The retina is light-sensitive eye tissue responsible for vision, but little is known about the genetic regulation of retinal gene expression. Investigating key drivers of gene regulation in the retina in healthy and diseased individuals remains a fundamental challenge in macular degeneration research, especially given the difficulty of accessing human retinal tissue. Deciphering the effects of genetic variation on retinal gene expression will underpin the development of novel treatment avenues for otherwise untreatable diseases causing blindness. A method to investigate these further focuses on the effects of genetic variants on gene expression levels derived from transcriptomic data. This type of ‘omics analysis, known as expression quantitative trait (eQTL) analysis integrates genotype and gene-expression data. The genotyping data for this thesis was generated in collaboration with scientists from the TIGEM, Italy, who first assembled the retinal transcriptome. We aimed to identify the genetic variants that modulate gene expression using a cohort of 41 individual donors of healthy retinal tissue. We performed retinal eQTL analysis using this independent cohort and compared our results with recently published retinal eQTL studies. After observing a weak eQTL signal potentially due to the small sample size, we explored potential strategies to mitigate the multiple testing burden so as to improve statistical power. To this end, we performed eQTL power analyses and limited both the set of variants and genes under consideration by thresholding on allele frequency and gene transcriptional abundance as well as disease relevance. Further, eQTL analysis was used to interpret the genetics of Macular Telangiectasia II, a blinding retinal degenerative disease. This included genome-wide and targeted interrogation of the signals from the largest genome-wide association study to date for this disease.

Signalling through the B cell antigen receptor (BCR) governs outcomes for the B cell at most fate-determining decision points during development as well as the broader adaptive immune response. The mechanisms underlying the nuanced differences in timing and strength of BCR signalling that bring about these diverse outcomes are poorly understood. This is largely due to the lack of structural information on the BCR’s architecture, a problem shared by most receptors of the single-pass membrane protein class by virtue of their transmembrane domains (TMDs) that are notoriously difficult to study by traditional biophysical techniques. In a lateral approach to study these elusive TMDs, our lab previously used a combination of cysteine scanning, nuclear magnetic resonance (NMR) and molecular dynamics simulations on the BCR’s counterpart in T cells, the T cell receptor (TCR), to generate a TM model of its central alphabeta subunit. This model was borne out by the later published cryogenic electron microscopy (cryo-EM) structure of the TCR, the first atomic-resolution structure of an intact single-pass receptor, reinforcing the power of the approach for challenging membrane-embedded complexes. Here, we have used a similar combination of experimental and computational techniques to map out the TM arrangement of the BCR’s constituent subunits: the ligand-binding membrane-bound immunoglobulin (mIg) and signalling CD79AB dimer, yielding the first experimentally-guided structural look into its assembly and identifying a common structural element in both BCR and TCR TMDs.

RNA-binding proteins (RBPs) are classically regarded as facilitators of gene expression. In recent years, however, RNA-protein interactions have also emerged as a pervasive force in the regulation of homeostasis. The compendium of proteins with provable RNA-binding function has swelled from the hundreds to the thousands astride the partnership of MS-based proteomics and RNA Sequencing. At the foundation of these advances is the adaptation of RNA-centric capture methods that extract protein that has been crosslinked in its native environment. These methods reveal snapshots in time displaying an extensive network of regulation and a wealth of data that can be used for both the discovery of RNA-binding function and the molecular interfaces at which these interactions occur. This thesis describes the development of an extraction method that purifies RBP-RNA complexes. This method differentiates itself from other RBP-discovery protocols in that it 1) purifies these complexes so completely that RBP identification can be conducted qualitatively and without differential abundance analysis, 2) permits transcript-targeted capture with sequence-specific oligos, 3) permits global, sequence-agnostic capture 4) both RBP and its bound RNA are isolated intact and 5) can reliably interrogate RBPs at depths that exceed present methods without metabolic or molecular labelling. The performance of this method is first assessed with a census of proteins that directly interact with global, or targeted, RNA transcripts from model cell lines. These efforts are then extended to investigate how protein-RNA interactions change during transition from quiescence to proliferation and then contraction in primary murine CD8+ T cells. Finally, these studies demonstrate how cellular responses provoke different proteins to moonlight as RNA binders and sheds light on a network of complex, co-evolved molecular machines.

Apoptosis is a form of programmed cell death. The intrinsic pathway of apoptosis is governed by the BCL2 family proteins. Targeting BCL2 proteins by small molecules that mimicking the BH3-only proteins to induce apoptosis has proven to be a successful strategy for cancer therapy. Venetoclax, a specific inhibitor of BCL2, has exhibited remarkable efficacy in treating cancers that rely on BCL2 for survival. However, the activity of venetoclax is often limited in other cancers whose survival relies on MCL1, another BCL2 family member. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials. However, the clinical development of these agents has been hampered by toxicity, especially cardiac toxicity. Potentially, another strategy to target MCL1 is by modulating NOXA, a BH3-only protein that selectively binds to MCL1 and mediates its degradation. I hypothesised that increased NOXA expression would prime cancer cells to venetoclax killing and that this would reduce their co-dependence on MCL1. In order to identify new targets to modulate NOXA expression, I generated and validated cell lines that report on NOXA transcription and then carried out CRISPR-Cas9 genetic screens in those NOXA reporter cell lines. In CRISPR-Cas9 loss-of-function screens focused on epigenetic regulators, I found several genes whose mutation or loss modulated NOXA expression, including CTBP1, CHTOP, ZMYM3, SPEN, HSPA1A, KEAP1, FOXA1, HDAC3 and SAP30. Some of these factors have been targeted for cancer therapies, for example KEAP1 and HDAC3, while the others have not yet been recognized for their therapeutic possibilities. Subject to their validation, my results have identified interesting novel mechanisms of NOXA regulation, thus providing the rationale basis for the development of new anti-cancer agents. In CRISPR-Cas9 tiling screens that focused on the NOXA promoter region, five cis-regulatory elements were identified that contributed to regulation of NOXA expression. Among them, a hypermethylated element on the NOXA promoter was found to be important for repressing NOXA expression across diverse cell lines derived from blood cancers. Disrupting this region led to NOXA induction. Potentially, the findings could provide a rational basis of combining hypomethylating agents with venetoclax in a range of haematological malignancies. In summary, several potential NOXA regulating proteins and DNA elements were discovered by the CRISPR-Cas9 screening approaches. Once validated, these findings should provide new insights into NOXA regulation.

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive T-cell malignancy that is frequently caused by the overexpression of oncogenic transcription factors. Like many cancers, T-ALL is a heterogeneous disease, with the acquisition of many genetic alterations resulting in multiple clones that contribute to cancer progression. This poses a challenge for therapeutic intervention, as different clones within a tumour can possess different genetic signature and thus possess different levels of sensitivity to therapeutic strategies. Therefore, gaining a better understanding of how these clones arise, is crucial to developing more targeted therapies aimed at these cells. LMO2 is a transcription factor that is overexpressed in approximately 9% of T-ALL cases. The CD2-Lmo2 transgenic mouse model overexpress Lmo2 in the thymus, resulting in a developmental block at the DN3 stage of T-cell development, and subsequent development into T-ALL after a long latency (approximately 10 months). Using these mice, our laboratory has shown Lmo2 confers self-renewal capacity to these developmentally blocked DN3 thymocytes many months before the overt presentation of T-ALL. These self-renewing DN3 thymocytes were termed pre-CSCs due to their ability to self-renew, their capacity to still develop into mature functional T-cells, and not initiate leukaemia for many months when transplanted into recipient mice. This thesis will focus on gaining a better understanding of the multi-step pathogenesis of Lmo2 induced murine T-ALL development. In Chapter Three, we explore the role cellular competition for thymic niche space and signals plays in the Lmo2 induced T-cell developmental block, and Lmo2 induced T-ALL. Using competitive bone marrow transplantation experiments we show that the presence of WT thymic progenitors in the thymus severely hinders the development of Lmo2 transgenic thymic progenitors past the DN2 stage of T-cell development, and their subsequent development into T-ALL. Interestingly, we found that overexpression of Bcl2 in Lmo2 transgenic thymocytes severely abrogated the self-renewal capacity of Lmo2 transgenic thymocytes, and hindered their development into T-ALL. Furthermore, we show that Lmo2 downregulates Il7r in DN2 thymocytes. In Chapter Four, we crossed CD2-Lmo2 transgenic mice with CD2-Il7r transgenic mice to create the CD2-Lmo2;CD2-Il7r double-transgenic mouse line, to investigate the role of Il7r overexpression in the Lmo2 induced developmental block. We found that overexpression of Il7r in Lmo2 transgenic thymocytes does not alleviate the Lmo2 induced DN2, or DN3 developmental block, but does increase the engraftment potential of Lmo2 transgenic DN3 thymocytes. Surprisingly, despite the increase in engraftment potential, Il7r overexpression in Lmo2 transgenic thymocytes resulted in a delay in T-ALL induced death, however Il7r overexpression promoted an immature T-ALL immunophenotype. In Chapter Five, we generated an inducible Lmo2 knockin mouse model in which Lmo2 expression can be inhibited by Dox administration. Using this mouse line, we show that while Lmo2 is still required for the self-renewal of Lmo2 transgenic DN3 thymocytes, Lmo2 is not required for T-ALL maintenance in the majority of Lmo2-Induced T-ALLs.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies by redirecting patient T cells to destroy cancer cells using engineered receptors. While CAR T cell therapies hold enormous potential as treatments in a wide range of tumour settings, treatments for non-B cell cancers have largely failed to significantly improve patient outcomes thus far. Furthermore, CAR therapies carry significant risk of inducing cytokine release syndrome (CRS), a potentially deadly toxicity caused by excessive release of inflammatory cytokines. The ability to minimize toxicity whilst maintaining adequate tumour cell-killing is therefore vital to the continued improvement of CAR therapies. We aimed to investigate the currently ill-defined relationship between CAR oligomeric state and potency using a novel protein engineering approach, with the aim of leveraging this knowledge to predictably modulate CAR activity. With de-novo protein design collaborators we identified synthetic transmembrane domain (TM) sequences that predictably formed defined homo-oligomeric structures. In addition to a previously validated trimeric TM sequence, I used X-ray protein crystallography to determine the structure of a dimeric TM peptide that agreed closely with its predicted structure. I inserted these novel oligomeric TM sequences into a well-established anti-HER2 CAR construct (comprising an anti-HER2 scFv attached via stalk/TM to costimulatory and stimulatory tail sequences) and validated their oligomeric state and signalling capacity in a mouse T cell line. When expressed in primary mouse T cells and incubated with HER2+ target cells, dimeric and trimeric CARs exhibited enhanced target cell killing compared to a reference anti-HER2 CAR. Using an in vivo mouse tumour model it was subsequently demonstrated that CAR oligomeric state correlates positively with CAR T cell anti-tumour efficacy. CARs encoding synthetic oligomeric TM’s also demonstrated a dramatic reduction in the release of inflammatory, CRS-associated cytokines within in vitro experiments. Using rational TM sequence mutations I identified lateral interactions between CARs and the endogenous T cell costimulatory molecule CD28 in primary mouse T cells as the key determinant of CAR cytokine release. These findings present an opportunity to improve efficacy and safety of CAR T cell therapies and warrant further validation in other clinically relevant CAR T cell disease models.

The heart is one of the first organs to develop in a vertebrate embryo. The intricate design of the asymmetric four-chambered organ is reflected in both its structures and mechanics. A healthy functional heart is an amalgamation of precisely aligned great vessels, continuous sheets of trilayered tissues that form the chamber walls and septum, a coordinated conduction system, and an uninterrupted supply of oxygenated blood from the coronary vasculature. At a cellular level, heart morphogenesis is a remarkable feat of tightly regulated cell proliferation, apoptosis, migration and differentiation. These events are driven by spatially and temporally coordinated gene expressions. Post-translational modifications of histone proteins such as acetylation and deacetylation are central to gene expression and repression. The genome site-specific activities of enzymes responsible for acetylation and/or deacetylation rely on protein domains that can recognize and bind to modified histone residues. These protein domains are present in either the histone modifying enzyme itself or adaptor proteins that are present within the histone modifying complex, known as histone ‘reader’ proteins. In this thesis, I have used knockout mice to investigate the roles of two histone reader proteins – ING4 and ING5, both present in histone acetyltransferase complexes in vivo, demonstrating the consequences of loss of these histone reader proteins in the patterning and structural development of the heart. In the first part of my thesis, I have demonstrated that loss of both Ing5 alleles alone did not incur any haematopoietic defects, unlike loss of the proposed histone acetyltransferase enzyme subunit present in the ING5 protein complex. Subsequently, I characterised the survival and appearances of mice with deletion of a combination of Ing4 and Ing5 alleles. I showed that ING4 and ING5 are functionally redundant, as mice died in mid-gestation in the absence of both proteins, whereas mice lacking only ING5 developed to term and mice lacking only ING4 were viable, healthy and fertile. However, mice lacking both Ing5 alleles and one Ing4 allele showed heart development abnormalities with full penetrance and died in utero. In the second part of my thesis, I explored mechanisms underlying the heart development defects observed in the compound mutants. I showed that the epicardium was most vulnerable to the loss of Ing4 and Ing5 alleles. I demonstrated that Ing4+/– Ing5–/– mutant mice were incapable of generating epicardium-derived cells and a proper coronary endothelial network. In addition, I have also demonstrated the redundancy of ING4 and ING5 with respect to gene expression changes and changes in global histone acetylation, further explaining how they possibly control heart development via regulating the functions of their associated histone acetyltransferase enzymes. Overall, my studies provide evidence that ING4 and ING5 are important reader proteins during embryonic development, in particular for the development of the heart and specifically in the epicardium tissue. ING4 and ING5 are likely to exert their functions by targeting the histone acetyltransferase activities of a MYST histone acetyltransferase enzyme to specific gene loci.

