Malaria remains responsible for an enormous health burden worldwide; considerable research effort is being devoted to finding ways to combat the disease and its transmission. Malaria is caused by Plasmodium species, and P. falciparum causes the most serious disease. The blood stage of the P. falciparum remains critically important to understand for development of treatments and vaccines. To initiate invasion, the P. falciparum merozoite recognises specific proteins on the host red cell membrane, known as invasion receptors. In order to study parasite–host interactions, laboratory adapted P. falciparum strains that invade mature human red cells have been used. Gene modification methods are well established for P. falciparum; however, genetic manipulation of the red cell has not been extensively applied because erythrocytes are not nucleated. The in vitro cultivation of erythroid cell lines facilitates both the scalable production of host cells to support P. falciparum invasion and editing of nucleated precursors that can be genetically modified in a precise manner. In this project, two erythroid cell lines – the Human Umbilical cord blood Derived Erythroid Progenitors (HUDEP-2) and the Bristol Erythroid Line- Adult (BEL-A), both of which can differentiate to more mature forms in vitro – were studied as possible host models. A FACS antibody panel, based on the stage-specific profile of HUDEP-2 and BEL-A cells, provided the means to analyse host invasion receptors as well as erythroid maturation markers. Band 3 is a red cell membrane protein with an uncertain role in merozoite invasion. A gene knockout was constructed in expansion stage BEL-A cells using the lentiviral CRISPR/Cas9 system, targeting band 3 which may be involved in merozoite invasion of human erythrocytes. Single-cell-derived clones were isolated and preliminary validation using PCR and flow cytometry was performed to verify disruption of band 3. Completion of work to validate and functionally characterise the band 3 knockout, and experiments to assess effects on invasion, were curtailed by COVID-19 stay-at-home orders issued to Melbourne between March and July 2020. In summary, a genetically editable in vitro erythroid model was defined to study the function of host invasion receptors for P. falciparum merozoite invasion. Clonal band 3- deficient BEL-A cells were generated, thus paving the way for studying their role as invasion receptors.

Globally, young children continue to die or fail to thrive from treatable and preventable causes including low birthweight (LBW), childhood undernutrition (wasting, stunting, underweight) and infectious diseases. Reducing the burden of these are an essential pillar of global child health and survival targets. These global trends are reflected in Papua New Guinea (PNG), a resource constrained setting that continues to observe high rates of illness and death in young children with LBW, childhood undernutrition and infectious diseases (especially malaria, pneumonia and diarrhoea) continuing to be the leading causes. Improving child health and survival in PNG requires evidence about the risk factors for LBW, sub-optimal growth and infectious diseases, as well as the inter-relatedness of risk factors, that can inform policies and identify appropriate interventions and strategies. This thesis aims to address critical knowledge gaps in this area and provide insights to inform interventions and strategies aimed at reducing low birthweight, childhood undernutrition and malaria in young children in PNG and globally. LBW is caused by a multitude of factors which are often inter-related and with that, it is often difficult to distinguish between independently direct and indirect effects. Moreover, current interventions targeted at preventing LBW generally assume a single dominant cause overlooking the inter-relatedness of risk factors and the possibility of factors exerting joint effects on LBW. These are difficult to establish with widely used standard statistical methods and have therefore been rarely investigated. Using structural equation modelling, we showed intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine (SP) plus azithromycin (AZ) to be independently directly associated with reduced probability of LBW. Unexpectedly, anaemia at enrolment was also directly associated with reduced probability of LBW. Maternal undernutrition at enrolment was independently directly associated with increased probability of LBW. No significant indirect associations between risk factors and LBW were established. After birth, children in lowlands PNG experience high rates of malaria, pneumonia and diarrhoea alongside undernutrition. Infections and undernutrition are believed to have a bi-directional relationship and whilst the effect of undernutrition on risks of illness and death is well known, little is known about the effects of malaria, pneumonia and diarrhoea on growth faltering, particularly among PNG children. By using multivariable regression and distributed lag models for data analysis, we observed malaria pneumonia and diarrhoea to have a differential impact on child growth. The effect of acute malaria on child growth was observed to be long-term while pneumonia and diarrhoea had short-term effects lasting up to 3 months. Of these three diseases, malaria was once ranked the top leading infectious cause of childhood morbidities and mortality in PNG, especially in lowlands PNG. The nationwide scale-up of malaria control interventions significantly reduced overall malaria transmission between 2008 and 2014 but a detailed understanding of the impact of this changing transmission on the epidemiology and risk profile of malaria infections and disease due to the two main species in young children was lacking. By analysing three consecutive longitudinal child cohorts (1-5-year-old children) conducted over the period of improved control (2013), we observed a differential impact of improved control on P. falciparum and P. vivax. Additionally, we showed that with declining malaria transmission, burden of malaria infections and illness were highly spatially localised to areas that had the highest burden prior to scale-up, highlighting potential hotspots of transmission. Collectively, the findings from this thesis provide important insights for improving child health in PNG and globally.

