzheimer's disease, Down's syndrome, and cancers. There are three isoforms of human PFK; it is assumed that these evolved to fulfil specific metabolic niches both within cells and between tissue types. However, the differences between isoforms have never been systematically compared. Understanding these differences is an essential prerequisite for developing novel therapeutic agents targeting human PFK. Trypanosomatid parasites are a major global cause of neurological morbidity and mortality. Neglected tropical diseases caused by trypanosomatid parasites include African Sleeping Sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania spp.). There is increasing interest in targeting the metabolic enzymes of these parasites, including PFK, which greatly differ from mammalian counterparts. This thesis describes biochemical, bio-physical, and bio-informatic studies on the three human PFK isoforms (PFK-M, PFK-L, and PFK-P), expressed in S. cerevisiae. Biophysical studies showed that the active conformation was tetrameric, with activity regulated by time and concentration dependent dissociation into smaller inactive species. The propensity to dissociate differed between isoforms, with PFK-M being most stable and PFK-P least stable. Dissociation was synergistically slowed by the addition of substrates and reducing agents, indicating different mechanisms of action. Kinetic studies were performed with respect to both substrates (ATP and F6P) in the presence of natural metabolites hypothesised to act as modulators of enzyme activity. Each isoform conformed to an allosteric sigmoidal kinetic model and had differing kinetic properties, with PFK-M being the most active and PFK-P the least active. ATP was found to act as both substrate and allosteric inhibitor, with activity showing a biphasic response to ATP concentration. Each isoform showed different susceptibilities to both ATP inhibition and regulation by allosteric modulators. The reverse reaction was shown to be possible under certain conditions. Bio-informatic data on intra-cellular and inter-cellular locations were determined using the Human Protein Atlas and the FANTOM5 datasets. PFK-M localises to the cytosol and may co-localise with endoplasmic reticulum; PFK-L associates with nucleoli and mitochondria; and PFK-P is cytosolic. Splice variants were not shown to be physiologically significant. Each isoform had different tissue expression levels, with overall PFK expression varying by tissue type. PFK-P was the principal isoform in cancers, whereas PFK-L was dominantly expressed in immune cells. Activated macrophages switched rapidly from PFK-L to PFK-P. PFK-M and PFK-P were the dominant isoforms in the brain, although there were differences between brain areas. Neurons expressed less PFK than astrocytes, in keeping with the lactate shuttle theory. PFK from each of the three main pathological trypanosomatid species were compared (T. brucei, TbPFK; T. cruzi, TcPFK; L. infantum, LmPFK); expressed in E. coli. Biophysical analysis showed each PFK to be tetrameric; no evidence of time or concentration dependent dissociation or inactivation was found. Kinetic properties differed between isoforms, with TcPFK being most active and LmPFK being least active. LmPFK was very poorly active with regard to F6P titrations unless AMP was present. No other modulators were shown to affect activity, although GTP was an alternate substrate. The reverse reaction was shown to be possible and may be compatible with physiological concentrations of ADP and F16BP in the trypanosomatid glycosome.

This thesis is composed of two topics both concerning diverse and obligatory trypanosomatid parasites. First part deals with identification of new Trypanosoma species identified in blood meal of tsetse flies caught in Dzanga-Sangha Protected Areas, Central African Republic, and identification of feeding preferences of tsetse flies. The second part concerns extraordinary monoxenous trypanosomatid Paratrypanosoma confusum which constitutes the most basal branch between free-living Bodo saltans and parasitic trypanosomatids. This thesis helped to elucidate morphology and biology of this deep branching trypanosomatid. Using genome and transcriptome sequencing and comparative bioinformatics approaches enabled search for ancestral genes shared with free-living bodonids and confirmed genome streamlining in trypanosomatids.

