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Trypanosoma brucei undergoes developmentally regulated morphological and biochemical changes during its life cycle, being transmitted between the mammalian host and the tsetse fly. It is generally recognized that cellular responses to environmental changes are mediated through signalling pathways, but our understanding of trypanosome signal transduction during differentiation is limited. Protein Tyrosine Phosphatase 1 (TbPTP1) is the one of the few factors identified to be responsible for differentiation from stumpy to procyclic form parasite, whereby TbPTP1 inhibition stimulates transition to insect-form cells (Szoor et al., 2006). In order to characterize the TbPTP1 signalling pathway, a substratetrapping approach was used, which identified a phosphatase TbPIP39 as substrate of TbPTP1. TbPIP39 interacts with, and is dephosphorylated by TbPTP1 in stumpy form cells. Additionally, it has been shown that upon citrate/cis-aconitate (CCA) treatment, phosphorylated TbPIP39 localizes to the parasite glycosomes, the organelles responsible for bloodstream forms metabolism, thereby promoting cellular differentiation to procyclic forms (Szoor et al., 2010). With the aim of further dissecting the TbPTP1 signalling pathway, the substrate-trapping approach was used, which identified one novel TbPTP1 substrate candidate, potentially involved in regulation of differentiation. In addition, the effect of other differentiation triggers, namely protease treatment or mild acid exposure, on the level of TbPIP39 phosphorylation was analyzed, to determine whether these stimuli operate via the same TbPIP39–dependent pathway as CCA signalling. Specifically, changes in the phosphorylation status of TbPIP39 were visualized and quantitated by the use of antibodies detecting either TbPIP39 or the Y278 phosphorylated form of TbPIP39 generated during CCA-dependent differentiation. Both protease treatment and mild acid exposure generated a different pattern of TbPIP39 phosphorylation, thus suggesting a different mechanism of action than CCA. Finally, the possibility of using piggyback strategies targeting TbPTP1 was investigated, as a means to decrease the number of the fly-transmissible stumpy form cells in the bloodstream, thereby controlling parasite transmission. For this purpose, natural and synthetic inhibitors of human PTP1B were tested against the parasite enzyme, since they are being developed by pharmaceutical companies for the treatment of diabetes and obesity. The compounds tested showed a moderate in vitro inhibitory activity against recombinant TbPTP1 and mainly a non-competitive type of inhibition, similarly to that observed for human PTP1B. However, none of the compounds showed in vivo specificity for TbPTP1, indicating that further studies will be needed to identify more specific inhibitors.

At least three major mitotic processes are regulated by the PP2A-B56 phosphatase complex: the Spindle Assembly Checkpoint (SAC), kinetochore-microtubule attachments and sister chromatid cohesion. We show here that these key functions of PP2A-B56, which require its localization to either the kinetochore or centromere, are split between distinct subsets of B56 isoforms. PP2A-B56γ and PP2A-B56δ localize to the outer kinetochore (via BUBR1), whereas PP2A-B56α and PP2A-B56ε localize to the centromere (via Sgo2). The differential localization observed is due to a difference in affinity for the receptors: PP2A-B56γ has a reduced affinity for Sgo2 compared to PP2A-B56α and, vice versa, PP2A-B56α has a reduced affinity for BUBR1 compared to PP2A-B56γ. Given that the known binding interfaces for both BUBR1 and Sgo2 are highly conserved in all B56 isoform, we generated a series of chimeras between B56α and B56γ to uncover isoform specific interactions. This led to the identifications of two distinct regions within B56α and B56γ that regulate the binding to Sgo2 and BUBR1. Furthermore, site directed mutagenesis has revealed that proper holoenzyme assembly has a role in regulating the localization of B56: it is needed for centromeric accumulation and it interferes with kinetochore accumulation of B56α. We will present a model to explain how this differential localization could be linked to post-translational modifications of PP2AC. Together, these results help to clarify how individual PP2A-B56 isoforms achieve subcellular specificity during mitosis.

"March 2003" Bibliography: leaves 206-233. The experimental data presented in this thesis provide evidence that PTP-Pez is an active phosphatase that interacts with and dephosphorylates the adherens junction protein ℓ-catenin. PTP-Pez also associates with proteins that form part of the tight junction complex, the scaffolding protein ZO-1 and the transmembrane protein occludin.