Structural Maintenance of Chromosomes Hinge Domain-containing protein 1 (SMCHD1) has been established as an epigenetic regulator, with critical roles in X-chromosome inactivation, autosomal gene silencing and genomic imprinting. Recently, variations in SMCHD1 have been associated with two human conditions: facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). There has therefore been a growing interest in unveiling SMCHD1’s atomic structure and the molecular mechanisms underlying its function in both a healthy and diseased state. To provide a better understanding of Smchd1’s molecular structure and function, I successfully expressed and purified the full-length 2007-amino acid mouse Smchd1 protein. Electron microscopy analyses of the Smchd1 dimer revealed an elongated rod-like structure that displays a high conformational flexibility, similar to that of other structural maintenance of chromosomes (SMC) proteins. This flexibility is largely conferred by the intermediate region of the protein that connects Smchd1’s two functional domains: the N-terminal GHKL ATPase and the C-terminal SMC hinge domain. In follow-up studies of the two individual domains, we revealed the first atomic-resolution structure of Smchd1’s hinge domain, providing a novel insight into its DNA-binding and dimerisation modes. Contrary to previously suggested models describing the DNA interaction mode of canonical SMC proteins, I showed that nucleic acids are not threaded through the central pore region of the Smchd1 hinge domain. Subsequent immunofluorescence studies additionally revealed that the hinge domain targets full-length Smchd1 to chromatin, and that a functional hotspot within the hinge is required for chromatin localisation in cells. SMCHD1’s ATPase domain has been of particular interest due to the identification of disease-related variants that are frequently located within this region of the protein. However, the mechanisms by which some of these pathogenic variants affect SMCHD1 function are poorly understood. Using analytical ultracentrifugation, I demonstrated that the wild-type SMCHD1 ATPase undergoes dimerisation, which was reliant on the inclusion of both the UBL domain and the presence of substrate, ATP. Follow-up cellular studies revealed that Smchd1’s catalytic activity, as well as the presence of the newly- identified UBL domain, are both necessary for the localisation of full-length Smchd1 to chromatin. Together, these studies provide an insight into the molecular basis of Smchd1 function and highlight how chromatin binding may be compromised in human disease. Future studies will further investigate the cellular localisation and dimerisation properties of disease-associated SMCHD1 variants, contributing towards our ongoing drug development program aimed at developing therapeutic treatments for FSHD patients.