Cancer is a major public health issue globally, ranking as the second most common cause of death. Molecularly targeted therapies, focused on exploiting tumour cell dependency on certain oncogenic driver mutations for growth and survival, have greatly improved patient outcomes. However, despite these advances, some of the most frequent oncogenic mutations in cancer, such as those found in KRAS, are extremely challenging to target directly. One promising strategy to expand the range of actionable targets for cancer drug development is the exploitation of synthetic lethal interactions. Synthetic lethality is the term used to describe the death of cells in response to the co-existing disruption of two genes, neither of which is lethal alone. In this setting, targeting a gene that is synthetic lethal with a cancer-relevant mutation has the potential to induce the death of vulnerable cancer cells while leaving healthy cells unaffected. With this background in mind, my lab participated in a focused ENU mutagenesis screen in zebrafish with the aim of identifying genes that are essential for high rates of cell proliferation during endodermal organ development but not required by quiescent tissues. This yielded mutants that exhibited either ‘cell death’ or ‘growth arrest’ phenotypes in the liver, intestine and pancreas. I investigated two of the underlying mutant genes, ahctf1 and rnpc3, for their capacity to engage in synthetic lethal interactions with the kras oncogene. In Chapter 3, I investigated the impact of ahctf1 heterozygosity on the growth and survival of KrasG12V-expressing hepatocytes in a zebrafish model of hepatocellular carcinoma (HCC), TO(krasG12V). ahctf1 encodes Elys, a multifunctional nucleoporin with essential roles in nuclear pore assembly and mitosis. I found that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of Tp53-dependent and Tp53-independent cell death pathways which reduced tumour burden. Importantly, ahctf1 heterozygosity did not impact normal liver development, advancing ELYS as an attractive target for cancer therapy with a viable therapeutic window. In Chapter 4, I examined if rnpc3 heterozygosity also reduced tumour burden in the TO(krasG12V) model. rnpc3 encodes 65K, a unique protein component of the U12-dependent spliceosome, a specialised splicing machinery required for the correct splicing of a very small percentage (3.7%) of genes. In hepatocytes expressing krasG12V, rnpc3 heterozygosity reduced the number of cells in S phase of the cell cycle and increased cell death, together reducing tumour burden, without affecting normal tissue. In Chapter 5, I demonstrated that the zebrafish model of HCC is a powerful platform for testing novel therapeutics. I evaluated the efficacy of PRMT5 and KAT6A/B inhibitors early in their development, and showed that they were effective in reducing tumour growth and worthy of future investigation. In conclusion, my studies revealed two promising new targets for cancer treatment. I also demonstrated that the zebrafish HCC model is highly amenable to pharmacological inhibition and provides a valuable system for the pre-clinical examination of drug treatments in vivo.

T cells are an essential component of the vertebrate adaptive immune system. In concert with the innate immune system, T cells protect the host from any number of pathogens that could be experienced over an organism’s lifetime. The hallmark of a T cell is its distinctive T cell receptor generated by somatic gene rearrangement. Variability in the T cell receptor repertoire arises during thymic T cell differentiation, which is then subjected to strict selection processes. Mature T cells in the periphery can undergo further differentiation upon the activation of naive cells to mount immune responses to pathogens. These differentiation events are accompanied by significant proliferative bursts, followed by the clearance of defective or superfluous cells. It follows then, that cell death is also an essential component of T cell differentiation and homeostasis. This PhD thesis explores the molecular mechanisms regulating the differentiation, proliferation and death of T cells, and how interplay among these mechanisms gives rise to immune homeostasis. This study examines how distinct cell death pathways, including the intrinsic and extrinsic apoptotic pathways and necroptosis, are differentially regulated through T cell differentiation and in the various subsets of mature T cells. We found that only inhibition of the intrinsic pathway of apoptosis overcomes failure of beta-selection in the absence of preTCR signalling or proliferation, enabling further differentiation. We also discovered that caspase 8 plays an important pro-survival role in inhibiting necroptosis in recent thymic emigrant T cells and regulatory T cells, and that this feature can be exploited in the case of regulatory T cells for therapeutic intervention in infection settings. In summary, this thesis defines context-specific roles of cell death modalities in controlling T cell differentiation and homeostasis, revealing the potential for immune interventions using targeted therapies.

Necroptosis is a form of programmed cell death that is controlled by a defined set of protein effectors, despite displaying the morphological characteristics of unregulated lytic cell death (necrosis). Recently, there has been increasing interest in this type of cell death, ignited by studies demonstrating that necroptosis is involved in the pathophysiology of various diseases – including inflammatory conditions, degenerative conditions, infectious diseases and cancers – and further kindled by the progression of small molecule necroptosis inhibitors into clinical trials. The best studied form of necroptosis is driven by the tumour necrosis factor (TNF) signalling pathway, which is initiated by TNF binding to its cell surface receptor TNFR1. Importantly, TNF-induced necroptosis is regulated by three key proteins: the kinases RIPK1 and RIPK3, and the pseudokinase MLKL, which acts as the cell death executioner. To identify novel inhibitors of necroptotic cell death, several small molecule screens were performed at WEHI. This PhD thesis details the investigation into the mechanism of action of two small molecule necroptosis inhibitors identified from these screens. I employed a suite of chemical biology, biochemistry and cell biology approaches to deduce the cellular targets of these small molecules and investigate their anti-necroptotic activity. Chapter 2 examines the identification and mechanism of action of Compound 2, a more potent necroptosis inhibitor than its parent compound, Compound 1, which was identified from a small molecule screen against MLKL. I determined that Compound 2 targets all three necroptotic effector proteins – MLKL, RIPK1 and RIPK3 – in vitro and in cells, to potently block necroptosis in human and murine cells at nanomolar concentrations. Moreover, this study highlights that necroptosis can be potently inhibited by targeting multiple effectors, suggesting that targeting multiple proteins in the pathway may be an ideal strategy for inhibiting necroptosis in a therapeutic context. Chapter 3 explores the cellular activity and cellular targets of ABT-869, an inhibitor of necroptosis identified from a phenotypic screen using a cell line expressing a constitutively active MLKL mutant. I determined that ABT-869 blocks necroptotic cell death by targeting RIPK1 and possibly RIPK3, although whether ABT-869 targets RIPK3 directly or indirectly, as a result of RIPK1 inhibition, remains to be elucidated. Furthermore, this study raises some interesting questions regarding the involvement of RIPK1 downstream of MLKL activation, which could contribute to an improved understanding of how necroptosis is regulated at the molecular level. Together, these two novel inhibitors of necroptosis identified from small molecule screens were found to block necroptotic cell death by targeting known components of the TNF-induced necroptosis pathway. This research provides insight into how small molecules can modulate necroptotic signalling by interacting with key necroptotic proteins to inhibit cell death.

Background: Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer and early disease eradication is critically important for long-term cure. ALL remains a leading cause of cancer death in children and young adults because of treatment toxicity and relapsed disease. Increased delineation of the key biological drivers in ALL as well as therapeutic response will present new opportunities to rationally combine existing and novel agents to improve outcomes whilst minimising toxicities. Blocks in apoptosis are now widely recognised as a hallmark of ALL but also a mechanism of resistance to standard chemotherapeutic agents. Co-targeting aberrant cell survival pathways, using novel combinations of BH3-mimetics, are an emerging therapeutic option. My thesis centres on translational and mechanistic studies of combining venetoclax (BCL-2 inhibitor) and S63845 (MCL-1 inhibitor) in high-risk (HR) ALL subtypes. Aim: To inform the clinical utility of BH3-mimetic combinations in HR-ALL by identifying synergistic combinations with each other as well as standard and targeted agents in vitro, evaluating the efficacy and tolerability of combination venetoclax (BCL-2 inhibitor) and S63845 (MCL-1 inhibitor) in vivo, and investigating mechanisms of therapeutic resistance. Methods: BH3-mimetics were combined with each other as well as dexamethasone and targeted tyrosine kinase inhibitors (TKI) to identify the most potent combinations across kinase-activated ALL cell lines and patient derived xenografts (PDX) in vitro and in vivo. The tolerability of combinations venetoclax and S63845 was detailed in NOD-SCID-IL7R (NSG) mice as well as healthy donor blood ex vivo. Loss-of-function mutations that confer resistance to TKIs and BH3-mimetics in a Ph+ALL cell line were identified using an unbiased genome-wide CRISPR-Cas9 loss-of-function screen. Differences in cell signalling and survival pathways following treatment with TKI and BH3-mimetics in vivo in PDX models of Ph-like ALL were identified using mass cytometry. Results: BCL-2 and MCL-1 protein expression remained high in kinase-activated B-ALL cell lines treated with targeted TKI despite up-regulation of BIM pro-apoptotic protein expression. Co-inhibition of BCL-2 and MCL-1, with combination venetoclax and S63845, induced synergistic killing in vitro and was comparable or superior to steroid or TKI combined with each BH3-mimetic, across a range of kinase-activated B-ALL cell lines and PDXs, including the Ph+ and Ph-like subtypes. The combination also had potent anti-leukaemia activity in vivo, which was demonstrated by rapid cytoreduction, but also acute tumour lysis syndrome (ATLS) in some PDX models of Ph-like B-ALL. Combining venetoclax and S63845 appeared tolerable, however, histologic evidence of haematopoietic toxicity was observed, at higher doses, in NSG mice, and synergistic cytotoxicity was observed in lymphocytes of healthy donor blood. Loss of function mutations in NOXA and BAX were identified from the CRISPR screen as important causes of resistance to dasatinib and venetoclax in Ph+ALL. Lastly, CyTOF demonstrated distinct single cell variability in response to TKI or BH3-mimetic treatment of Ph-like ALL, including differences in surface marker expression (CD38, CD179a, and CD34), and cell signalling pathways (pSTAT5). Conclusion: Co-inhibition of BCL-2 and MCL-1 induces synergistic killing in vitro and rapid cytoreduction in vivo in a range of HR B-ALL models, including the Ph+ and Ph-like subtypes. The combination of BCL-2 and MCL-1 inhibition was comparable or superior to steroid or TKI combined with each BH3-mimetic. Although this combination of BH3-mimetics was tolerable in vivo at lower doses, histologic evidence of haematopoietic toxicity and tumour lysis syndrome was observed in NSG mice and PDX models, respectively. The expression level of BCL-2 family anti-apoptotic genes (BCL-2 and MCL-1), BH3-only class of pro-apoptotic genes (NOXA and BAX), surface markers (CD38, CD179a, and CD34) and cell signalling pathways (pSTAT5), predicted treatment resistance. Co-targeting BCL-2 and MCL-1 warrants evaluation in clinical trials that incorporate supportive care including infection prophylaxis and tumour lysis precautions. The findings from this research may be exploited for future studies to test novel combinations of drugs, on the basis of their ability to act on non-overlapping mechanisms, which are likely to result in synergistic anti-leukaemia efficacy.

Innate lymphoid cells (ILCs) are a specialized arm of the innate immune system responsible for protecting the mucosal barrier and defending against infection. ILC activation and cytokine production occurs in response to stimuli in the local environment making them an important initiators of the early immune response. However, they also contribute to inflammatory diseases and thus must be tightly regulated. The development and function of ILCs is dependent on the tight regulation of transcription factors. Two transcription factors GFI1 and GFI1B are involved in this differentiation but have been poorly studied and thus how they shape differentiation is not clear. GFI1 and GFI1B was differentially expressed throughout ILC development. GFI1B expression was limited to the early progenitors whereas GFI1 was expressed in both early progenitors and in all mature ILC subsets in the peripheral organs. GFI1B expression identified the early progenitors in the bone marrow with the capacity to give rise to all ILC subsets. GFI1B expression is also required for the development of progenitors in the bone marrow and lung ILC2. This impacted the lung inflammatory response induced in mice treated with papain as the eosinophil recruitment by ILC2s was impaired. In contrast, GFI1 was highly expressed in mature NK cells and was vital for development and maintenance of these cells. GFI1 regulated multiple NK cell pathways, including proliferation, homing to peripheral organs and effector functions. Defects in these functions impaired the protection mediated by NK cells against metastatic melanoma cells. Together, GFI1 and GFI1B are both key regulators of ILC development, although they regulate different stages of ILC development and function. ILCs are enriched at the mucosal barrier, particularly at the gastrointestinal tract. They play an important role in protecting against infections but also have the capacity to promote inflammation when dysregulated. Their role in the development of colorectal cancer (CRC) is unclear as individual ILC subsets have been associated with both protecting against or promoting tumorigenesis. Characterisation of the immune infiltrate of mice with CRC showed that there was an accumulation of IL-5-producing ILC2 within the colon and this was strongly correlated to the tumour burden. ILC2-deficient mice developed a higher tumour burden compared to control mice, which indicated a protective role for ILC2s against colorectal cancer.

