The immunological responses in hosts infected with Trypanosoma lewisi were examined during the course of infection and after recovery. Peak antibody levels coincided with the time of parasite elimination, but remained significantly elevated for over one year after the end of the infection The antigen repertoire recognized by antibodies demonstrated that some were revealed only by sera taken during the infection, and other antigens were revealed for the first time only by post-recovery sera. Immunomodulatory protective and suppressive factors were demonstrated in the plasma of irradiated, infected rats. These factors were identified as parasite-derived exoantigens which are shed in vivo and in vitro; exoantigens are complexes of proteins, lipids and polysaccharides and are membrane-surface coat associated, as shown by phase-partitioning and surface-labeling studies. The suppressive activity of the exoantigens was dose-dependent, probably mediated by a suppressor substance(s) produced by macrophages that subsequently inhibits production of interleukin 2 by T helper cells.

Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity.

Includes bibliography / 118, [78] leaves, [1] leaf of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1984

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 141-163.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph.D.) - University of Glasgow, 2007. / Ph.D. thesis submitted to the Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin, 1955. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 124-125).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Sep 19