Maintenance of the haematopoietic system is controlled by intercellular signalling molecules known as cytokines. Cytokines function by binding to receptors at the surface of target cells and activating a number of signalling pathways inside the cell that lead to changes in gene transcription and ultimately a cellular response, whether it be differentiation, division, cell death or other. These processes need to be tightly controlled and regulated, and so there are cellular mechanisms for controlling both the duration and intensity of the signal produced by cytokines. This thesis investigates the structure and function of two regulators of cytokine signalling; PTP1B and LNK. PTP1B is a protein tyrosine phosphatase that interacts with and dephosphorylates the JAK proteins, returning them to their inactive state. Similarly, LNK, a lymphocyte adaptor protein also interacts with JAK2, and negatively regulates a subset of cytokines by an unknown mechanism. The work presented in this thesis details the study of the interactions between PTP1B and LNK with the JAK proteins using structural and biochemical techniques, allowing for the characterisation of the mode of substrate recognition, and substrate preference. The studies in chapter 3 revealed that the PTP1B phosphatase can directly dephosphorylate JAK kinases, inducing their transition to an inactive state. The interaction between PTP1B and the motif on JAK that is its target was studied in detail. While previous studies of PTP1B substrate recognition have defined a second aryl binding site that allows PTP1B to interact with substrates containing two consecutive phosphotyrosine residues, here it is shown that there is a second mode of binding where this second binding site is not used. Given that PTP1B is the prototypical phosphatase in the category I phosphatases, this may warrant redefining this category. Moreover, the accessibility and position of the JAK activation loop to phosphatases was shown to be critical for dephosphorylation rate, and so it can be postulated that some conformations of the JAK proteins may inhibit their dephosphorylation by phosphatases. The results presented in chapter 4 study the interaction between the LNK SH2 domain and various phosphorylated targets, including JAK. It was shown that LNK can interact with several sequence motifs from proteins involved in haematopoiesis but that JAK2 and JAK3 are its favoured targets. Structural studies were performed to uncover the molecular basis for this high-affinity interaction with JAK. In addition, studies of mutations found in the LNK SH2 domain which were identified in patients with myeloproliferative neoplasms revealed how these changes to the SH2 domain reduced the ability of LNK to bind to its targets, which may contribute to disease. In summary, the information presented in this thesis add to our understanding of the regulation of the JAK-STAT pathway, and how changes to essential protein- protein interactions may be involved in disease.

Dengue fever is a mosquito-transmitted disease of the tropics and sub-tropics that is caused by dengue virus (DENV). There are an estimated 60-100 million clinical cases of dengue fever per year, resulting in at least 10,000 deaths. Most clinical cases of dengue are characterised by flu-like symptoms. However, for unknown reasons, a small proportion (1-2%) of clinical cases progress to a life-threatening form of disease referred to as “severe dengue”. Severe dengue is characterised by cytokine storms, heightened endothelial permeability and associated sequelae such as shock and haemorrhage. During the onset of severe dengue, viraemia and viral antigenaemia are sharply declining or absent. Therefore, it is logical to deduce that dysregulated host signalling is the underlying cause of the cytokine storm phenotype and symptoms of severe dengue. However, although many host factors have been characterised in the context of DENV infection, the root cause of this signalling dysregulation is still poorly understood. Furthermore, there are currently no drug treatments available for the treatment of severe dengue, and although there is a licensed dengue vaccine, it confers only moderate protection, and administration of this vaccine to dengue naive individuals is contraindicated by the World Health Organisation. In the first part of this thesis, I characterised how genetic disruption of key host signalling pathways altered the response of macrophages and mice to DENV infection. I found that infection of cells and mice that had a co-deletion of genes encoding cellular inhibitor of apoptosis proteins (cIAPs) resulted in decreased production of virus, and an exaggerated production of inflammatory cytokines. In the second part of this thesis, I determined whether clinical stage cancer therapeutics could be repurposed as treatments for severe dengue. To investigate this, I established an in vivo mouse model of severe dengue and treated these mice with anti-inflammatory compounds. However, these drug treatments did not reduce clinical manifestations of infection or improve the survival of the infected mice. These studies suggest that cIAPs facilitate the efficient replication of DENV. In addition, I hope that the negative results from my therapeutic experiments can inform future experimental plans, and contribute to reducing the worldwide burden of severe dengue.