Interleukin-1 beta is a potent proinflammatory cytokine that requires inflammasome-associated caspase-1, a cysteine protease, to cleave inactive precursor IL-1beta, to its active secreted p17 fragment. Excessive IL-1beta activation and release promotes autoinflammatory diseases, such as Cryopyrin associated periodic syndromes (CAPS), gout and cancers. Despite this role in inflammatory disease, the post-translational regulation of IL-1beta is poorly understood. This thesis studies the post-translational modifications (PTMs) of IL-1beta, with a particular focus on the ubiquitination of precursor IL-1beta and its role in modulating IL-1beta activity.

T follicular helper cells (TFH) are specialised CD4+ T cells that promote B cells maturation into antibody secreting plasma and memory cells. Most of the current vaccines generate protection via the induction of long-term antibody responses and circulating TFH are reliable predictors of vaccine response. Conversely, dysfunctional TFH cells are associated with the pathogenesis of immunodeficiency, systemic autoimmunity and allergy. Despite their importance, we have an incomplete understanding of how TFH cells differentiate and function in distinct inflammatory settings. Answering this question has broad health implications to stimulate rational development of vaccines and therapeutics against diverse infections, autoimmune and allergic disease. In this thesis, I investigated the transcriptional and migration control of TFH differentiation during viral infection. In this setting, TFH cells differentiate in parallel with T helper 1 (TH1) CD4+ T effector cells. I investigated two canonical TH1 factors, T-bet and CXCR3, to understand their roles in TFH cells differentiation. Comparing two viral infections, I demonstrated a context-dependent role for T-bet in TFH differentiation and identified the cytokine and chemokine factors that underlie distinct T cell differentiation. Combined, this work demonstrates that there are multiple paths that direct TFH differentiation. This study has led to further investigations into pathogen-specific TFH programs, which will help us understand how TFH orchestrate tailored B cell responses in diverse infections.

Abstract: Apoptosis is a conserved cellular process of programmed cell death. The intrinsic pathway of apoptosis is principally regulated by three functional and structural subgroups of the BCL-2 protein family: pro-survival proteins, pro-apoptotic effector proteins and apoptotic initiator BH3-only proteins. The interactions between these proteins on the mitochondrial outer membrane (MOM) determine whether a cell survives or dies. In healthy cells, pro-survival proteins inhibit the pro-apoptotic effector proteins BAK and BAX. In response to death stimuli, BH3-only proteins are activated to promote apoptosis either by activating BAK and BAX or neutralising pro-survival proteins. As well as BCL-2 family proteins, non-BCL-2 proteins such as VDAC2 are emerging as important regulators of apoptosis. However, the different impacts are observed in different contexts – with VDAC2 promoting apoptosis in some cases and restricting apoptosis in others. For BAK-dependent apoptosis, VDAC2 inhibits BAK function by forming complexes with BAK on the MOM. Therefore, loss of VDAC2 sensitises the cells to apoptotic stimuli in some contexts. This suggests that there may be proteins that accelerate BAK-dependent apoptosis in VDAC2-deficient cells. A previous genome-wide CRISPR-Cas9 library screen was performed in Bax-/-Vdac2-/-Mcl-1-/- MEFs to identify genes that when deleted inhibited the response to BH3-mimetics. In this screen, the absence of MCL1 enabled BH3-mimetic drug ABT-737 that inhibits BCL-2, BCL-XL and BCL-W to drive apoptosis in these cells, while the absence of BAX and VDAC2 ensured that apoptosis was mediated by BAK and controlled independently of VDAC2. From this screen, three proteins involved in ubiquitin signalling were identified as potential candidates: MARCHF5, UBQLN1 and USP24. In Chapter 3, I focused on validating each of these candidates in BAK-dependent apoptosis and identified that deletion of Marchf5 in Bax-/-Vdac2-/-Mcl-1-/- MEFs had the greatest impact on BAK-driven apoptosis. The effect of Marchf5/MARCHF5 on BAK function was further explored across different contexts using both Mcl1+/+ and Mcl1-/- MEFs and two human cell lines (HeLa and KMS-12-PE). Consistent with the initial validation results, deleting Marchf5/MARCHF5 in both murine and human cells provided long-term protection from BAK-driven apoptosis induced by BH3 mimetic drugs. Having successfully identified MARCHF5 as an important regulator of BAK-dependent apoptosis in Chapter 3, I further investigated how MARCHF5 modulated BAK apoptotic function in Chapter 4. My research indicated that BAK adopted an activated conformation and formed stable Mode 2 inhibitory complexes with MCL-1 and BCL-XL in MARCHF5-deficient cells, thereby providing protection from BH3 mimetic drugs. Given that MARCHF5 is an E3 ubiquitin ligase, I further identified that the drug resistance observed upon MARCHF5 deletion could be phenocopied by abrogating MARCHF5 enzymatic activity. Finally, in Chapter 5 I performed proteomics and functional CRISPR/Cas9 genetic screens to identify substrates of MARCHF5 that could account for the mechanism driving BAK into Mode 2 complexes in MARCHF5-deficient cells. Through these complementary approaches, several potential candidates were revealed, that may represent novel mediators of BAK apoptotic activity.

Receptor Interacting Serine/Threonine Kinase-3 (RIPK3) is essential for necroptosis, an inflammatory form of programmed cell death pathway implicated in innate immunity, kidney ischemia reperfusion injury, and systemic inflammatory response syndrome. In the classical model, cells committed to necroptosis phosphorylate RIPK1, which in turn drives RIPK3 phosphorylation and oligomerisation. Active RIPK3 oligomers subsequently phosphorylate mixed lineage kinase domain-like protein (MLKL) pseudokinase which induces its translocation to the plasma membrane. The necroptosis pathway culminates in MLKL perforating the plasma membrane as a prelude to cellular rupture and release of inflammatory cytokines and damage-associated molecular patterns to the extracellular milieu. In addition to being a pro-necroptotic kinase, RIPK3 is also capable of triggering apoptosis when its kinase activity is restrained. Moreover, numerous death-independent roles of RIPK3 have been described in the context of inflammation such as arthritis, viral infection, or colitis whereby RIPK3 either promotes or dampens the secretion of pro-inflammatory cytokines. Understanding the molecular regulation of RIPK3 will thereby facilitate the ongoing pre-clinical development of RIPK3 inhibitors. Like most proteins, post-translational modification (PTM) is a critical fine tuner of RIPK3 activities. Ubiquitylation, in particular, has recently garnered attention in the cell death field as loss of this PTM may result in hyperactive RIPK3 which consequently accelerates death and inflammation. However, the post-translational control of RIPK3 signalling is not fully understood. Using mass-spectrometry, I identified a novel ubiquitylation site on murine RIPK3 on lysine 469 (K469). Complementation of RIPK3-deficient cells with a RIPK3-K469R mutant demonstrated that the decoration of RIPK3 K469 by ubiquitin limits both RIPK3-mediated caspase-8 activation and apoptotic killing, in addition to RIPK3 autophosphorylation and MLKL-mediated necroptosis. Unexpectedly, the overall ubiquitylation of mutant RIPK3-K469R was enhanced, which largely resulted from additional RIPK3 ubiquitylation upstream on lysine 359 (K359). Loss of RIPK3-K359 ubiquitylation reduced RIPK3-K469R hyper-ubiquitylation and also RIPK3-K469R killing. Collectively, I therefore propose that ubiquitylation of RIPK3 on K469 functions to prevent RIPK3 hyper-ubiquitylation on alternate lysine residues, which otherwise promote RIPK3 oligomerisation and consequent cell death signalling. I further investigated the consequence of abolishing RIPK3 K469 ubiquitylation by generating Ripk3K469R/K469R mice. In agreement with in vitro findings, primary fibroblasts with mutant RIPK3-K469R enhanced apoptosis, and in vivo studies demonstrate that RIPK3-K469 ubiquitylation contributes to pathogen clearance. Specifically, when Ripk3K469R/K469R mice were challenged with Salmonella enterica serovar Typhimurium, bacterial loads in the spleen and liver were significantly increased relative to wildtype control animals. The increased bacterial burden in the mutant mice was consistent with reduced IFNg produced in the serum, while the elevated MCP-1 cytokine upon infection might be indicative of heightened immune infiltrates. Although necroptosis signalling clearly triggers cell death, how it might impact other cellular responses remains unclear. Therefore, to further delineate the functional outcomes of necroptotic activity I examined how its signalling impacts autophagy. The autophagy pathway is triggered when cells are deprived of nutrients. Although regarded as a pro-survival pathway which acts to recycle and remove damaged organelles, studies have recognised that autophagic pathways can impact cell death processes. In apoptosis, for instance, autophagy acts to limit pro-inflammatory IFN-b secretion, thus decreasing apoptotic immunogenicity. Nonetheless, little is known about the status of autophagy during necroptosis. I demonstrate through various genetic, imaging, and pharmacological approaches that active MLKL translocates to autophagic membranes during necroptosis. However, contrary to previous findings which reported the activation of autophagy upon necroptotic activity based on increased lipidated LC3B, a commonly used marker of autophagy induction, I challenged this conclusion by demonstrating that the accumulation of active LC3B during necroptosis is a consequence of reduced autophagic flux. Therefore, unlike apoptosis which proceeds in tandem with autophagy, the induction of necroptosis negates autophagy in an MLKL-dependent manner. While the function of MLKL-mediated autophagy inhibition warrants further investigation, I propose that attenuating autophagy during necroptosis contributes to the immunogenicity of this cell death modality by limiting the ability of the cell to clear damaged organelles and immunogenic molecules. Overall, my research has helped in outlining how a key necroptotic molecule RIPK3 is regulated post-translationally and how this is relevant in the context of microbial defence. I have also defined novel functional roles for necroptosis signalling in the regulation of autophagic responses. Understanding the molecular regulation of necroptosis signalling and how this cell death pathway is linked to other cellular responses, such as autophagy, is important for the accurate design of new therapeutics to target these pathways in pathological settings.

Genomic profiling has revealed that acute myeloid leukaemias (AMLs) tend to have a relatively low mutation rate, but that many different driver genes may contribute to the development of the disease, complicating efforts to identify similarities between cases. Three AMLs (of whom 2, WEHI-AML-1 and WEHI-AML-2, were siblings) underwent detailed genomic analyses using different molecular techniques supplemented by sequencing data from international databases. The results from the siblings identified a novel inherited predisposition to AML involving the base excision repair protein, MBD4. AMLs deficient in MBD4 have a unique mutational signature characterised by elevated CG>TG mutations and all 3 acquired mutations in driver genes in a conserved order. The importance of MBD4 in maintaining genomic integrity was confirmed in other cancers, through the analysis of large public cancer datasets, and a genetically modified mouse model. The third AML, WEHI-AML-3, had DDX3X-MLLT10, a translocation that has only been reported in T-acute lymphoblastic leukaemias. As expected, analyses of the three AMLs at different clinical timepoints, showed the leukaemias underwent clonal evolution, providing further insight into their disease biology. For example, the leukaemia in WEHI-AML-3 acquired new mutations resulting in mechanisms for therapy resistance and subsequent poor clinical outcome. The results from the 3 AMLs have broader implications for other haematological malignancies and cancers.