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates. This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 new trypanosomatids and compared them with 8 previously published maxicircle genomes of other trypanosomatids. Transcripts of ~13 of the 20 total genes in these maxicircles undergo post-transcriptional modifications involving the insertion and deletion of U residues at precise sites, to yield the final, fully-edited, translatable mRNA. We have deciphered the diverse patterns and extents of RNA editing of each edited gene in the maxicircle of each organism, and inferred the sequences of the putative fully edited mitochondrial transcripts and proteins. Using a binary value - based strategy (1/0), we quantified the RNA editing in all these trypanosomatids and estimated the evolution of RNA editing in the group. Additionally, we conducted phylogenetic analyses using a subset of unedited maxicircle genes to predict the relationships between the various trypanosomatids in this project, and compared them to the previously published nuclear gene-based phylogenies. For convenience of analysis, the 28 total trypanosomatids in this work were divided into two groups: the first group consisting of the endosymbiont-bearing and related insect trypanosomatids, which constitute the first half of the project, and the second group consisting of trypanosomatids of the Trypanosoma genus, including T. cruzi-related and unrelated parasites, constituting the latter half of the project. In summary, most of the trypanosomatid maxicircles showed a syntenic panel of 20 protein-coding genes (excluding any guide RNA genes), beginning with the mitochondrial ribosomal genes and ending with the gene encoding NADH dehydrogenase-5. Although some genes were partially or completely absent in the maxcircles of some species, the remaining genes were completely syntenic. The total number of genes edited and their editing patterns varied considerably among the first group of insect trypanosomatids, but were remarkably similar in the second group of the Trypanosoma genus. On a broad scale, the mitochondrial phylogeny reflects the nuclear phylogeny for these trypanosomatids, except within the T. cruzi population. Similarly, RNA editing appears to have evolved in parallel with the nuclear genes, although subtle differences are again noticeable within the T. cruzi family.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Trypanosoma evansi is a pathogenic trypanosomatid with world distribution, which may cause big economic losses and affect almost all mammalian species. One of the most affected species is horses, which may develop a disease known as Mal das cadeiras. There are several clinical signs and pathologies resulting from this parasite infection. The acute phase is characterized by intermittent fever, subcutaneous edema, progressive anemia, blindness, lethargy and hemostatic abnormalities. In the chronic phase, animals exhibit cachexia, edema, incoordination and paralysis. The most common treatment for this infection is the use of drugs; however, these drugs have a moderate efficacy, and they may present toxicity, especially to kidney and liver. Thus, it is important to study new therapies for the treatment of the disease caused by T. evansi. The aim of this study was to investigate the anti-trypanosomal effect of the treatment with 3‟deoxyadenosine (cordycepin - adenosine analogue) combined with deoxycoformycin (pentostatin - inhibitor of the adenosine deaminase (ADA) enzyme and deoxyadenosine analogue) in mice experimentally infected with T. evansi. Furthermore, we also verified the influence of the therapy in the hematologic, biochemical and ADA activity parameters, makers of cell viability and toxicity, oxidative stress and histopathology analyses. These analyses were divided into three experiments. The first one showed that the combination of cordycepin (2mgkg-1) and pentostatin (2mgkg-1) was 100% effective in the T. evansi-infected groups; however, the treatment increased significantly the liver enzyme levels, which were accompanied by histological lesions in the liver and kidneys. The second experiment showed a reduction in the levels of plasma total protein in healthy mice and treated with 1mg/kg cordycepin or 1mg/kg pentostatin. The animals treated with pentostatin alone or associated with cordycepin showed an ADA activity significantly reduced. The third experiment showed that the combination of cordycepin (2.0 mg kg-1) and pentostatin (0.2, 0.5, 1.0, 2.0 mg kg-1) is effective in the clearance of T. evansi, although at higher concentrations (cordycepin 2mg kg-1 and pentostatin 2mg kg-1), toxicity was observed. Therefore, the dose cordycepin 2.0 mg kg-1 in combination with pentostatin 0.2 mg kg-1 was recommended as a therapeutic option. This combination showed to be 100% effective in the experimentally infected animals and presented no toxicity to the animals. / Trypanosoma evansi é um tripanossomatídeo patogênico de distribuição mundial, causador de grandes prejuízos econômicos, podendo afetar várias espécies de mamíferos, principalmente equinos, espécie na qual produz uma doença conhecida como Mal das cadeiras . Muitos são os sinais clínicos e as patologias decorrentes da infecção por este parasito. A fase aguda da doença é caracterizada pelo surgimento de febre intermitente, edema subcutâneo, anemia progressiva, cegueira, letargia e alterações hemostáticas. Na fase crônica os animais apresentam caquexia, edema, incoordenação motora e paralisia de membros posteriores. O tratamento para essa infecção é medicamentoso, no entanto, os produtos químicos disponíveis possuem eficácia moderada e toxicidade, especialmente para os rins e fígado. Assim, é importante a pesquisa de terapias alternativas para o tratamento da doença causada pelo T. evansi. O objetivo deste estudo foi investigar a susceptibilidade do T. evansi à 3′-deoxiadenosina (cordicepina - análogo da adenosina) associada a deoxicoformicina (pentostatina - inibidor da adenosina deaminase (ADA) e análogo da desoxiadenosina) em camundongos infectados experimentalmente e verificar a influência desta terapia nos parâmetros hematológicos, bioquímicos, de atividade da ADA, marcadores de viabilidade e toxicidade celular e de estresse oxidativo e análise histopatológica. Essas análises foram divididas em três experimentos. O primeiro experimento demonstrou que a combinação de cordicepina (2 mg kg−1) e pentostatina (2 mg kg−1) foi 100% efetiva na cura dos animais infectados, mas esse tratamento ocasionou um aumento significativo nos níveis de enzimas hepáticas e produziu lesões histológicas no fígado e rins. O segundo experimento demonstrou uma reducão nos níveis de proteínas plasmáticas totais nos roedores sadios e tratados com 1mg/kg de cordicepina ou 1mg/kg de pentostatina, tendo os animais pertencentes aos grupos tratados com pentostatina isolada ou associada a cordicepina, uma diminuição da atividade da ADA. O terceiro experimento mostrou que as combinações de cordicepina (2,0 mg kg-1) e pentostatina (0,2; 0,5; 1,0; 2,0 mg kg1) foram eficazes na cura de animais infectados, mas na dose mais alta (cordicepina 2mg kg-1 e pentostatina 2mg kg-1), foi observado toxicidade elevada. A dose de cordicepina 2.0 mg kg-1 associada a pentostatina 0.2 mg kg-1 foi recomendada como opção terapêutica, com 100% de cura dos animais infectados experimentalmente sem apresentar toxicidade aos mesmos.