The generation of protective antibody is one of the most important parts of the humoral immune response and is the basis for the vast majority of successful vaccination strategies. Antibody is produced by rare populations of differentiated B cells, known as plasmablasts and plasma cells. The differentiation of B cells into antibody secreting cells (ASCs) is complex and highly orchestrated by vast array of mechanisms, including epigenetic regulation. Broadly speaking, epigenetics describes all non-genetic regulation of gene expression. Hence, modifications to the chromatin, but not the underlying DNA sequence, result in altered gene expression. In recent years, epigenetic modifying compounds (EMCs) have emerged as potential therapeutic agents for the treatment of haematological malignancies and immune disorders. However, it is now clear that EMCs also modulate the immune response via both direct and indirect mechanisms. Despite the extensive studies on EMCs, the precise functional role of many of these compounds remains unknown. This thesis explores the effects of two EMCs that have previously been shown to affect the antibody response. Specifically, the Brd4 inhibitor JQ1 and GSK126, an Ezh2 inhibitor. Using quantitative analysis, I examined the effects of each EMC on different parameters that combine to control the magnitude of the antibody response. By combining functional analysis with transcriptomic and epigenomic studies, I investigated the precise molecular mechanism and gene targets of these EMCs. Thus, these studies provide the opportunity to identify novel regulators of antibody secreting response. I showed that JQ1 treatment dampens the antibody secreting response by targeting multiple parameters of B cell function, including cell proliferation and survival. The effects on B cell function were the result of global Brd4 displacement as opposed to previously suggested gene specific mechanisms. In addition, I identified the pro-apoptotic molecule Bim as the molecular target of JQ1 directly responsible for inducing apoptosis in stimulated B cells. Conversely, inhibiting Ezh2 increases B cell differentiation and antibody production of B cells. I showed that Ezh2 inhibition causes global downregulation of H3K27me3 without altering the genome accessibility. Genome-wide studies identified a number of novel regulators of Ezh2 inhibition induced ASC differentiation, including the Blimp-1 target Atoh8. Results from this thesis illustrate the strength of in vitro reductionist systems that combine functional analysis of cell biology with genomics to isolate epigenetic mechanisms that regulate immunity. JQ1 has a significant effect on B cells and has the potential to be used as a therapeutic agent to dampen the antibody secreting responses in autoimmunity, particularly those involving increased antibody production. In contrast, pharmacological inhibition of Ezh2 increases ASC differentiation and antibody production. Thus, it could potentially be used to boost antibody responses that could be applied to treat immunodeficiency or as a differentiation therapy in cancer models.

Nucleotide-binding oligomerization domain-containing (NOD) proteins NOD1 and NOD2 are intracellular pathogen recognition receptors (PRRs) that recognize bacterial peptidoglycan (PGN) fragments. Aberrant NOD signaling is associated with a diverse range of inflammatory disorders, and recent findings suggest that compounds that inhibit the NOD pathways could be beneficial in the treatment of inflammatory bowel disease, allergic asthma, and diabetes mellitus. NOD signaling requires the polyubiquitylation of receptor-interacting serine/threonine-protein kinase 2 (RIPK2), the central adaptor kinase that coordinates downstream responses. Multiple drugs targeting RIPK2 have been developed. However, none of them have made it into the clinics yet. Accordingly, many molecular mechanisms of RIPK2 activation have only recently been discovered and are not yet fully understood. A significant reason for this is the lack of biochemical tools to study the NOD pathways at endogenous levels and the use of overexpression of pathway component, which leads to the formation of artefactual interactions and pathway autoactivation independent of PGN binding. My Ph.D. aimed to close this gap and establish novel biochemical tools for the study of NOD2 signaling mechanisms at endogenous levels. Therefore, we generated endogenously FLAG-tagged RIPK2, and HA-tagged NOD2 knock-in mice using CRISPR/Cas9 technology. Using