Tumour Necrosis Factor (TNF) is one of the most potent pro-inflammatory cytokines and it is secreted in response to danger signals, such as those caused by pathogen infection. High levels of TNF have been associated with many chronic and inflammatory diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and psoriasis. To prevent high TNF levels and uncontrolled inflammation, Tnf mRNA is degraded when not required, making post-transcriptional regulation a central mechanism to control Tnf expression. Post-transcriptional control operates through cooperation between cis-elements present in the 3’UTR and trans-acting proteins such as RNA-binding proteins. Until recently, knowledge about post-transcriptional regulation of Tnf was limited to the role of the AU-Rich Element (ARE), and to a lesser extent to that of the Constitutive Decay Element (CDE). In 2015, our group identified a New Regulatory Element (NRE), which changed the view on the post-transcriptional regulation of Tnf. Importantly, we have discovered a cooperative mechanism between two or more elements to regulate Tnf mRNA stability in vitro. In this thesis, I have translated our previous in vitro observations, in vivo, using the CRISPR/Cas9 technology to generate mice with deletions of one or two regulatory elements in the Tnf 3’UTR. I showed that the variety of phenotypes of the mice changed greatly in severity, including a concomitant deletion of the ARE and the CDE causing embryonic death. This suggests that not only different cis-elements cooperate to destabilise Tnf mRNA efficiently, but the mechanisms involved also appear to operate in a cell- and tissue-specific manner. Furthermore, I aimed to characterise a new trans-acting RBP called ZC3H12C that was previously identified by our group as involved in the post-transcriptional regulation of Tnf in vitro. To study the physiological role of ZC3H12C and the consequences of its loss, and its expression in vivo, I engineered a mouse deficient in Zc3h12c, in which the green fluorescent protein GFP that can be used as a marker of expression replaces ZC3H12C. Zc3h12c-deficient mice are found as adults at the expected Mendelian frequency and look outwardly normal. In particular, they do not present with any phenotype related to an excess of TNF (like cachexia or arthritis), even at an advanced age. However, loss of Zc3h12c causes aberrations in the structure of secondary lymphoid tissues, and hypertrophic skin-draining lymph nodes with supernumerary B cells and inflammatory dendritic cells in ageing mice. Flow-cytometry analysis of our GFP-reporter mouse showed that dendritic cells (DCs) are the immune cell type expressing ZC3H12C the most. RNA-seq analysis on splenic DCs suggested that loss of Zc3h12c affected the anti-viral immune response. Accordingly, when challenged with chronic LCMV, Zc3h12c-deficient mice presented with an abnormally exaggerated immune response. I characterised the impact of the loss of Zc3h12c in the context of psoriasis to confirm previous Gene Wide Association Studies (GWAS) suggesting that Zc3h12c was one of the risk genes involved in psoriasis incidence in human. I found that loss of Zc3h12c did not impact psoriasis’ development, but this observation could be due to the limits of the psoriasis model used in my study. I further characterised the role of Zc3h12c in skin homeostasis by mimicking the Toxic-Epidermal Necrolysis disease using subcutaneous injection of SMAC-mimetics to induce TNF-dependent cell death in the skin. In this context, loss of Zc3h12c appeared to be beneficial and reduced the lesions and the cell death induced by the SMAC mimetic compound. Finally, to evaluate the potential role of TNF in this phenotype, I generated mice lacking both Tnf and Zc3h12c. While double-deficient (DKO) mice never developed lymphadenopathy, around 30% of the Tnf/Zc3h12c DKO mice developed severe multiorgan inflammation, including pancreatitis, myocarditis, otitis, myositis, pyelonephritis, anaemia, extramedullary haematopoiesis and bone marrow failure. Histopathological analysis suggested that concomitant loss of Tnf and Zc3h12c rendered the mice immunocompromised and potentially sensitive to the opportunistic pathogen Pasteurella pneumotropica, for which they tested positive. To evaluate the role of the TNF-TNFR2 signaling in the phenotype, and given the widely known role of TNFR2 in autoimmunity development, I generated Tnfr2 and Zc3h12c double-deficient mice. While I failed to observe a single Tnfr2/Zc3h12c DKO mouse falling sick, I also observed an absence of disease development in the Tnf/Zc3h12c DKO mice and this coincided with the clearance of P. pneumotropica from the animal facility. These observations raise new questions on the role of Tnf and Zc3h12c in the control of immune responses and inflammation, and further investigation will have to be conducted. Overall, my work suggests that Zc3h12c might be a risk factor in the context of anti-TNF treatment leading to autoimmunity in some patients.

The persistence of a replication-competent HIV reservoir necessitates life-long antiretroviral adherence and precludes the possibility of a HIV cure via conventional therapy alone. Furthermore, recent clinical studies have made it increasingly clear that the predominant strategy for reservoir elimination, enforced transcriptional reactivation, does not diminish the size of the latent reservoir or reduce the time to viral rebound following treatment interruption. A novel approach seeks to purge the HIV reservoir by activating apoptotic pathways in latently infected cells and shifting the balance away from survival and towards cell death. Several lines of evidence implicate Bcl-2 family proteins in the long-term survival of memory CD4+ T cells – the major reservoir for HIV. Bcl-2 antagonism thus represents a viable strategy for sensitizing latent cells to death and delaying viral rebound. The development and clinical progression of BH3-mimetics, which induce apoptosis by binding pro-survival Bcl-2 homologs, has resulted in a well- characterised class of inhibitors with relatively few unknowns regarding toxicity, side effects and dosage. In this thesis, I hypothesise that there are apoptotic blocks in place, specifically a greater dependence on pro-survival Bcl-2 proteins, which prevent a minority of infected CD4+ T cells from dying during active infection. I hypothesise that latently infected cells are distinct from other infected or healthy cells, and that this pro-survival phenotype allows them to persist in such a way that renders them susceptible to pro- apoptotic therapeutics which target the intrinsic pathway, such as BH3-mimetics. In Chapter 3, I infect primary human CD4+ T cells with HIV in vitro to assess the ability of BH3-mimetics to kill actively infected cells. I demonstrate that ABT-737 and Venetoclax, but not the Mcl-1 inhibitor S63845, preferentially kill activated, HIV infected CD4+ T cells in the setting of productive viral replication. These results shed light on the pro-survival role of Bcl-2 proteins during active HIV infection, and inform our progression into a preclinical model of HIV latency. Chapter 4 uses a humanized mouse model of HIV latency to further interrogate the importance of Bcl-2 pro-survival proteins in reservoir survival. I investigate the ability of Venetoclax, a clinically-approved Bcl-2 antagonist, as well as S63845, a preclinical Mcl-1 inhibitor, to delay viral rebound following analytical treatment interruption. This work provides the first compelling evidence that BH3-mimetics, either as monotherapy or in combination, can eliminate latently infected cells in vivo. In Chapter 5 I perform a tat/rev Induced Limiting Dilution Assay (TILDA) on CD4+ T cells from latently infected mice in order to quantify the impact of Venetoclax on the magnitude of the latent HIV reservoir. I confirm the existence of an inducible reservoir in our mouse latency model, although I do not observe a significant effect of Venetoclax treatment as measured by TILDA. I also use single-cell RNA sequencing to characterize peripheral CD4+ T cells from ART-suppressed human donors following Venetoclax treatment ex vivo, arriving at the suggestion that Venetoclax may target CD4+ T cells that are enriched for a gene signature associated with activation and cell metabolism. This work lays the foundation for furthering our understanding of which cells may contribute to HIV persistence and which may be susceptible to death- inducing compounds. Overall, this thesis represents a comprehensive assessment of the ability of BH3-mimetics to kill HIV active and latently infected cells, offering a strong justification for the translation of pro-apoptotic therapeutics such as Venetoclax into a clinical setting where reservoir eradication is the goal.

During an adaptive immune response activated B and T lymphocytes undergo rapid clonal expansion and generate extensive cellular heterogeneity. How lymphocytes guarantee the emergence of functional diversity amongst responding cells is not fully understood. In this thesis, the strategies utilised by the adaptive immune system for the diversification of B and T cells is investigated at the cellular, molecular and clonal levels in a quantitative manner. Activated B cell heterogeneity is predominantly driven by two critical programs. Firstly, the differentiation of antibody-secreting cells (ASCs) and secondly, the diversification of antibody isotype by class switch recombination (CSR). The regulation of these two processes was investigated through combined clonal and molecular analysis using a high-throughput proliferative lineage tracing approach to study ASC differentiation and CSR across thousands of clones. Two distinct fate programs emerged. Firstly, the timing of ASC differentiation within clones was strongly correlated. Diversity in commitment to the ASC lineage is established early and could be traced to the naive founder cell, from where it is transmitted to all progeny during clonal expansion. In striking contrast, isotype switching was highly variable across related cells irrespective of common ancestry, revealing a highly stochastic, cell-autonomous process regulated late within activated single cells. Further analysis demonstrated that single cells faced with a choice of two heavy chain isotypes solve the conflict using stochastic selection that is independent of their clonal lineage. As the principle molecular drivers of CSR are well known, their variation amongst single cell within clonal families was measured. Extensive variation was demonstrated in the expression of both activation-induced cytidine deaminase (AID) and the transcription of the germline noncoding RNAs. Furthermore, there was no correlation between AID expression and germline transcription, nor was the expression of distinct germline transcripts correlated. Thus, the net effect of stochastic influences over these two components can account for the single cell autonomy governing CSR. This stochastic molecular mechanism of CSR was developed into a quantitative model that accurately described and predicted B cell fate decisions across cell division and under varying experimental conditions. Quantitative analysis was applied to multi-parameter data of CD8 T cell heterogeneity, generated in response to diverse external stimulation. Using a combination of novel and established analytical techniques, the influence of time and division progression on T cell diversification, and their control by external signals, was accurately measured. The results of this investigation was subsequently used to construct a kinetic model of time- and division-dependent expression patterning for the molecule CD69 under varying external conditions. This model accurately described the expression dynamics of CD69 over time and division and highlighted the utility of a quantitative modelling approach to understanding CD8 T cell heterogeneity. Collectively, the work presented in this thesis represents a set of quantitative principles that describe lymphocyte fate decisions.

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic modifier that plays an important role in X chromosome inactivation, autosomal gene silencing, and is also implicated in several diseases in humans (Blewitt et al., 2008, Mould et al., 2013, Gendrel et al., 2013, Lemmers et al., 2012, Gordon et al., 2017, Shaw et al., 2017, Jansz et al., 2018). Thus far the majority of work on Smchd1 has been carried out on its zygotic form. Therefore, I partnered allele-specific genomics with conditional deletion of Smchd1 in the oocyte to test the role of maternally derived Smchd1 in imprinted gene expression. I found that Smchd1 is a novel maternal effect gene involved in genomic imprinting. When Smchd1 is maternally deleted loss of imprinting is observed at ten imprinted genes, without affecting DNA methylation imprints. Additionally, I also discovered seven imprinted genes where zygotic Smchd1 plays a dose dependent role. Interestingly, almost all the imprinted genes affected by the loss of maternal Smchd1, including Xist possess H3K27me3 imprints. This, together with Smchd1’s known role in long range chromatin interactions and function as insulator protein(Chen et al., 2015, Jansz et al., 2018), lead me to hypothesise that maternal Smchd1 may carry out its function secondary to H3K27me3 imprints and establish a chromatin state required for imprinted expression. To narrow down Smchd1’s behaviour in its native environment and how this behaviour relates to its structure and function, I established a series of robust microscopy experiments. I used fluorescence recovery after photo bleaching (FRAP) and Lattice LightSheet microscopy to demonstrate for the first time, Smchd1 dynamics in the inactive X during interphase and mitosis. Through study of our unique gain of function mutant MommeD43 I found that the act of unbinding and reloading likely has a role in Smchd1’s insulation effects. By using 3D-direct stochastic optical reconstruction microscopy (3D-dSTORM) I discovered that Smchd1 is approximately a 40 nm long protein in the nucleus. These novel techniques open up new avenues to explore higher order structures that Smchd1 may form in the nucleus related to its role in chromatin architecture. Together these data not only revealed a new function for maternal Smchd1 but also established novel methods to unravel Smchd1's molecular mechanism.

The functionally and phenotypically diverse cell populations that make up the immune system arise from the expression of a select part of a genome at a given time. The mechanisms governing such differential transcription are still, surprisingly, not fully understood. Only recently has the three-dimensional organisation of chromatin in the interphase nucleus been acknowledged to play a crucial role in modulating transcription. For instance, distal cis-regulatory elements like enhancers can form long-range chromatin loops with promoters to drive transcription, and these chromatin loops are in turn harboured in topologically associating domains (TADs), shielded from interference by outside elements. These three-dimensional structures can be lineage-specific and their roles during cellular differentiation are beginning to be uncovered. The chromosome conformation, or genome architecture, in B- and T-lymphocytes, captured previously as Hi-C data in the lab, has provided immense information about lineage-specific DNA interactions that might be critical during differentiation. Based on this resource, the work herein aimed to develop an approach to identify, characterise and functionally dissect any novel and critical regulatory elements. Using this strategy, I have identified several putative T- and B-cell specific elements and subsequently adopted the CRISPR/Cas9 platform in generating large deletions as to dissect these elements. The approach has identified and confirmed the enhancers of T cell-specific transcription factors Bcl11b and Gata3. Upon closer inspection an uncharacterised long non-coding RNA (lncRNA) Gm13218 was uncovered to associate with the enhancer of Gata3. Given the recent recognition of lncRNAs as important regulator of the 3D genome, I have retrieved the full-length sequence and characterised its expression pattern. It was found that expression of Gm13218 is highly correlated with that of Gata3 during early T cell development in thymus as well as T helper 2 (TH2) cell differentiation. Knockdown and overexpression of Gm13218 transcripts, CRISPR-mediated silencing, activation, demethylation of the locus as well as interference of transcription elongation suggest that Gm13218 may be involved in the establishment, but not the maintenance of Gata3 expression. By utilising Hi-C, RNA-seq, cell division and cell cycle indicators, the spatiotemporal dynamics of genome architecture during B cell activation and terminal differentiation into antibody-secreting cells was examined. It was revealed that genome organisation exhibit two discrete waves of restructuring – the first occurs just prior to the first cell division, with the resulting genome architecture being inherited through the subsequent rapid clonal expansion for many days until the second wave of restructuring upon differentiation into plasmablast. In addition, the first restructuring event was shown to precede the first DNA replication phase, suggesting that genome reorganisation is independent of, and well partitioned from, DNA synthesis and mitosis. In contrast, transcription underwent very early burst and was altered throughout the entire differentiation process. Further analysis suggests that transcription is intricately intermingled with genome organisation in a reciprocal fashion. Overall, the work in this thesis has revealed a number of important findings regarding how the 3D genome controls the development and function of the immune system.

