Medical Biology - Theses
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ItemCharacterization of plasmepsin X as a cross-species antimalarial target( 2019)The emergence and spread of drug resistance have hindered the campaign for malaria eradication. The development of new drug targets is critical for our anti-malarial arsenal of interventions. Plasmepsins, which are aspartic proteases expressed by malaria parasites, serve important functions for parasite survival. Among the 10 members of this enzyme family, plasmepsin X (PMX) is essential for P. falciparum growth and has been shown to be involved in the egress of merozoites from infected red blood cells and the invasion of merozoites into red blood cells. Several aspartic protease inhibitors have anti-malarial activity on P. falciparum and are proposed to target PfPMX. The aim of this project was to investigate if these compounds affect P. knowlesi growth and whether PMX is a cross-species target for antimalarial development. This work showed that two aspartic protease inhibitors, 49c and 1SR, caused inhibition of P. knowlesi parasite growth. In further studies, live cell imaging demonstrated that these compounds inhibit P. knowlesi parasite growth by blocking parasite egress. Next, the optimal condition for protease activity was characterised after the expression and purification of a functional recombinant P. knowlesi plasmepsin X (rPkPMX). Using a fluorogenic protease assay, both 49c and 1SR were shown to inhibit the activity of rPkPMX. Furthermore, rPkPMX was able to cleave synthetic substrates, which were based on the predicted cleavage sites of PfSUB1, PfRAP1, PfRh2, TgROP1 and TgMIC6 predicted cleavage sites. By screening a panel of aspartic protease inhibitors, the BACE1 inhibitor, LY2886721, was identified as an inhibitor of rPkPMX activity as well as P. knowlesi and P. falciparum parasite growth. Therefore, PMX can be used as a cross-species target for antimalarial drug development.
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ItemGM-CSF regulation in inflammatory arthritis( 2019)Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of the joints, affecting 0.5% to 1% of global population. Current targeted therapies antagonize the debilitating effects of key inflammatory mediators and immune cells. However, few patients achieve complete remission, prompting novel therapeutic approach. Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) was first identified as a haemopoietic growth factor but is now recognised as a proinflammatory cytokine in a number of autoimmune inflammatory diseases, including RA and Multiple Sclerosis (MS). GM-CSF promotes destructive joint inflammation by priming pro-inflammatory phenotypes of myeloid cells, such as neutrophils, monocytes and macrophages. Accordingly, therapies targeting GM-CSF or its receptor are currently under clinical evaluation in RA and MS and seem promising. However, much remains to be explored how GM-CSF is dynamically regulated during arthritis, especially in autoantibody-mediated inflammation as seen in seropositive RA patients. This work aims to characterize the cellular source of GM-CSF during antibody-induced arthritis and evaluate the cell-intrinsic negative regulation of GM-CSF signalling in myeloid cells and arthritis. I utilized the autoantibody-driven, immune complex-mediated serum transfer induced arthritis (STIA) murine model, which mimics the effector phase of seropositive RA patients. Using the novel GM-CSF dual reporter mice, joint-infiltrating Natural Killer (NK) cells were found to be main GM-CSF-producing cells during STIA. By using NK-deficient (Mcl1fl/fl:Ncr1-Cre) mice, NK-depleted (anti-NK1.1 antibody-treated) mice or specifically deleting GM-CSF production by NK cells (Csf2fl/fl:Ncr1-Cre), I was able to show the importance of NK cells and their GM-CSF-producing function in maintaining arthritis (Chapter 3). GM-CSF on myeloid cells induced the Cytokine Inducible SH2-containing (CIS) protein, a member of the suppressor of cytokine signalling (SOCS) protein family. Using Cish-/- mice, I showed that CIS negatively regulated GM-CSF signalling post activation, which is evident in intracellular signalling pathways, effector cell functions and in antibody-induced arthritis (Chapter 4). Taken together, this study provides new insights into the pathogenesis of antibody-driven, GM-CSF-mediated autoimmune inflammation and provide a rationale towards designing novel anti-inflammatory agents such as NK modulator or CIS mimetics for RA.