these mice, I assessed the tissue distribution of NOD2 and RIPK2, as well as their signaling mechanisms, interaction networks, and post-translational modifications (PTMs) during PGN-induced immune signaling. NOD2 and RIPK2 were largely co-expressed and most abundant in organs that naturally harbor large immune cell populations. Our knock-in mice showed a normal response to NOD2 stimulation by activating NF-KB and MAP kinase pathways and the production of pro-inflammatory cytokines. Using bone marrow-derived macrophages (BMDM), I was able to show that NOD2, RIPK2, and the ubiquitin E3 ligase X-linked inhibitor of apoptosis protein (XIAP) interact at endogenous levels in a partially stimulation-dependent manner. Furthermore, I optimized a mass spectrometry workflow that is tailored for the detection of interaction networks of endogenously expressed, affinity-tagged proteins. My work suggests that immunoprecipitation of endogenous proteins harboring a single FLAG or HA tag is not specific enough for the analysis by mass spectrometry and that instead, triple tags or more specific affinity tags must be used. Furthermore, I was able to precisely map the post-translational modifications on endogenous RIPK2 during NOD2 signaling. I characterized the function of the individual modifications using reconstitution experiments in RIPK2 knock-out cell lines. I found that single phosphorylation and ubiquitylation sites on RIPK2 are redundant and can be compensated for by other modifications. In the course of those experiments, I discovered a novel regulatory interface on the C-lobe of the RIPK2 kinase domain, which mediates binding of the ubiquitin E3 ligase x-linked inhibitor of apoptosis protein (XIAP) as well as RIPK2 oligomerization. Mutation of single amino acids within this interface completely blocked NF-KB activation and cytokine production after NOD2 stimulation. I hypothesize that this interface, which is composed of a hydrophobic pocket, can potentially be exploited for the generation of a novel and specific class of RIPK2 inhibitors that block NOD signaling without targeting the RIPK2 ATP-binding pocket. I further report that, like RIPK2, NOD2 is polyubiquitylated in an XIAP-dependent manner. NOD2 ubiquitylation occurs exclusively after pathway activation and includes K63 and M1-linked chains, as well as possible multiple mono-ubiquitylation sites. This suggests that the ubiquitylation of NOD2 exerts a functional role, such as the regulation of NF-KB and MAP kinase activation or endosomal trafficking of NOD receptor complexes. To ultimately determine the role of NOD2 ubiquitylation during immune responses to PGN, further studies are required.

DNA methyltransferase 3a (DNMT3a) is a de novo DNA methyltransferase that can establish DNA methylation signatures in cells. Recently, germline mutations in DNMT3a were found to cause an intellectual disability and overgrowth disorder named Tatton-Brown-Rahman syndrome and somatic mutations in DNMT3a constitute one of the most common mutations in haematological malignancies. The findings presented in this thesis inform on the role of the most common DNMT3a mutation, R882H, using a novel murine model with an emphasis on ageing, haematopoiesis and hematopoietic malignancies. The mutant Dnmt3a mouse model was created using CRISPR/Cas9 genome editing technology to introduce the most common R882H mutation into the murine Dnmt3a locus at residue R878H (murine homologue of R882). Breeding of Dnmt3aR878H/+ mice revealed an inability of female Dnmt3aR878H/+ mice to deliver healthy offspring. This was a result of a maternal defect as surrogate mice could produce viable Dnmt3aR878H/+ pups through IVF. Dnmt3aR878H/+ mutant mice also had a shorter lifespan compared to their wt littermates when aged. The Dnmt3aR878H/+ aged mice were more susceptible to liver disease that was characterised by extensive hepatocyte steatosis and hepatocyte carcinoma and were also more likely to develop leukaemia with B cell morphology compared to their wt littermates. To determine whether the Dnmt3aR878H/+ mutant mice had defects in haematopoiesis before overt haematological malignancy, the haematopoietic system was analysed under steady state conditions and in haematopoietic competition assays. There was evidence of a defect in early T cell development in the thymus characterised by significantly fewer immature T cell progenitors in Dnmt3aR878H/+ mutant mice compared to their wt littermates. To resolve whether Dnmt3aR878H/+ mutant haematopoietic stem