Histone acetylation affects the way DNA and associated proteins are packaged in the cell nucleus and regulate chromatin organisation and gene expression. Acetylation of core histones has been broadly correlated with initiating and maintaining open chromatin, poised or active gene transcription, DNA damage repair, as well as chromosome decondensation during mitosis and meiosis. The acetylation of lysine residues is catalysed by histone lysine acetyltransferases (KATs) and deacetylases (HDACs), which are tightly regulated. Dysregulation of KATs and aberrant lysine acetylation has been associated with tumorigenesis and negative prognoses in a wide range of cancer, presenting a new area of potential therapeutic targets. Potential acetylation targets of KATs catalysing the acetylation of histones have predominantly been studied in cell-free assays, where the enzymes show little substrate specificity. In contrast, histone acetyltransferases appear to acetylate surprisingly specific residues in whole cells. In this thesis I investigate the effects of acute deletion of the MYST lysine acetyltransferase TIP60 (KAT5) using inducible cre-recombinase and CRISPR/Cas9-mediated deletion in human cells and mouse cells, as well as mouse embryos and its potential role in cancer cells. I found that loss of TIP60 caused complete cell growth arrest in human and mouse cells. In the absence of TIP60 cells displayed cell cycle arrest in G1 and G2/M phase with increased endoreplication, accompanied by chromosomal segregation defects. Remarkably, the proliferation arrest caused by loss of TIP60 also occurred in the absence of the tumour suppressors p53, INK4A and ARF and therefore was independent of these. In contrast, cell survival was not affected. Growth arrest independent of major tumour suppressors flags TIP60 a potential target for novel cancer therapeutics. TIP60 was found to be essential for of H2AZ acetylation, particularly, lysine 7 acetylation. In contrast, global chromatin bound H2AZ levels were not reduced. H2A and H4 acetylation was reduced slightly in TIP60 depleted cells. Identifying H2AZ lysine 7 acetylation as a biomarker for TIP60 activity is a major step in developing TIP60 as a drug target. The mRNA levels of 6236 human and 8238 mouse genes, including many metabolic genes, were dependent on TIP60, supporting a role for TIP60 as a key transcriptional co-activator. Characterization of key mechanisms causing chromosomal aberrations and identifying approaches that can be used to reduce likelihood of genome instability due histone acetylation defects will pave the way for better detection of early changes in cancer and development of therapeutic applications in the future. This work represents important steps towards the development of histone lysine acetyltransferases as drug targets. A comprehensive analysis of histone acetylation activity of TIP60 shines light on the potential of its many proposed roles.

The differentiation of B cells into antibody secreting cells (ASCs) and the production of protective antibodies is a critical part of the adaptive immune response to infection. ASCs are also important for the formation of immunological memory which provides protection against reinfection and the generation of ASCs is the goal for almost all current vaccination strategies. Despite the importance of these cells, we still lack a complete understanding of the factors that control B cell differentiation into ASCs, ASC survival and antibody secretion, all of which must be tightly regulated to ensure an optimal immune response. Here, I have developed a CRISPR/Cas9 mediated arrayed screening approach for the identification of novel positive and negative regulators of primary mouse B cell proliferation, survival, differentiation into ASCs and antibody secretion. By interrogating multiple gene sets I have identified all elements within the ASC gene signature that are essential for the in vitro generation of ASCs. I have also identified several novel negative regulators of the B cell differentiation process (AB124611, Arhgef18, A430078G23Rik, Fam43a, Pold1, Ripk3, Rnf130 and Rps6ka5). This work has also uncovered a novel role for 6 genes, (Cdv3, Hspa5, Sec61a1, Selk, Sumo2, Vcp) in driving the proliferation of B cells. One of these genes, Cdv3, has no previous association with proliferation in any cell type and presents an exciting new candidate for further investigation. I have demonstrated that within the ASC gene signature there are 35 genes which are essential for efficient antibody secretion. Interestingly, many of these genes are components of the ER protein processing pathway, however, not all elements of this pathway appear to be essential for antibody secretion. These results raise the possibility of there being a specific pathway for antibody secretion, or that the genes identified in this thesis may represent weak links in the ER protein processing pathway which could potentially be exploited therapeutically to inhibit antibody secretion in disease settings. Finally, I have used an Irf4 deficient mouse model to uncover a novel role for Irf4 in the development of the peritoneal B-1a population. I have shown that Irf4-/- mice lack peritoneal B-1a cells and by examining multiple stages of B-1a cell development I have demonstrated that in the absence of Irf4, B-1a cell development is blocked at the transitional B-1a stage. By employing RNA sequencing to analyse the transcriptional profiles of the remaining Irf4-/- B-1 cells and analysis of previously published ChIP sequencing data, I have revealed a potential role for Irf4 in directly activating the expression of Bhlhe41, a transcription factor that is required for B-1a cell development and homeostasis. Together, the results from this thesis build upon decades of previous work on the genetic regulation of B cell biology. Integrating the novel regulators of B cell proliferation and differentiation that I have identified in this thesis into the current model of ASC generation will improve our understanding of how the decision between undergoing differentiation or maintaining the B cell fate is made. A detailed understanding of how this fate decision is made has far reaching implications for human health and disease as this information can be used to inform vaccine design, reveal the causes of immunodeficiencies or highlight novel avenues for targeting pathogenic ASCs in autoimmunity and cancer.

Regulatory T (Treg) cells are critical for the maintenance of immune homeostasis and peripheral tolerance. Different subsets of Treg cells have recently been described with many studies showing the importance of context-specific differentiation of Treg cells, in particular within non-lymphoid organs. These non-lymphoid organ Treg cells have a fully suppressive Treg cell phenotype with an effector function and are termed effector (e)Treg cells. However, the ontogeny of eTreg cells have not yet been fully described. Additionally, molecular determinants of the eTreg cell program remain incompletely understood. My thesis examines the transcriptional events that regulate the generation of eTreg cells during their thymic development, their homeostasis and response to infection. Using different gene targeted mouse models at steady state and in viral infection models, I studied the intrinsic molecular mechanisms that contribute to eTreg cell differentiation. In particular, I focused on follicular Treg (TFR) cells, which constitute the eTreg cell subset of the germinal centre. The molecular control of eTreg cell fate and function converges on the transcription factors IRF4 and Blimp-1. IRF4 is induced by antigen receptor signals and cooperates with AP-1 factors, BATF and JUN, to regulate transcriptional networks involved in lymphocyte differentiation, function and metabolism. For example, these factors regulate genes important for antibody class switch recombination in B cells and functional differentiation of distinct CD4 T helper (Th) subsets, including Th2, Th9, Th17 and T follicular helper cells. IRF4 expression in Treg cells is critical for effector differentiation, yet the precise mechanisms of how IRF4 regulates the transcriptional program of TFR cells remains unknown. Using a novel transgenic IRF4 reporter mouse we found that IRF4 is highly expressed in TFR cells. Using IRF4 knockout mouse models, we demonstrate that IRF4 is necessary for TFR cell generation in a Treg cell-intrinsic manner. IRF4 controls important aspects of the transcriptional program that drives TFR cell differentiation, including genes essential for Treg cell migration. Furthermore, I identified the transcription factor c-Maf to be essential for TFR cell generation and demonstrated its central role in maintaining a follicular program in Treg cells. Subsequently, using ribonucleic acid (RNA)-sequencing, we generated a “follicular signature” of gene expression from the combined analysis of TFH and TFR cells. Integrated transcriptional analyses showed that in the absence of either IRF4 or c-Maf, the majority of the follicular signature genes were downregulated, indicating that these two transcriptional regulators, aside from Bcl6 are indispensable for follicular TFR cell development. Finally, analyses of IRF4 and c-Maf DNA binding sites, identified by chromatin induced precipitation (ChIP)-sequencing, in combination with open chromatin regions in follicular T cell specific loci, we showed that the precise orchestration of distinct sets of genes is required to promote conserved aspects of the follicular T cell fate. In conclusion, my thesis describes how a key transcriptional network orchestrates fundamental steps in TFR cell differentiation and function, which contributes to the understanding of eTreg cell biology.

The fast development of single cell RNA sequencing (scRNAseq) presents new challenges in data analysis and opportunities for protocol development. To address challenges in data preprocessing, I developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols. Results from scPipe can be used as input for downstream analyses and can be easily incorporated into R-based pipelines with other tools. In order to compare different computational methods for scRNAseq data, I generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create `pseudo cells' from up to five distinct cancer cell lines. Multiple datasets were generated using both droplet and plate-based scRNAseq protocols and processed by scPipe. We found pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps. Finally, I developed single cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, I performed a comprehensive characterization of the full-length isoform landscape in single cells of different types and species and found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. The datasets, protocols and tools that I developed and generated are useful resources for the single cell research community.

Colorectal cancer (CRC) remains one of the leading causes of cancer deaths worldwide. Advances in therapy have resulted in significant gains in survival, particularly in the metastatic setting. While the discovery of biomarkers, such as RAS mutations, have helped refine treatment selection to some degree, more accurate biomarkers are urgently needed. Comparison of existing treatments, as well as the evaluation of the efficacy of new therapies, are informed by randomised controlled trials (RCTs), which form the evidentiary backbone of clinical practice guidelines and represent the gold standard of assessment. Despite their high internal validity, they can lack generalisability due to their highly selective inclusion criteria. Prospective, registry-based, randomised controlled trials (RRCTs) have the potential to bridge the gap between RCTs and real-world clinical practice in oncology. The objective of this thesis is to explore how clinical registries can help to advance biomarker research. This thesis applies real-world data to examine the clinical utility and validity of emerging CRC biomarkers, and explores the feasibility of RRCTs in the oncology setting. In a cohort of 99 metastatic colorectal cancer (mCRC) patients, the role of the epidermal growth factor receptor (EGFR) and its ligands, amphiregulin and epiregulin, as potential prognostic and predictive biomarkers for mCRC patients is explored (Chapter 5). This study examines protein expression by immunohistochemistry and includes patients who were not treated with EGFR inhibitors, representing the largest such cohort reported to date. The real-world validity of biomarker trials is explored in Chapter 6, where the characteristics of patients enrolled in these studies are compared to real-world patients. Using an established multi-centre CRC registry as the reference real-world cohort, clinical data was analysed for participants in three types of biomarker trials (retrospective, prospective observational and prospective interventional). This study provides novel insights into recruitment to, and potential validity of, biomarker trials. Finally, Chapter 7 examines the feasibility of an Australian-first RRCT in oncology. This ongoing study is exploring chemotherapy sequencing in first-line treatment of mCRC and leverages an established multi-centre registry as the data collection platform. This study demonstrates the potential of RRCTs to accelerate progress in optimising patient treatments and outcomes. This thesis demonstrates the power of high-quality clinical registries to facilitate prospective randomised trials, while providing opportunities to investigate and validate biomarkers in real-world settings.