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ItemCharacterisation of the Plasmodium aspartyl proteases DNA-damage inducible protein 1 (DDI1) and Plasmepsin VII (PMVII)( 2019)Plasmodium falciparum resistance to artemisinin-(ART) based combination therapies (ACTs) and other antimalarials poses a major threat to malaria control and elimination. Current efforts are aimed towards identifying potent antimalarials which inhibit multiple stages of the parasite lifecycle or discovering novel drug targets which may help overcome ART-resistance. This work aimed to characterise two aspartyl proteases of P. falciparum which may hold promise as antimalarial targets. One strategy recently proposed to overcome ART-resistance is the synergistic use of a parasite-selective proteasome inhibitor to sensitise ART-resistant parasites to artemisinin. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the P. falciparum ubiquitin-proteasome system (UPS) could yield a combination therapy to tackle ART-resistance. DNA-damage inducible protein 1 (DDI1) is a previously uncharacterised essential aspartyl protease in P. falciparum. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPS in mammalian cells. In other organisms, DDI1 plays a role in shuttling proteins to the proteasome for degradation via its ubiquitin-like domain. We hypothesise PfDDI1 is an active aspartyl protease and plays a role in the parasite’s UPS. To investigate the role of DDI1 in the UPS and parasite survival, we identified a DDI1 orthologue in P. falciparum and characterised this using several strategies. We utilised CRISPR-Cas9 to knock out, tag and inducibly knock down DDI1 across the asexual lifecycle of P. falciparum, and study the effect of this on parasites. Expression of recombinant DDI1 proteins provided insight into the protease activity and substrate repertoire of PfDDI1. Together these studies provide insight into the domain architecture, essentiality and function of PfDDI1 and clues into its potential as an antimalarial target. Development of an antimalarial to block parasite transmission between humans and mosquitos is also a viable strategy to reduce malaria burden. In this study, we also explore a potential transmission-blocking target, Plasmepsin VII (PMVII) and create tools to enable further study of this aspartyl protease in sexually reproductive gametocytes. These tools are vital to determine the function and substrate repertoire of PMVII and elucidate its potential as an antimalarial target.
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ItemIdentify new regulators of TNFR1-induced necroptotic pathway( 2019)Acute Myeloid Leukaemia (AML) is a vastly heterogeneous blood disorder with a poor prognosis for patients older than 65. Our group has been focused on developing new treatments for AML to replace the standard intensive chemotherapy. Previous data showed that the SMAC-mimetic birinapant in combination with the caspase inhibitor IDN could kill different types of AML both in vitro and in vivo through activation of necroptosis cell death pathway. However, over 50% of the patient samples tested in study showed resistance to necroptosis. This project aims to determine the molecular mechanisms that mediate necroptosis resistance in AML and identify new regulators of necroptotic pathway. The results obtained in this study will expand the knowledge of necroptosis signalling in leukaemia and will contribute to the optimal clinical use of birinapant/IDN drug combination. This project contains 2 parts; (1) We will use human AML cell lines that are resistant to necroptosis to determine the molecular changes involved in cell death resistance. (2) We will use CRISPR/Cas9 knock out screen in human AML cell lines that are sensitive to necroptosis, trying to identify new regulators of TNFR1-induced necroptotic pathway. Together these experimental approaches will allow a better understanding of the regulation of TNF-necroptosis signalling in AML. By overexpression wild-type RIPK3 in the KG-1 cell line, we successfully sensitised KG- 1 cells to necroptosis, which indicates that the KG-1 endogenous RIPK3 is dysfunctional. By cDNA sequence of KG-1 endogenous RIPK3, we detected several mutant base pairs, which may lead to the dysfunction, but this result needs further prove by genome sequence, which is undergoing. By CRISPR knock out screen, we found several targets that may lead to the necroptotic resistant, and MAGE3 is the most research-worthy one. Knockout MAGEB3 in the MV4;11 cell line led to the downregulation of RIPK3 and the necroptotic resistance. However, this result could not be repeated on the U937 cell line, and the mechanism of how MAGEB3 regulates RIPK3 is still unclear. Further research will be done on MAGEB3 to have a better understanding of the role of MAGEB3 in the necroptotic pathway. Together, all these results gave a better understanding of the necroptotic pathway and may contribute to the treatment of AML.