and progenitor cells (HSPCs) had a competitive advantage over wt HSPCs, HSPCs from the Dnmt3aR878H/+ mutant mice were competitively transplanted alongside wt HSPCs into lethally irradiated wt recipient mice. It was shown that Dnmt3aR878H/+ HSPCs and their descendants outcompeted their wt counterparts after 6 months, with some evidence that Dnmt3aR878H/+ HSPCs had already begun to accumulate after 3 months. These findings were extended to show that Dnmt3aR878H/+ HSPC-derived cells can also outcompete wt HSPC-derived cells in other haematopoietic tissues, such as the thymus and spleen. Furthermore, it was also shown that Dnmt3aR878H/+ HSPCs also have an increased serial transplantation capacity compared to their wt counterparts. To better understand how the Dnmt3aR878H mutation promotes the development of haematological malignancies, a model of g-irradiation induced thymic lymphoma was employed where the cancer cell of origin arises from a HSPC. It was shown that Dnmt3aR878H/+ mutant mice developed thymic lymphoma at a significantly faster rate than their wt littermates. Gene expression changes in Dnmt3aR878H/+ HSPCs that might account for their increased predisposition to leukaemogenesis revealed that Dnmt3aR878H/+ LSK cells have an underlying disturbance in Notch signalling and that upon g-irradiation, they have a blunted induction of the p53 signalling network compared to wt HSPCs. Many other cellular pathways were also deregulated in Dnmt3aR878H/+ HSPCs, and they will be the subject of future experiments. Overall, it was shown that heterozygous Dnmt3aR878H mutations cause a vast array of abnormalities including problems in pregnancy, metabolic defects leading to obesity and liver pathologies as well as haematological disturbances leading to an accumulation of HSPCs in the bone marrow and a susceptibility to the development of haematological malignancies.

Toxoplasma gondii is an obligate intracellular parasite, which chronically infects one-third of the world’s population. Toxoplasma infection severely affects immune-compromised individuals, resulting in birth defects, blindness, or brain encephalitis. Individuals often become infected through the consumption of contaminated food and water sources. Toxoplasma has two life stages during the lytic life cycle, the fast disease-causing tachyzoites and the chronic bradyzoites. Following host cell invasion, Toxoplasma tachyzoites extensively manipulate their host cell by exporting a distinct repertoire of effector proteins across the newly-established parasitophorous vacuole. This process interferes with the host's transcriptional program and is thought to enable parasite persistence and dissemination in spite of the host’s immune response. Eventually, Toxoplasma forms bradyzoite cysts in the visceral organs, which are a reservoir for disease reactivation. In the current scientific literature, the disparity in our knowledge between tachyzoite- and bradyzoite-host interactions is large. Almost nothing is known on how this chronic-stage of infection persists post-cyst formation and what role host manipulation plays in latency. Therefore, in this thesis I aim to understand whether Toxoplasma bradyzoites modulate the host transcriptional response for their survival and, if so, what role could dense granule protein export have in this process. I explore the host transcriptional profile of bradyzoite containing cells using RNA sequencing to question what role host manipulation plays in latency. I show that bradyzoite-containing host cells have a unique transcriptional landscape when compared to tachyzoite infection, and, by pairing this technique with protein export deficient parasites, I show that many of these changes are dependent parasite protein export. Next, I investigate whether the known tachyzoite effector proteins have a function in chronic infection. IST, an inhibitor of host IFN-gamma signalling, was identified as the only known tachyzoite effector to be expressed, synthesised, and exported in bradyzoites, suggesting a role for this effector protein in the chronic stage. Furthermore, I demonstrate that effector proteins are critical in protecting bradyzoite infected host cells from undergoing cell death upon IFN-gamma-mediated cell death, purposing three models that enable cyst persistence. This thesis explores bradyzoite-host interactions to interrogate the possible mechanism behind Toxoplasma’s lifelong infections.

This thesis investigates the role of histone acetylation during embryonic development.