Tumor necrosis factor (TNF) is a master inflammatory cytokine that can, depending on the circumstances, promote survival and proliferation or induce cell death. Anti-TNF drugs have proven strikingly successful in treating inflammatory diseases such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD) but it is still unclear exactly why. For a long time, it was thought that they work solely by preventing TNF induced transcription of other inflammatory cytokines, but more recently it has been proposed that one of their major anti-inflammatory functions is to prevent TNF induced death. Therefore, understanding the mechanism by which TNF induced death is regulated may enable the conceptualization of newer or improved approaches in treating a variety of inflammation-associated pathologies. Binding of TNF to its receptor TNFR1 leads to the formation of two distinct signalling complexes. While most previous studies have focused on the membrane-bound, transcription-activating complex (complex-1), the composition and post-translational modifications of the cytosolic, caspase-8-containing, death-inducing complex (complex-2) remain far less well defined. To analyse TNFR1 complex-2 composition at endogenous levels, we decided to generate FLAG tagged caspase-8 knock-in mouse strains. The reagents for the FLAG tag enable very efficient and specific purification and identification of a FLAG tagged protein and its partners. After some preliminary tests and trials, I decided to use a 3x FLAG tag which has been reported to be 20–200 times more sensitive than other FLAG tags in immunoprecipitation and detection assays. Before generating the mouse strains, in Chapter 3 I performed extensive in vitro comparison of N-terminally or C-terminally 3x FLAG-tagged caspase-8 using a doxycycline (Dox)-inducible stably integrated lentiviral system. The results suggested that when expressed above endogenous levels, the expression and killing activity of caspase-8 was unaffected by a 3x FLAG tag. Interestingly, when expressed at physiological levels, C-terminally 3x FLAG tagged caspase-8 appeared to be equivalent to untagged caspase-8 and marginally more efficient in mediating TNF-induced death and complex-2 formation compared to N-terminally 3x FLAG tagged caspase-8. In addition, I immunoprecipitated TNFR1 complex-2 from cells expressing endogenous levels of 3x FLAG tagged caspase-8 and performed a mass spectrometry (MS) analysis. According to this analysis, Tankyrase-1 (TNKS1/PARP5a/ ARTD5), a member of the poly ADP-ribose polymerase (PARP) superfamily, appears to be a novel interactor of complex-2. Based on our in vitro data, we generated N-terminally or C-terminally 3x FLAG tagged caspase-8 knock-in mice using CRIPSR/Cas9 technology and these mice were characterized in Chapter 4. Homozygous N-terminally or C-terminally 3x FLAG tagged caspase-8 knock-in mice were viable, fertile and developed normally, indicating that N-terminally or C-terminally 3x FLAG tagged caspase-8 were expressed and active in vivo, at least to heterozygous caspase-8 levels. As expected, the expression of N-terminal or C-terminal 3x FLAG tagged caspase-8 was detectable in tissue and cells from knock-in mice by Western blot and immunofluorescence stain using an anti-FLAG M2 antibody. The 3x FLAG tagged caspase-8 displayed similar tissue distribution and comparable expression levels as endogenous caspase-8. The cell death assay suggested that the primary cells and transformed cells from 3x FLAG tagged caspase-8 knock-in mice responded similarly as wild-type cells to apoptotic and necroptotic stimulations. Moreover, by performing anti-FLAG immunoprecipitation, I successfully purified endogenous TNFR1 complex-2 from knock-in mice derived cells. These data indicated that 3x FLAG tagged caspase-8 knock-in mouse strains are useful tools to study caspase-8 and caspase-8-containing protein complexes at physiological levels. In Chapter 5, I characterized tankyrases-mediated poly(ADP-ribosyl)ation (PARsylation) as a novel checkpoint that limits TNF-induced cytotoxicity. Using primary cells from the 3x FLAG tagged caspase-8 knock-in mice described in Chapter 4, I found that the enzyme tankyrase-1 (TNKS1/TNKS/PARP5a/ARTD5), which was identified by mass spectrometry in Chapter 3, is recruited to the endogenous TNFR1 complex-2. Western blot data indicates that tankyrase-2 (TNKS2/PARP5b/ARTD6) may also be recruited. Tankyrases are poly ADP-ribose polymerases and belong to an ancient group of enzymes that post-translationally modify proteins with ADP-ribose. I found that during TNF signalling, complex-2 becomes poly(ADP-ribosyl)ated (PARsylated) in a tankyrases-dependent manner. Furthermore, tankyrases-specific inhibitors sensitized cells to TNF-induced cell death, which correlated with increased levels of complex-2. This suggested that normally tankyrases help limit TNF induced death. Mechanistically, I showed that tankyrases may modulate the stability of complex-2 by recruiting the E3 ubiquitin ligase RNF146, that in turn promotes ubiquitylation and degradation of complex-2. Moreover, inactivation of tankyrases dramatically increased the killing of the clinical Smac-mimetic (SM) birinapant in a primary acute myeloid leukemia (AML) model. Taken together, this thesis describes 3x FLAG tagged caspase-8 knock-in mice as new tools to study caspase-8 and caspase-8-containing protein complexes at physiological levels. Furthermore, this study identifies tankyrases-mediated PARsylation as a novel checkpoint in TNF signalling that expands our understanding of how TNF induced death is regulated and provides a rationale to use tankyrases inhibitors for cancer therapy.

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.

AML (Acute Myeloid Leukemia) is a rapidly progressing cancer of the blood and bone marrow where the accumulation of abnormal myeloid cells crowds out healthy blood cells. Despite the improvements in understanding the biology of AML, these advancements are not reflected in the trajectory of the survival rate. One reason for the poor translation of research results into novel therapies, is the genetic and epigenetic heterogeneity of the disease, which hampers the development of reliable AML models. Mouse models of AML are designed to mimic human disease by expressing known oncogenes in the hematopoietic system of mice. Although the oncogene-specific disease pathology appears to reflect what is observed in human patients, the translation of novel treatments into the clinic has often been unsuccessful. Expression of the hematopoietic transcription factor EVI1 has been identified as a marker for poor prognosis in human AML patients. In this thesis, I aimed to develop a mouse model, that recapitulates a particularly aggressive and treatment-resistant form of AML by overexpressing EVI1 together with the fusion-oncogene MLL-AF9. Although I successfully generated EVI-expressing AMLs, the presence of the transcription factor did not affect disease features such as latency, disease pathology, morphology and immunophenotype of leukemic cells. However, when cultured in vitro, leukemic cells expressing EVI1 were more resistant to Smac mimetic combination treatments and the chemotherapeutic agent Ara-C, therefore reflecting the treatment resistance observed in human patients. Although some targeted treatments are now available, chemotherapy remains the standard of care therapy for AML. Therefore, to improve disease outcome, novel treatments are desperately needed. The IAPs (Inhibitor of Apoptosis Proteins) have been identified as an attractive therapeutic target in a number of cancers including AML. Smac mimetics, a class of drugs that specifically inhibit IAPs, have been found to selectively induce cell death in leukemic cells when combined with an inhibitor of the MAP kinase p38 both in vivo and in vitro. To understand the molecular mechanisms behind this synergistic killing, I studied the phosphoproteome of treated cells using mass spectrometry and revealed that the PI3K/Akt/mTOR survival pathway was activated following Smac mimetic treatment. Upon p38 inhibition, this survival signaling did not occur. Furthermore, CSF1R was identified as a potential regulator of the PI3K/Akt/mTOR pathway. In support of this finding, the combination of a Smac mimetic and CSF1R inhibitor, resulted in synergistic cell killing in vitro. To study proteins, gene tagging provides a valuable tool for a range of applications including real-time monitoring of target protein activities, modifications of proteins and protein-protein interactions. The CRISPR/Cas9 technology is a powerful tool for modifying any DNA of interest and enables the tagging of endogenous genes. However, the targeted insertion of foreign DNA into cell lines is challenging. I aimed to generate endogenously FLAG-tagged proteins in cell lines by targeting components of the TNF pathway using the CRISPR-Cas9 knock-in technology. Although ssDNA containing the FLAG sequence was inserted into the genome of mouse dermal fibroblasts (MDFs) at the correct site, next generation sequencing revealed that this process was both inefficient and error-prone and resulted in the introduction of mutations. Nonetheless, I was able to enrich for tagged RIPK1 following FLAG pull-down, which was detectable via both immunohistochemistry and mass spectrometry.

High-Grade Serous Ovarian Carcinoma (HGSOC) is the most common subtype of ovarian cancer, and is the leading cause of ovarian cancer death. This subtype is molecularly characterised by frequent loss of Homologous Recombination (HR) DNA repair, making it susceptible to Poly-ADP ribose polymerase (PARP) inhibitor treatment. Despite the efficacy of PARP inhibitors (PARPi) in treating HGSOC, disease recurrence is common. A poor understanding of PARPi toxicity and resistance mechanisms has limited improvements in overall survival of patients. This project utilised HGSOC Patient-Derived Xenograft (PDX) and in vitro models to determine underlying mechanisms of PARPi sensitivity and resistance. Characterisation of the HGSOC PDX cohort led to the discovery of 2 models with HR gene secondary mutations, thereby explaining the poor PARPi responses of these tumours. Loss of the Classical Non-Homologous End-Joining (C-NHEJ) DNA repair pathway was also investigated but was not supported as a mechanism of resistance in PDX, or in a genome-wide PARPi-resistance CRISPR screen using a BRCA2-mutant cell line. The CRISPR screen revealed PARP1 mutations as the top hit, but no C-NHEJ mutation hits. CRISPR screens have also been optimised for a BRCA1-mutant cell line and a unique BRCA1 methylated cell line, to enable future validation in different HRD contexts. HR gene methylation analysis in the HGSOC PDX models revealed distinct BRCA1 promoter methylation profiles, subsequently found to represent “homozygous” and “heterozygous” methylation states. Our group demonstrated that all copies of BRCA1 must be methylated for gene silencing and PARPi response, and that methylation can be lost under treatment pressure in patients. I then investigated this mechanism in two HGSOC PDX models with RAD51C promoter methylation. RAD51C promoter methylation was found to have homogeneous or heterogeneous patterns, and both caused gene silencing and PARPi response in the PDX, that could be lost under treatment pressure. One third of patient samples tested appear to have a heterogeneous RAD51C methylation profile and, like the PDX, some responded to chemotherapy. To assess whether global methylation profiles influenced HR gene methylation stability, I carried out genome-wide methylation array analysis of PDX models and clinical samples. Analysis of cyclically PARPi re-treated RAD51C methylated tumours from one PDX model demonstrated increasing global losses in methylation with each treatment cycle. Through characterisation of the HGSOC PDX cohort and in vitro CRISPR screens, I have uncovered both well-established mechanisms of resistance (e.g. secondary mutations), and less studied mechanisms of sensitivity and resistance, such as loss of RAD51C methylation and PARP1 loss. These models provide a platform for further study of these PARPi resistance mechanisms, including potential therapeutics to prevent or overcome their development in the clinic.

Necroptosis is a form of inflammatory programmed cell death. Thought to have evolved to combat pathogen infection, necroptosis can be triggered via signalling through pattern recognition receptors or cytokines from the TNF family. The final stages of necroptosis signalling are characterised by the kinase RIPK3 oligomerising with various RHIM domain containing proteins, which may assemble together into large functional amyloid complexes. Within these complexes RIPK3 is thought to auto-phosphorylate and then recruit and phosphorylate the final known effector of the pathway Mixed Lineage Kinase domain-Like protein (MLKL). Once the pseudokinase MLKL is phosphorylated, it transforms from an inert cytoplasmic monomer, to an oligomeric membrane associated killer. The means by which phosphorylation of MLKL’s pseudokinase domain by RIPK3 acts as a switch to activate the protein is unknown, as is the stoichiometry of the oligomer. Once MLKL reaches the plasma membrane, it disrupts the membrane by an unknown mechanism and this is thought to be the final blow in cell death. The study of MLKL as the final effector protein of necroptosis has also been hampered by differences between the mouse and human protein, the details of which are only emerging. This thesis details three complementary studies each aimed to elucidate the molecular details of a particular step in MLKL’s mechanism of action. Chapter 3 examines the compatibility between MLKL orthologues from different species. Through functional and structural characterisation of MLKL orthologues stringent requirements for RIPK3 compatibility were found, as well as new evidence for the molecular switch mechanism. Chapter 4 describes the identification of specific residues of the MLKL second brace helix that are essential for the formation of MLKL oligomers, and that the second brace helix is essential for communicating regulatory signals through the protein. Chapter 5 has the first study of MLKL associating with Giant Unilamellar Vesicles using confocal microscopy, and the first exploration of MLKL’s effect on membranes through neutron reflectometry. Together the separate studies constitute an in-depth exploration of MLKL’s entire mechanism of action, and a comprehensive examination of the differences between mouse and human MLKL.

Primary Immunodeficiencies (PIDs) are a heterogeneous collection of several hundred disorders, that have in common deficient or dysregulated immunity. This leads to an increased susceptibility to infections, autoimmune disease, or to uncontrolled inflammation; in some PIDs there may be features of all three components. Understanding the pathogenesis of PIDs has led to insights into the immune system broadly, and this knowledge has aided improvements in the treatment of many more common infective and inflammatory diseases. Many PIDs are monogenic, and advances in technologies that enable the interrogation of the genetic basis of these conditions, have led to a steep increase in the number of diseases now recognized as PIDs. In this project we aimed to establish a cohort of individuals with the most common form of PID, Predominantly Antibody Deficiency (PAD). We sought to characterize the diagnostic and clinical features of these individuals, and apply genomic sequencing methods for more precise diagnoses, as well as to identify new genetic etiologies of PAD. One of the most striking findings regarding the clinical features of the patient cohort, was the morbidity associated with PADs. In particular we observed very frequent complications of immune dysregulation, that manifest as autoimmune disease, particularly affecting the haematological system as cytopenias, or gastrointestinal tract as enteropathy, or malignancies. These complications are challenging to manage in the setting of PID, hence understanding the mechanism of disease is crucial to improving outcomes. In a group of PAD patients we identified monogenic causes of disease, that led to some individuals receiving precision treatments. In the realm of gene discovery, we identified several novel genetic variants, two in genes that had not previously been recognized as disease-causing in PAD. The first, NFKB1, is now recognized as the most common genetic etiology of PAD. The importance of NF-kappaB signaling and its regulation was further highlighted by the discovery of other disease-causing variants in the NF-kappaB pathway. The effect of NFKB2 mutations on the immune response was investigated in a kindred with two sisters affected by PAD, who demonstrated disparate clinical and immunological features. Further studies were performed in a mouse model of NF-kappaB2 deficiency, whereby B cell and CD8+ T cell behaviours were studied in a quantitative manner, and revealed the cell-type specific effect of loss of this component on the adaptive immune response. Finally, an entirely new monogenic PID was identified. In a young man with a clinical diagnosis of PAD, complicated by autoimmune disease, a homozygous, frameshift mutation in NFKBID was identified. The mutation was demonstrated to cause loss of expression of IkappaBNS, an inhibitor of the NF-kappaB pathway, and with that, dysregulated NF-kappaB signaling. The finding of a second patient with PAD and compound heterozygous variants in NFKBID has added further evidence that this is a bona-fide novel PID.