The mitotic spindle checkpoint plays a critical role during the cell cycle by protecting the faithful transmission of chromosomes during mitosis. If chromosomes are improperly bound to the spindle microtubules the checkpoint will prevent progress to anaphase by temporarily arresting cells in metaphase until all the chromosomes are correctly aligned. Bub1b is an essential component of the mitotic spindle checkpoint that transiently localises to kinetochores during mitosis and becomes phosphorylated, a response that is sustained during mitotic arrest. Bub1b has been implicated in other processes related to mitotic progression and is thought to regulate mitotic timing and have a role in caspase mediated cell death after prolonged mitotic arrest. The development of aneuploidy and cancer has been associated with mutations in the BUB1B gene and reduction in the level of Bub1b protein. To further our understanding of Bub1b function in the spindle checkpoint and mitosis, new protein interactions involving Bub1b were identified. This thesis describes the search for alternative proteins that interact with Bub1b, and their function in the mitotic spindle checkpoint and regulation of Bub1b activity. Using a yeast two-hybrid approach, members of the B56 family of regulatory subunits of serine-threonine protein phosphatase (PP2A) were identified as novel interacting partners of Bub1b. Substrate specificity of PP2A is determined by the regulatory subunits. There are five characterised isoforms of the B56 family, each encoded by separate genes. In addition, some isoforms have several recognised splice variants. Confirmation of interactions by alternative methods demonstrated that the isoforms B56γ and B56[epsilon] preferentially interact with phosphorylated Bub1b, whereas the interaction of the remaining B56 isoforms (α, β and [delta]) occurs at a lower affinity with no specificity for the phosphorylated form. It was further demonstrated that B56γ1 associated with phosphorylated Bub1b in vivo. Induced overexpression of splice variants of B56γ1 and B56γ2 demonstrated a significant reduction in levels of phosphorylated Bub1b during mitotic spindle checkpoint activation. In addition, an associated lower mitotic index was evident in cells with B56γ1 overexpression. Specific inhibition of PP2A activity with okadaic acid was shown to prolong Bub1b phosphorylation during normal mitosis and to restore the levels of phosphorylated Bub1b in arrested cells over expressing B56γ. These findings suggest a role for PP2A activity in regulation of Bub1b function that is mediated through substrate recognition by B56 regulatory subunits.

Enzymes play an important role in the environment, they breakdown natural-occurring and anthropogenic molecules so that they can be transported into cells and utilized. Enzyme assays are routinely used in soil science and oceanography to measure the activities of specific processes and to serve as general indicators of microbial activity. Conventional enzyme assays are conducted as batch incubation of sediment and water samples. During these assays the concentration of product is measured and enzyme activity is then determined as the rate of product formation. Few studies have measured enzyme activities of groundwater. This work investigates the use of β-glucosidase and phosphatase assays for quantifying in situ enzyme activities in groundwater. Improvements to conventional enzyme assays using p-nitrophenyl substituted compounds were made by developing a high performance liquid chromatography method to improve quantitation limits of the product and to quantify concentrations of both the substrate and the product. An in situ single-well push pull test was then conducted to measure β-glucosidase activity in situ and to estimate the Michaelis constant (K[subscript m]) and the maximum reaction velocity (V[subscript max]) in petroleum-contaminated groundwater at a field site near Newberg, Oregon. An important feature of the single-well push pull test is the nonlinear drop in pore water velocity that the test solution experiences as it moves out from the injection point. The nonlinear drop in pore water velocity is of particular interest because enzyme-mediated reactions are very fast and changes in the hydraulic properties during the test may give rise to mass-transport limitations. Fast reactions lead to the simultaneous depletion of substrate and accumulation of product at the site of the reaction so substrate and product concentrations near the enzyme can be different then the concentrations in bulk solution. And the rates obtained from a single-well push pull tests may be a combination of the rates at which substrate diffuses to the microorganism and at which the reaction occurs. Laboratory experiments with sediment-packed columns were conducted with a range of pore water velocities typically achieved in the subsurface during as push-pull test as a means for examining the potential effects of inhibition and diffusion on phosphatase enzyme kinetics. In this set of column experiments rates of phosphatase-mediated reactions were investigated instead of β-glucosidase, which is an inducible enzyme. Numerical investigations were then conducted to examine the importance of diffusion limitations for describing the influence of transport processes on the observed rates of reaction. The theoretical investigation was conducted by formally upscaling the proposed sub-pore-scale processes to develop a macroscale (or Darcy scale) description of the transport of the substrate. These results indicate that mass-transfer limitations due to the diffusion of the substrate to the enzyme cause an increase in the apparent K[subscript m] but have no effect on V[subscript max]. In this study an analytical method was developed to measure rates of enzyme-mediated reaction in situ so that the measured rates reflected actual rates of microorganism in their natural environment. More carefully controlled laboratory experiments demonstrated that rates of enzyme-mediated reactions measured at low substrate concentrations depended on the flow properties of the test solution. / Graduation date: 2006

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Research Doctorate - Doctor of Philosophy (PhD) / This study described in this work examines the potential for derivatives of the potent PP1 (IC50 9.0 µM) and PP2A (IC50 3.0µM) inhibitor, norcantharidin, the demethylated cantharidin analogue, and their protein phosphatase inhibition, namely PP1 and PP2A and their cytotoxicity across a range of human cancer cell lines. A variety of derivatives were examined, paying particular attention to modifications to the anhydride moiety. These included a series of ring opened and ring closed cantharimides, a series of α-hydroxylactams, a series of lactone analogues and derivatives, and a series of heteroatom substituted analogues. Of the analogues developed, the ring opened and ring closed cantharimides displayed moderate to excellent activity, in cases, an improvement over the lead compound norcantharidin was observed. The ring closed dodecyl-linked bis analogue (63) was the most potent analogue displaying µM potent cytoxicities against all the cell lines examined. Of the ring opened analogues, the morpholino analogues proved most active.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2003. / "March 2003" Bibliography: leaves 206-233.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 149-157). Also available in electronic version.