Malaria is caused by Plasmodium parasites and Plasmodium vivax is the dominant Plasmodium spp. in low-transmission regions outside of Africa. Due to the unique biological characteristics of this parasite, such regions often feature asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. Serology is a promising tool for monitoring transmission levels, estimating past and recent exposure and identifying populations at risk of infections. However, due to key gaps in our knowledge of naturally acquired antibody responses to P. vivax, the full potential of serology has not yet been reached. This thesis aimed to establish antibody kinetics against a large panel of P. vivax antigens following infections in western Thailand, and investigate the factors potentially associated with the acquisition and development of antibody responses. A multiplexed bead-based assay was first established and antibody measurements against more than 50 antigens were taken in P. vivax-infected individuals from western Thailand following symptomatic and asymptomatic infections. I found that most P. vivax antigens followed a highly similar post-infection kinetic pattern in the absence of any boosting infections. The magnitude and longevity of antibody responses varied between antigens, antibody subclasses and subtypes. An assay quantifying the antigen-specific memory B cell responses was established and verified to determine the role of memory B cells on antibody kinetics for future experiments. Lastly, I reported that the genetic diversity of an antigen sequence had a significant impact on antigen-specific antibody responses, and such impact increased in individuals with more past exposure and mature immunity. The findings presented in this thesis provide novel insights into naturally acquired immunity development to P. vivax and support the decision of taking genetic diversity of antigen sequences into consideration for the development of highly efficacious sero-surveillance tools and vaccines.

Platelets are numerous small, anucleate cells circulating in the blood. Their most well recognised role is in coagulation and haemostatic maintenance in response to vascular injury. However, they are versatile cells containing a reservoir of several hundred bioactive molecules and surface receptors and functions beyond their canonical roles are being rapidly discovered. One of their most well-studied non-haemostatic roles is in supporting haematogenous cancer metastasis. Experimentally, platelet depletion or functional inhibition of platelets slows metastatic progression in mouse models of cancer, and in clinical cohorts, elevated platelet count is correlated with a survival disadvantage in several cancer types, including lung cancer. These studies, however, do not account for heterogeneity between lung cancer subtypes. Consequently, the role of platelets in the major lung cancer subtypes (adenocarcinoma (ADC), squamous cell carcinoma (SqCC), and small cell lung cancer (SCLC)) is not fully understood. Lung cancer is the leading cause of cancer death in Australia and worldwide, and the poor prognosis for the majority of patients highlights the importance of developing improved treatment options. Obtaining a greater understanding of how platelets contribute to tumour progression in lung cancer may aid in the development of new therapies. In this thesis, I have utilized an autochthonous KrasLSL-G12D/+;p53flox/flox genetically modified mouse model (GEMM) of lung ADC, and a p53flox/flox ;Rb1fl/fl GEMM of SCLC, together with genetic models of thrombocytopenia, to interrogate the role of platelets in lung cancer growth and progression. While thrombocytopenia failed to impact primary tumour growth, in experimental metastatic models thrombocytopenic mice displayed significantly extended survival. Inhibiting platelet function with pharmacological agents elicited a similar outcome and highlighted the potential for combining anti-platelet agents with other anti-cancer agents in SCLC therapy. Additionally, retrospective analysis of a lung cancer patient cohort revealed thrombocytosis was predictive of poor survival in ADC patients with metastatic disease. Interestingly, this association was not apparent in SqCC or SCLC patients, highlighting the necessity of patient stratification if thrombocytosis is to be used as a clinical prognostic biomarker. Finally, the impact of lung ADC or SCLC cancer on platelet reactivity was investigated in murine experimental metastasis models. I identified a defect in GPVI signalling in platelets in SCLC tumour bearing mice, which I found to be associated with thrombocytopenia and an increased proportion of immature platelets. Overall, this thesis has contributed in several ways to the broader understanding of platelets in lung cancer, has highlighted the importance of separate analysis of lung cancer histological subtypes when interpreting clinical and experimental data, and provides insights supporting potential novel treatment strategies which combine anti-platelet and lung cancer therapies.