Severe Plasmodium falciparum malaria has been attributed to cytoadhesion and sequestration of infected erythrocytes (IEs) to microvascular endothelium, and rosetting of IEs with other non-infected erythrocytes. These mechanisms are mediated by the interaction between variant surface antigens (VSAs) on the IEs and host receptors on the endothelial cells and erythrocytes. There are at least three VSAs important for this process, which are P. falciparum Erythrocyte Membrane Protein-1 (PfEMP-1), Repeat Interspersed Family protein (RIFIN), and Subtelomeric Variant Open Reading Frame (STEVOR). This study aimed to investigate whether the polymorphisms in the host genome that encode VSA receptors may influence the pathogenesis and mediate protection against severe malaria. Secondly, as PfEMP1, RIFIN, and STEVOR have been shown to mediate rosetting, the potential co-expression among these three VSAs were investigated by comparing their expression on the erythrocytes infected by rosetting and non-rosetting P. falciparum parasites in two different host environments based on ABO blood types (A+ vs O+ erythrocytes). Thirdly, the development of antibody against rosetting-mediating VSAs were screened in longitudinal cohort children from Papua New Guinea, and determined whether they were associated with protection against severe malaria. For the host genetic aspects, this study focused on investigating the role of the polymorphisms on the Endothelial Protein C Receptor (EPCR) encoding PROCR gene and the ABO blood types, which were shown to be associated with severe malaria, in determining the protection against severe malaria in a cohort of very young children from Papua New Guinea (PNG). In children with the PROCR rs867186 polymorphism, there was a risk-reduction trend for severe malaria incidence but it was not significant which was likely due to small number severe malaria cases (n=24). However, the ABO blood group was not found to be associated with protection against severe and clinical malaria. In addition, the significantly higher levels of antibodies to rosetting-associated than EPCR binding PfEMP-1-CIDR domains in children carrying the PROCR polymorphism suggested the preference towards parasites expressing non-EPCR binding VSAs, such as rosetting mediating VSAs. The in vitro cultures for rosetting parasites in A+ and O+ erythrocytes showed distinct patterns of upregulated genes where more VSAs consisting mainly PfEMP-1 and RIFIN were seen in A+ than O+ rosetting parasites, and consequently a higher rosetting rate in the former. This is consistent with previous studies showing that individuals with O blood type tended to have low rosetting rate and were protected from severe malaria. The antibody detection using the sera from the PNG cohort children against rosetting associated RIFIN and STEVOR identified in this study showed a consistent pattern indicating the role of ABO blood group as well as the PROCR polymorphism in determining the selection of VSA subtypes. In conclusion, this study is the first to link host genetic polymorphisms with differential exposure to malaria antigens and highlights the importance of considering the diverse environment in which natural infections occur. This study has provided a better insight into the complex host-parasite interactions during P. falciparum pathogenesis, which is crucial to form a basis to further develop the most effective approach to interrupt this process. Future studies are mainly directed to validate the findings using a study population with more severe disease cases and higher level of immunity, as well as replicating the in vitro rosetting study in different parasite isolates to confirm whether VSA expression is conserved across different P. falciparum rosetting parasite strains.

Sexual stage development in Plasmodium spp. is essential for transmission through the mosquito and to the human host. It represents objects to study a broad range of biological processes, including stage conversion and parasite/host co-adaptation. After the bloodmeal, male and female gametes emerge from intracellular gametocytes and zygote formation follows fertilization. Ookinetes develop from the zygote and traverse through the midgut epithelial cell layer to the basal lamina side of outer wall and develop into oocysts, the only parasite developmental stage that grows extracellularly and this growth and development creates thousands of sporozoites. Once fully developed and egressed, these sporozoites are released into the mosquito hemocoel and they migrate to the salivary gland ready to infect next mammalian host and continue their life cycle. This sexual stage also represents a major bottleneck during the life cycle of Plasmodium as, in mosquito midgut, parasites have to persevere for up to 24 hours outside host cell, exposed themselves to various risk factors such as components of human immune system included within bloodmeal, natural midgut microbial flora in mosquito midgut, and mosquito innate immune system. This exposure can lead up to an approximate 300-fold decrease in parasite survivability during the transmission to mosquito. Due to this unique feature, sexual stage is prime target for transmission blocking intervention strategies aimed to inhibit spread of the disease by the mosquito. Protease enzymes are essential during many steps of malaria parasite development in the blood and transmission stages and an important group of these enzymes are the plasmepsins, of which there are 10 in Plasmodium acting at various points through the life cycle. So far, only 4 plasmepsins are identified to be involved in critical processes and required for transmission. Firstly, plasmepsin VI is highly expressed during sexual stages and was previously shown to be involved in sporozoite development in P. berghei. Secondly, plasmepsin VIII is expressed in mature sporozoite and responsible for sporozoite motility in P. berghei. Finally, PMIX and X are found to be essential in both blood and mosquito stages, making them stand out as promising drug targets. In this study, we attempted to determine the biological functions of plasmepsin VI, IX, and X during transmission of malaria parasites. We found that plasmepsin VI is required for transmission of P. falciparum and might plays an important role in sporozoite egress process instead of sporozoite development as observed in P. berghei. We also found that our dual inhibitor that target both plasmepsin IX and X is able to block the transmission of P. falciparum to mosquito while another antimalaria compound that target only plasmepsin X is enough to block transmission of P. berghei from mouse to mosquito suggesting that both plasmepsin IX and X are essential for transmission. Taken together, our data has identified 3 plasmepsins that play important roles in sexual stage of malaria parasites and more works are needed in order to determine the mechanism of action of these 3 proteases.

Acute myeloid leukaemia (AML) is an aggressive disease with a current 5-year survival rate of only ~30%, therefore development of more effective treatments is urgently needed. Overexpression of inhibitor of apoptosis (IAP) proteins, responsible for the regulation of tumour necrosis factor (TNF)-mediated apoptosis, have been correlated with cancer progression and treatment resistance. Natural IAP antagonists exist, termed second mitochondria-derived activator of caspases or Smac, leading to the pharmaceutical development of Smac-mimetics. Smac-mimetics, such as the drug birinapant, are currently in clinical trials for a range of cancers, including AML. Although, birinapant has shown promising anti-cancer effects, its efficacy as a single-agent and in combination with numerous anti-cancer therapies, has been variable and limited. Therefore, overcoming patient resistance to Smac-mimetic therapy and potentiating treatment are still major challenges. Using a library of more than 5,700 bioactive compounds, we examined the emergence of resistance to birinapant in AML. Here I present the identification of multidrug resistance protein 1 (MDR1/ABCB1) inhibitors as a class of clinical drugs that can overcome Smac-mimetic resistance in cancer. Inhibition of MDR1 with 3rd generation specific inhibitors increased intracellular levels of birinapant and sensitised AML and ovarian cancer patient cells to birinapant killing. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, I show that MDR1 is a predictor of response to birinapant-based therapies in cancer cells. Moreover, the combined therapy of MDR1 inhibitors and birinapant effectively killed leukaemic stem cells (LSCs) and prolonged survival of AML burdened mice in vivo (Chapter 3). The findings observed in Chapter 3 in AML and ovarian cancer cells, were extended to a chronic hepatitis B virus (HBV) infection model (Chapter 4). Birinapant has previously shown efficacy at eradicating HBV infection, however due to toxicity concerns, in-human clinical trials have ceased. In Chapter 4, I demonstrate that a lower concentration of birinapant can be combined with the MDR1 inhibitor zosuquidar, to potentiate birinapant-mediated HBV infected liver cell death and HBV-DNA clearance in vivo. An investigation into the remaining high throughput screen hit compounds, revealed half were psychotropic and inhibited MDR1 to synergise with birinapant. In Chapter 5, I focus on the serotonin receptor 7 (5-HT7) agonists, LP-44 and LP-12, as well as the psychoactive compounds Fluphenazine dihydrochloride, SDZ 21009, GR-127935 and CGP-71683. My identification that these compounds inhibit MDR1 to synergise with birinapant underlines the dependence cancer cells have on MDR1 to evade Smac-mimetic therapy. This thesis presents a comprehensive analysis of birinapant as a substrate for MDR1 and identifies MDR1 as a biomarker of Smac-mimetic therapy. Furthermore, this study provides evidence towards a novel combination regimen combining birinapant with 3rd generation MDR1 inhibitors to potentiate Smac-mimetic therapy for the treatment of AML, ovarian cancer and HBV. Altogether these findings suggest a therapeutic opportunity for Smac-mimetic plus MDR1 inhibitor combination therapy in cancer and infectious disease.

Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.

The production of antibodies, with their potential to recognise unique targets and prevent repeat infections, is an important aspect of immune health. In order to generate free antibodies, the cells responsible, B cells, must undergo a differentiation step to transform from lymphoblast to antibody secreting cell (ASC). This differentiation step prevents further antibody modifications and hence the timing for optimal immunity requires a delicate balance between expanding useful clones and providing early protection. How differentiation is controlled to achieve this balance for an effective immune response is of great interest. In this study, the progression from naive B cells to ASC was investigated in the context of an emerging model for competing cell fates. By this model, alternate cell fates, such as division, death and differentiation, are pursued independently in individual cells but are in competition such that events which occur earlier prevent those that require more time from being observed. Evaluation and testing of this model requires careful measurement of distributions of times to fates which is only possible with single cell fate tracking. Here I have developed and applied methods for live cell imaging and analysis for assessing and evaluating cell fate changes over time. Using these methods, several modes of regulating differentiation times were revealed. Low levels of stimulation through CD40 produced a greater proportion of antibody secreting cells per generation as division is slowed and more time is allowed for differentiation, consistent with competing cell fates. A second mechanism was found where increasing division numbers directly reduced the amount of time required for cells to differentiate, without modulating division times, ensuring the natural development of ASC during the ongoing immune response. A direct method of uncensoring was explored where cell cycle inhibitors were used to prevent division, with the hypothesis that more cells would go on to differentiate in the absence of competition. Various inhibitors were assessed for their suitability to this task, and a panel of compounds were found to be suitable for uncensoring underlying differentiation and cell death times. Findings from this study are consistent with the model of independent and competing cell fates, and significantly advance our understanding of how antibody responses are controlled and can be modelled at the cell population level.

Gastrointestinal parasitic infections, in particular soil-transmitted helminths (STHs), are a major cause of morbidity in tropical developing countries worldwide. Globally, infections are estimated to cause disease-related morbidity of approximately 3.4 million disability adjusted life years and an infection prevalence of around 1.5 billion people. More than 3.5 million people manifest acute clinical symptoms, although the majority of infections represent asymptomatic cases. Severe infections cause acute clinical symptoms including malnutrition, abdominal pain, diarrhoea, fever, skin conditions and iron deficiency anemia. Most importantly, long-term sequelae in pre-school and school-aged children and pregnant women can lead to stunting, wasting and an impaired cognitive development. Inadequate sanitation, impoverished conditions, poor public health systems and population overcrowding all contribute to high infection prevalence levels. STH infections occur predominantly in sub-tropical to tropical regions worldwide, such as South America, Sub-Saharan Africa and South-East Asia. Diagnosis is confirmed by microscopy-based tools (Kato Katz Thick Smear method), which is both, cost-effective and applicable in remote settings, but has significant drawbacks in terms of labour intensity and diagnostic accuracy. The World Health Organization recommends annual or biannual mass drug administration of the front-line benzimidazole drugs, albendazole and mebendazole, to reduce overall infection prevalence and disease-related morbidity. However, there is an increasing concern that ongoing mass drug administration may lead to the emergence of drug resistance. There is an urgent need for accurate and streamlined diagnosis of STH infections, in particular among subpopulations harbouring low-intensity infections in order to effectively treat populations at risk. Additionally, an improved evaluation of drug treatment efficacy including a subsequent monitoring of potentially emerging drug resistance is crucial for future studies. A global multi-factorial intervention approach is required to effectively move towards transmission interruption and ultimately eradication of STH infections. The here presented thesis has resulted in the successful development of a molecular diagnostic tool for qualitative and quantitative estimation of STH infections. A novel, semi-automated qPCR-based tool (multiplexed-tandem qPCR (MT-PCR)) was developed and validated using two cross-sectional cohorts from South-East Asia (Timor-Leste, Cambodia). The MT-PCR subsequently was used to investigate the infection prevalence and intensity in a cross-sectional cohort study of Karen ethnic pre-school and school-aged children from the Tha Song Yang district, Tak province, in Thailand. Furthermore, the effectiveness of three different preservation methods for faecal samples was compared in terms of soil-transmitted helminth infection recovery and gut microbiome characterisation. The impacts of infection on overall and gut microbial health were investigated. Thirdly, faecal DNA samples from three cohorts (Cambodia, Vietnam, Thailand) were screened for single nucleotide polymorphisms at codons Phe167Tyr, Glu198Ala and Phe200Tyr, that may be associated with the emergence of benzimidazole drug resistance. Using this amplicon sequencing approach, the variant allele frequency of the beta tubulin isotype 1 gene was analysed.

