School of Biomedical Sciences - Theses

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    Characterization of cyclic nucleotide-mediated signal transduction pathways in Toxoplasma gondii
    Yang, Luning ( 2017)
    T. gondii is amongst the most common human pathogens, chronically infecting more than 30% of the human population. T. gondii has a complex life cycle, often spanning multiple hosts. T. gondii parasites invade target host cells to establish infection. Residing inside host cells, parasites are immotile and active replicate. After multiple rounds of replication, T. gondii activates their motility and egress from host cells. Invasion and egress relies on parasites motility, which is strictly controlled by a number of second messenger-mediated signalling transduction pathways. The signalling pathways that controls parasite motility have become a focus of investigations in the past two decades due to its potential as a therapeutic target. Previous studies showed that activation of Ca2+-mediated signalling pathways and cGMP-mediated signalling pathways drives parasites microneme secretion and motility. Conversely, cAMP-mediated signalling pathways may work as suppressors on parasites motility. In Chapter 3, I evaluated the function of a putative guanylyl cyclase in T. gondii asexual growth. TgGC accumulates at apical tip of the parasites and alters its localization during intracellular growth and extracellular motility. TgGC is critical for T. gondii growth and it plays an important role in parasites microneme secretion, invasion and egress, highly suggestive of its function in cGMP generation. We showed that cGMP-mediated signalling pathways localize upstream of cytosolic Ca2+ rise and could be occurring by activation of phosphatidylinositol-specific phospholipase C. In Chapter 4, I investigated the regulation of cAMP signalling. I identified the cAMP-dependent protein kinase A regulatory domain TgPKAr that regulates activity of catalytic domain TgPKAc1, which has been suggested as a negative regulator of motility and Ca2+ signalling. TgPKAr and TgPKAc1 localize at the parasite periphery, which occurs likely through palmitoylation and myristoylation on the N-terminal TgPKAr sequence. Overall, my work highlights the pivotal role signal transduction pathways play in T. gondii parasites and sheds light on the complex hierarchy and interplay between second messengers-mediated signalling cascades that regulate parasites motility and infectivity.
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    Investigating the role of NK cells in allogeneic haematopoietic stem cell transplantation
    Jiao, Yuhao ( 2017)
    Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is used to treat a range of haematological malignancies. However, the use of allo-HSCT is limited due to significant toxicity related to conditioning intensity, opportunistic infection, graft failure, and graftversus host disease (GVHD). Standard conditioning regimen including myeloablative total body irradiation (TBI) is considered to be the trigger for pro-inflammatory cytokine release and further induce acute GVHD (aGVHD). To decrease the risk of aGVHD, a reduced intensity conditioning (RIC) regimen was introduced to clinical practice because of its lower toxicity. However, the insufficient suppression of host immune system caused by RIC might lead to higher chances of graft failure. In this case, to identify the risk factors to graft rejection after RIC regimen was crucial. And by targeting the major contributor to graft failure post-transplantation, RIC can be utilised to prevent aGVHD while high donor engraftment can still be achieved. Recipient natural killer (NK) cells were found to be significantly more radio-resistant than other cytotoxic lymphocytes following TBI and thus could be a considerable contributor to acute allograft rejection. MHC-mismatched transplantation allo-BMT mouse models were utilised in this project to determine the extent by which radio-resistant recipient NKs are involved in causing acute allograft rejection. By using wild-type (WT) and the NK cell deficient (Bcl2fl/fl Ncr1-iCre) mice as recipients, donor cells were rapidly rejected in WT recipients with RIC-allo-BMT, whereas Bcl2fl/fl Ncr1-iCre recipients that were irradiated with the RIC 2×400 rad TBI achieved long-term engraftment and lower aGVHD clinical scores. This positive outcome with reduced TBI dose was attributed to lesser donor T cell expansion, lower pro-inflammatory cytokine levels and higher myeloid cell reconstitution in the NK cell deficient recipients, compared to recipients that had undergone transplantation with myeloablative TBI (2×600 rad). These findings clearly indicate that recipient radio-resistant NK cells are the main cause of graft failure and can be targeted to lower conditioning intensity and promote engraftment. Besides the Bcl2fl/fl Ncr1-iCre mouse models, other NK cell deficient or aberrant models were also studied in order to understand further how NK cells can regulate aGVHD and donor engraftment. Using an alternative approach with greater clinical relevance, WT mice administered with the BCL2-inhibitor S63845 and/or the MCL1-inhibitor ABT-199 exhibited similar outcomes as the BCL2- and MCL1-deficient mouse models. Inhibitor treatment prior to allogeneic transplantation with RIC regimen reduced the number of host residual NK cells. And in this case, mice that had the inhibitor treatment and RIC-allo-BMT had reduced risk of graft failure and could also be free from aGVHD. Another key finding of this study, which has significant potential for clinical translation was validation of the therapeutic graft-versus-leukaemia (GVL) effect in the RIC-allo-BMT models. Either genetically modified NK cell deficient mouse RIC-allo-HSCT models or inhibitor treatment mouse RIC-allo-HSCT models were proven to have intact GVL effect. Therefore, the less sufficient anti-neoplastic efficacy of RIC regimen could be compensated by the intact GVL effect. And most importantly, the overall mortality related to leukaemia relapse of the NK cell deficient RIC-allo-BMT models was significantly lower than the WT RIC-allo-BMT models.