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ItemIdentifying Plasmodium vivax encoded proteins that may prevent host cell death during liver development( 2019)Malaria is a major global health problem and a leading cause of death worldwide. The mechanism behind some parts of the parasite life cycle are still obscure, especially the liver stage which is essential for parasite development and maturation. It is likely that the parasite prevents the host hepatocyte from undergoing cell death during invasion. This is especially relevant for Plasmodium vivax as the hypnozoite can lay dormant in a liver cell for months, even years. We hypothesise that P. vivax encodes proteins to inhibit host cell death in liver. We used the computer algorithm I-TASSER to identify several P. vivax proteins which were predicted to have similar structures to human proteins involved in cell death. We expressed these P. vivax proteins in mammalian cells and performed functional tests to investigate their potential roles experimentally. Identification of P. vivax proteins that influence host cell death would improve our understanding of how P. vivax can survive for prolonged periods in the host cell during liver stage and may accelerate the development of new drugs for malaria liver stage, which is necessary for the ultimate goal of eliminating malaria.
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ItemStructural and functional characterisation of the molecular assembly of two pseudokinase scaffolds( 2019)The human pseudokinase SgK269, and its structurally related homologue SgK223, are oncogenic interacting scaffolds that promote the assembly of specific tyrosine kinase signalling pathways. SgK223 and SgK269, as well as the recently discovered PEAK3, belong to the PEAK family of protein pseudokinases. They are large, multidomain proteins that are comprised of an N-terminal region of unknown structure and function, a large unstructured PEST region containing tyrosine phosphorylation sites and a C-terminal domain comprised of a pseudokinase domain flanked by regulatory helices. SgK223 and SgK269 have been shown to localise to focal adhesions and their overexpression leads to increased cell migration and changes in cell morphology, hallmarks of cancerous cells. Recent studies from our lab and others have provided structural insight into the C-terminal domain and flanking alpha helices of SgK223 and SgK269. These structures highlighted a conserved mechanism of dimerisation that drives homo- and hetero-association of SgK223 and SgK269 and plays an important role in cell migration. Additionally, SgK223 and SgK269 were demonstrated to undergo homo- and hetero-oligomerisation through their pseudokinase domains. In contrast to the C-terminal domain, little is known about the function of the N-terminal domains of SgK223 and SgK269, although there is sequence conservation between them. In this study, we have begun characterising the N-terminal domains of SgK223 and SgK269 using biophysical and biochemical techniques, initially demonstrating that these domains are monomeric and appear to have no defined secondary structure. To further investigate SgK223 and SgK269 homo- and hetero-association we carried out single site alanine mutagenesis to determine the energetic hotspots at the dimerisation interface of SgK269. Furthermore, we carried out mutagenesis within the N-lobe of SgK223 and SgK269, to investigate the role of this interface in homo- and hetero-oligomerisation. Additionally, we characterised the PEAK family interactions with the critical interacting signalling adaptor protein, CrkII, using biophysical assays and X-ray crystallography. We found that each member of the PEAK family has a proline-rich motif within their PEST linker that interacts with CrkII N-SH3 domain with ~1-3 uM affinity. The crystal structure of the CrkII N-SH3 domain bound to the SgK269 proline-rich motif demonstrated the critical consensus residues for the PEAK family interaction with CrkII. To further investigate the role of SgK223 and SgK269 homo- and hetero-association in cells, these studies were complemented with localisation microscopy techniques. Utilising mutants of SgK223 and SgK269 that can no longer dimerise or oligomerise, we investigated the importance of SgK223 and SgK269 associations for their localisation and thus, role in signalling. Insights into the scaffolding functions of SgK223 and SgK269 will inform how they contribute to the assembly of signalling pathways and hence their role in cancer.
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ItemAdvancing BH3 mimetics to treat cancers( 2019)The evasion of apoptosis, one of the hallmarks of cancer, is observed in many cancers. This can also impair the efficacy of many conventional chemotherapies. The BCL2 protein family is the central regulator of the intrinsic apoptotic pathway and plays a vital role during tumor development. In particular, the levels of the pro survival family members are often elevated in some cancers. Venetoclax, a BH3 mimetic inhibitor that mimics the BH3-only proteins, natural inhibitors of the pro-survival BCL2 proteins, has proven to be effective for treating hematological cancers by selectively targeting BCL2. This has translated into regulatory approvals of venetoclax for treating a subset of chronic lymphocytic leukemia and acute myeloid leukemia. In addition to targeting BCL2, potent and specific BH3 mimetic inhibitors of its relatives, BCLxL and MCL1, are now also available. However, their full clinical utility is poorly defined. This thesis focuses on advancing the utility of the BH3 mimetic compounds as anti-cancer agents. Previous studies have suggested roles for BCLxL and MCL1 in many solid cancers (e.g. colon, breast, lung). In particular, colorectal cancers have elevated levels of the pro survival protein, one usually associated with chemo resistance. Furthermore, colorectal cancer patients with advanced disease or those who carry poor prognostic markers do not respond well to the current stand of care therapies such as surgery and adjuvant chemo/radiotherapy. Given the pressing need to find better treatments for these patients, we first utilized a panel of validated BH3 mimetics to assess the feasibility of using them for treating colorectal cancer. By using cancer cell lines and patient-derived organoids, we identified and validated BCLxL and MCL1 as the most important survival factors for colorectal cancer. We then validated them as potential targets by pharmacological inhibition in a mouse model in vivo. Moreover, we found that even those tumors that harbor poor prognostic factors respond as avidly as those do not, further highlighting the potential of this approach for treating patients with colorectal cancer. Even though the targeting BCLxL might be a possible approach to kill cancers that depend on it, the clinical use of BCLxL selective inhibitors is limited due to the toxicity of BCLxL inhibition on platelets. I have screened for novel regulators of BCLxL using the CRISPR/Cas9 technology, which might offer potential approaches to target BCLxL safely. The final goal of this thesis is to identify biomarkers that predict response to BH3 mimetics, given that there are few reliable tools to stratify patients that might respond well to these novel anti-cancer agents. By using large scale transcriptomic datasets from publicly available RNA sequencing studies, I was able to identify a few candidate genes and achieve reasonable prediction performance.
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ItemImproving differential expression analysis of single-cell RNA-seq data: method and application( 2018)Single-cell RNA sequencing (scRNA-seq) technology enables high-throughput transcriptome profiling at single-cell resolution, providing researchers with an unprecedented opportunity for dissecting heterogeneous biological systems. However, the distinct features of scRNA-seq data also present us a variety of analytical challenges. This thesis focuses on the bioinformatic analysis of scRNA-seq experiments. We first describe the design of a novel statistical framework for differential gene expression (DE) analysis. Through explicit modelling of the molecule capturing process, we are able to perform DE analysis on the inferred pre-dropout molecule counts. Benchmarking using simulated and real data showed improved performance compared with existing methods. Then we show a case study of a breast cancer tumour infiltrating T cell dataset. Leveraging several newly developed methods for scRNA-seq data, including our DE method, we identified and characterized a novel cell population, tissue-resident memory T cells, in the tumour infiltrating T cells.