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    Defining and developing novel host targeted therapies to eliminate Mycobacterium tuberculosis infections
    Zhang, Yisheng ( 2017)
    Tuberculosis, caused by Mycobacterium tuberculosis (M.tb), is a massive global health problem with one third of the world’s population infected. Aproximately two billion people have been killed by tuberculosis over the last two centuries. With the growth in our understanding of tuberculosis disease and production of the Bacillus Calmette–Guérin (BCG) vaccine, the tuberculosis incidence and mortality have been dramatically reduced over the past century. However, the number of newly diagnosed tuberculosis cases per year is still very high in some parts of world. Complex issues confound our ability to tackle this disease including problems with detecting and differentiating infection and disease, inadequate sanitation, low BCG vaccination rates, poor vaccine efficacy and the emergence of drug resistant strains. These issues increase the pressure, need and urgency of developing better preventive management techniques to stop the spread of new infections and better agents to treat infection, particularly resistant infections. A characteristic of intracellular M.tb infection is its ability to cause or promote lytic forms of host cell death (e.g. necrosis). The major shortcoming of necrosis is that organisms are not killed and they escape the intracellular environment enabling them to infect more host cells. One mechanism that assists in the killing of intracellular M.tb is the activation of host cell inducible nitric oxide synthase (iNOS). Additionally, recent work in our laboratory has shown that apoptosis can be promoted during M.tb infection to help kill intracellular M.tb and clear infection. This was done using a drug that antagonises the inhibitor of apoptosis proteins (IAPs). IAPs activate a cell survival pathway downstream of TNF signalling. If IAPs are antagonised with small molecule inhibitors, TNF no longer activates a host cell survival signalling pathway but rather it activates an apoptotic signalling cascade. In my studies, I combined the IAP antagonists’ with bosentan, a clinical stage drug that has been developed to promote iNOS activity. I found that bosentan was not efficacious in reducing M.tb burdens in lung and this may be due the drugs ability to increase oxygenation in the lung. Perhaps the increased oxygenation promoted the ability of aerobic M.tb to proliferate. Interestingly, IAP antagonists reversed the negative effects of bosentan presumably by promoting apoptosis of infected cells. As well as killing intracellular M.tb, apoptosis is also thought to promote antigen presentation and hence immunity. If we are able to promote apoptotic cell death during BCG vaccination we may be able to promote more robust immunity to M.tb. So I examined the ability of IAPs antagonists to promote BCG efficacy and I found that these drugs, when administered at the correct time, did promote BCG vaccine efficacy. I additionally sought to examine the role of a subset of T cells called mucosal associated invariant T cells (MAIT cells) that have been implicated in facilitating the acquisition of adaptive immunity. MAIT cells are found in the mucosa and several organs including lung. These cells become activated when they encounter host cells presenting bacterially derived vitamin B metabolites in association with MHC class I-like molecule (MR1). I activated MAIT cells with bacterial vitamin B metabolites in both wild type and MR1 gene knock out mice and then immunized animals with BCG vaccine prior to M.tb challenge. I found that promoting MAIT cell activation had no significant effect on promoting BCG vaccine efficacy.