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ItemIdentification and characterisation of monoclonal antibodies against malaria( 2018)Plasmodium falciparum and Plasmodium vivax are responsible for the majority of malaria-related deaths globally. The clinical symptoms of malaria are caused by the blood-stage infection. Malaria parasites invade erythrocytes using multiple parasite ligand-host receptor interactions. For P. vivax, its infection is strictly limited to human reticulocytes which are young erythrocytes. Recent work showed that P. vivax reticulocyte binding protein 2b (PvRBP2b) was identified to bind reticulocyte receptor transferrin receptor 1 (TfR1) to mediate the invasion into human reticulocytes. For P. falciparum, one of these ligand-receptor interaction is between P. falciparum reticulocyte binding protein-like homologue 4 (PfRh4) and the erythrocyte receptor complement receptor 1 (CR1). The PfRh4-CR1 invasion pathway is responsible for the majority of sialic acid-independent invasion. Anti-PvRBP2b mouse monoclonal antibodies obtained by hybridoma fusion method, anti-PfRh4 antibodies purified from either mouse or rabbit immunisation, or from immune individuals in Papua New Guinea inhibit the PvRBP2b-TfR1 and PfRh4-CR1 interaction respectively, and block parasite invasion. These results proved that PvRBP2b-TfR1 and PfRh4-CR1 interactions play critical roles in P. vivax and P. falciparum invasion respectively, and also suggest the potential application of anti-PvRBP2b and anti-PfRh4 monoclonal antibodies in parasite invasion research and malaria treatment. I aimed to isolate, express and characterise mouse monoclonal antibodies using the single B cell expression cloning method that combines antigen-specific B cell sorting and antibody gene cloning. I enriched B cells from PvRBP2b-immunised mice and sorted-PvRBP2b-specific B cells with B cell surface markers and fluorescent PvRBP2b. From a single B cell, I synthesised the respective cDNA and amplified the antibody genes with nested-PCR. The antibody chains were cloned into expression plasmids for expression in human embryonic cells followed by antibody purification. I characterised ten antibodies and identified three monoclonal antibodies that are able to inhibit PvRBP2b binding on reticulocytes. I also planned to use the same technology to isolate anti-PfRh4 human monoclonal antibodies from individuals in the P. falciparum endemic area. I was unable to adapt this technology because of the presence of CR1, the receptor for PfRh4, on human B cells. However, I optimised PfRh4 functional validation assays as a screen for antibody reactivity and functional inhibition for future researches. Overall, my work demonstrates the successful adaption of single B cell expression cloning techniques for isolation of antigen-specific monoclonal antibodies against P. vivax and P. falciparum targets, which will deepen our understanding of parasite invasion into erythrocytes and provide potential immunotherapy for malaria treatment.
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ItemAssociation between antibody responses to blood stage parasitic antigens and protection from Plasmodium falciparum and Plasmodium vivax Malaria in Timika, Indonesia( 2018)Malaria is a major health problem causing over 212 million clinical cases every year around the world. In Indonesia, Timika is one of the highest malaria-endemic areas with a high annual parasite incidence (876 per 1,000 people per year) and high co-endemicity of P. falciparum as well as P. vivax. Moreover, a high rate of antimalarial drug resistance has also been reported in Timika, illustrating the need for developing other therapeutic tools such as vaccines to prevent disease. Clinical immunity to malaria only develops after many years of constant exposure to the parasites. This form of protection does not result in sterilising immunity but prevents clinical cases by significantly reducing parasite density. Naturally acquired immunity predominantly targets blood- stage parasites and is known to require antibody responses, but despite the key role that antibodies play in protection the antigenic targets of immunity are not completely defined. The aim of this study was to investigate associations between antibody responses and protection from symptomatic malaria in a field study conducted in Timika to identify targets of naturally acquired immunity to malaria. Naturally acquired immunity to seven Plasmodium falciparum and one Plasmodium vivax merozoite-stage vaccine candidate antigens were analyzed by ELISA, parasite growth inhibition functional assays (GIA) and ELISPOT. The main findings revealed that high antibody levels to P. vivax Duffy binding protein (PvDBP) were associated with high parasitemia. This finding suggests that PvDBP could be used as a serological marker for recent exposure. In contrast, high antibody responses to P. falciparum erythrocyte antigen 175 (PfEBA-175) Region IV-V, P. falciparum reticulocyte binding protein-like homologue 4 (PfRh4), PfRh5 and P. falciparum Rh5 interacting protein (PfRipr) predict protection from symtomatic malaria. Furthermore, PfRh5-specific memory B cells could be detected among protected malaria-exposed healthy controls as well as asymptomatic individuals, suggesting a role for these cells in sustaining long-term immunity. Together, these findings highlight that PfEBA-175, PfRh4, PfRh5, and PfRipr are important targets of naturally acquired immunity to malaria and promising vaccine candidates.