The mammary gland is a fascinating organ that develops after birth and is capable of remodelling through multiple rounds of reproduction. The behaviour of mammary epithelial cells and how these interact dynamically with their environment are poorly understood. Cell morphology and arrangement can be addressed by three-dimensional (3D) confocal imaging to provide large-scale, subcellular resolution views of tissue architecture. Further insight can be gained from intravital imaging that allows direct observation of cell behaviour in vivo, but this has rarely been implemented for the normal mammary gland. Mammary ducts are embedded in adipose tissue, making in vivo imaging of mammary ducts extremely challenging. Chapter 3 provides a detailed protocol for an intravital imaging method that was adapted and optimised for the mouse mammary gland. This technique enables high-resolution, 3D intravital imaging of the mammary gland for up to twelve hours. The skin flap surgical technique was modified to expose the entire inguinal mammary gland, allowing rare accessible epithelial structures to be identified. Additional fine microdissection of connective tissue maximised the resolution of imaging. Significant measures were taken to achieve as near to physiological conditions as possible, including creating a sealed environment over the exposed tissue. Strategies used for image analysis are then discussed, including image stabilisation, cell tracking and 3D visualisation. This technique advances our ability to observe mammary cell behaviour in vivo and will enable future investigation of rare events that are spatially and temporally regulated, such as stem cell behaviour, tumour initiation and microenvironment interactions. Mammary gland morphogenesis occurs by migration of terminal end buds through the mammary fat pad. Terminal end buds are large, club-like structures comprising a cap layer and a multi-layered body that give rise to bilayered ducts. Epithelial progenitors within terminal end buds generate mature cells of ducts but how these behave and cooperate to generate the bilayer is not well understood. Chapter 4 describes the lineage-specific behaviours of terminal end bud progenitors as observed by intravital microscopy. Cap cell migration into the body was recorded at high resolution in vivo for the first time. High-dimensional image quantification of cap cell behaviour showed that most cap cells that migrate into the body die rapidly but a small proportion survive long term. Progenitors for the luminal lineages were observed to have contrasting behaviours, with hormone-sensing progenitors being highly migratory. Single cell transcriptomic analysis of terminal end buds is described, providing possible molecular drivers of the distinctive behaviour of hormone-sensing progenitors. This work provides an unprecedented view of mammary stem cell behaviour, making an important contribution to our understanding of how cellular behaviour drives organogenesis. Chapter 5 describes a previously uncharacterised population of resident intra-epithelial macrophages that were revealed by 3D confocal imaging. These cells, termed mammary ductal macrophages, are regularly positioned over the entire mammary gland at all stages of development. They do not migrate but monitor the epithelium by dendrite movement, allowing them to rapidly sense and respond to epithelial damage. Ductal macrophages proliferate in pregnancy to maintain their density on the epithelium in lactation. During involution following weaning, they rapidly phagocytose dying alveolar cells to facilitate remodelling. Breast tumour-associated macrophages are pro-tumorigenic and strongly resemble ductal macrophages, not stromal macrophages. Macrophages are emerging as important targets for breast cancer treatment, therefore, better understanding of parallels between DM function in healthy and perturbed tissue may enable development of improved cancer therapies. Finally, in Chapter 6, the presented results are summarised and their context within the field, wider implications and possible future directions are discussed. Overall, this thesis presents original research that advances our technical ability to address questions of cell dynamics in the mammary gland, provides important insights into mammary stem cell behaviour during morphogenesis, and characterises a novel tissue-resident macrophage population, finding a key role for these in mammary gland remodelling.

Colorectal cancer (CRC) is the third most common cancer worldwide, and one of the most common cancers in Australia. When detected early, there are multiple treatment options for CRC; however, patients often relapse and ultimately succumb to metastatic disease. 5-Fluorouracil (5-FU)-based chemotherapy is a common first-line treatment for CRC. However, chemotherapy response rates remain low, often due to the development of resistance, which is one of the main limitations in the management of the disease. Our understanding of the progression of CRC, and the development of new therapeutics for CRC, has been facilitated in part by animal models. Unfortunately, many murine models of CRC are adenomas, with few patient-derived models, or models of metastatic disease available. As a result, the opportunity to improve our understanding of the pathogenesis of advanced disease, or test the efficacy of novel therapeutics for advanced disease, is limited. The overarching aim of this PhD thesis was to generate new models of CRC to facilitate the study of this devastating disease. A biobank of 16 CRC patient-derived xenografts (PDXs) was successfully established in immunocompromised mice. These PDX lines recapitulated the histopathological, molecular and genetic features of the original patient tumours. These PDXs represent new pre-clinical tool that will allow for testing the efficacy of potential new therapeutics. A selection of four PDX lines underwent serial 5-FU treatment to generate a library of resistant PDXs, and their matched non-resistant chemonaive controls. The 5-FU resistant PDX tumours underwent an upregulation of the IL-11R/STAT3/Bcl-2 pathway in response to 5-FU. This suggests that IL-11 signalling is elevated in response to 5-FU to promote tumour cell survival. Thus, targeting the IL-11/IL-11R signalling may be a promising strategy to overcome chemoresistance. Finally, four different genetically-engineered mouse models (GEMMs) were established to generate a reproducible model of metastatic CRC. It was found that mutations in Apc (or dysregulation of Wnt signalling) restricted to the colonic epithelium lead to the formation of adenomas, as did the addition of mutations in Tp53. The combination of Apc mutations with Tp53 and Kras mutations lead to an earlier tumour onset, but did not result in metastasis, contrary to previous reports in the literature. It was found however, that mutations in Tp53 and Kras in the stem cell compartment, combined with dysregulation of Wnt signalling, lead to potential metastasis to the liver. However, this did not occur in 100% of the animals, and is thus not amenable to therapeutic studies. Future studies will incorporate alterations to TGFb signalling, in an effort to increase the reproducibility of metastasis. These studies highlight the lack of our understanding of the drivers required for tumour cell metastatic potential. Taken together, the research described in this thesis has led to the generation of a number of new animal models of CRC, that may be of use to future studies of the pathogenesis and treatment of this disease.

Necroptosis is one type of caspase 8-independent programmed cell death. Mixed lineage kinase domain-like (MLKL) is the essential terminal effector in the necroptosis signalling pathway. Once receptor interacting serine/threonine protein kinase 3 (RIPK3) is activated by upstream cell death signals, it phosphorylates MLKL and triggers the oligomerization and membrane translocation required for MLKL induced membrane disruption. This leads to death and removal of infected or damaged cells. In 2015, the ubiquitylation of MLKL was reported in the context of lipopolysaccharide-induced necroptosis in bone marrow derived macrophages (BMDMs). Ubiquitin modification of MLKL has not been reported in any peer reviewed publication since then. Though MLKL-ubiquitylation was shown to be RIPK3-dependent, how it is regulated in the general context of necroptosis signalling is unclear. In Chapter 3, I present evidence that this distinctive ubiquitylation can be induced by a range of necroptotic stimuli, occurs on both mouse and human MLKL, and relies on activation of MLKL by upstream components of the necroptosis pathway. The correlation between MLKL ubiquitylation and oligomerization supports my hypothesis that MLKL oligomerization drives its ubiquitylation occurring on biological membranes. In Chapter 4, I investigate the role of MLKL-ubiquitylation related to cell death induction. MLKL species that undergo ubiquitylation but do not induce cell death demonstrate that ubiquitylated MLKL is not sufficient to drive plasma membrane disruption per se. A novel method of fusing MLKL to the pan-DUB (deubiquitylating enzyme) USP21 was developed and we found that when the ubiquitylation is constitutively removed, MLKL can still induce necroptosis. Since proteasome/lysosome inhibitor attenuates the turnover of ubiquitylated MLKL, and phosphorylated MLKL-USP21 accumulates in cells, we propose that the mechanism of corelated MLKL ubiquitylation and turnover exists to delay or to prevent necroptotic cell death below a certain threshold of MLKL activation. Understanding the ubiquitylation linkage type, the modified lysine residues and the potential E3 ligase can provide us with important clues to explore how MLKL ubiquitylation is formed and how it mechanically influences necroptosis. In Chapter 5, I describe evidence demonstrating that MLKL is short-chain ubiquitylated at multiple sites that are at least presented on its N-terminal 4HB domain. A method of enriching modified MLKL species from the crude membrane fraction via immunoprecipitation with M2-FLAG sepharose beads was developed, and we identified four modified lysines on MLKL, marking sites of ubiquitylation. Despite promising leads, the identity of any single obligate E3 ligase remains to be revealed. This investigation of oligomerization-driven MLKL ubiquitylation provides the cell death field with a new angle to study the regulation of necroptosis.

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.

Breast cancer is a heterogenous disease that can be stratified into at least six subtypes based on gene expression profiling. Each of these subtypes likely arise from a different cell of origin, through a repertoire of genetic aberrations that influences treatment decisions and subsequent resistance to therapy. Multiple mechanisms of resistance exist, including evasion of apoptotic cell death, which is a hallmark of cancer. The development of BH3 mimetics, which antagonise pro-survival proteins of the BCL2 family, is a new field of targeted cancer therapy. Initial studies observed that BCL2 is overexpressed in the majority of primary and metastatic estrogen receptor positive (ER+) breast cancer, and targeting BCL2 with ABT-199 (venetoclax), a potent BCL2 inhibitor, synergises with endocrine therapy in preclinical models of ER+ breast cancer. This thesis builds on these insights into the molecular events that are responsible for resistance to cell death. ER+ breast cancers also frequently exhibit deregulation of the retinoblastoma (Rb) pathway that includes cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/retinoblastoma (Rb), resulting in uncontrolled cellular proliferation. CDK4/6 inhibitors significantly augment tumor response when combined with endocrine therapy in ER+ breast cancer. Despite their potent antiproliferative activity, CDK4/6 inhibitors fail to induce apoptotic cell death. This thesis investigates the addition of the BH3 mimetic ABT-199 to endocrine therapy (fulvestrant) and the CDK4/6 inhibitor palbociclib in breast tumor organoids and patient-derived xenograft models of ER+ breast cancer. Triple therapy, which was well tolerated in vivo, produced a superior and more durable tumor response compared to single or doublet therapy. Increased apoptosis was observed in vitro and in vivo, with improved survival in PDX models. Notably, MHC class I and PDL1 expression increased in a syngeneic mouse model of ER+ breast cancer, accompanied by a reduction in regulatory T cells. Together, these findings provide a rationale for investigation of combination therapy in the clinic. The targeting of another pro-survival protein, MCL1, has been an intense area of interest as it is frequently dysregulated in cancer. In breast cancer, MCL1 is often amplified, particularly in triple negative and HER2-amplified subtypes, where high expression predicts resistance to treatment and poor outcomes. This thesis shows that a novel inhibitor of MCL1, S63845, demonstrated marked synergistic activity in preclinical models of breast cancer, supporting a rationale for clinical evaluation of MCL1 inhibitors in breast cancer. This work also explores how the accumulation of genetic hits to a key cell of origin influences the resulting breast cancer subtype. The precise initiating events that predispose the sequence from normal epithelium to neoplastic progression is poorly understood. Breast organoids were generated from human reduction mammoplasties, creating a tool to study the clonal evolution of breast cancer. CRISPR/Cas9 gene editing was used for targeted knockdown of four of the most commonly mutated tumour suppressor genes (P53, PTEN, RB1 and NF1) in cultured mammary progenitor cells. Mutated organoids gained long-term culturing capacity and formed tumours of luminal histology when transplanted into mice. Organoids were demonstrated to be responsive to endocrine therapy or cytotoxic chemotherapy, supporting the potential utility of this model to enhance our understanding of the cellular origins and genetic lesions underlying breast cancer.

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.

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.

Acute myeloid leukaemia (AML) is a cancer of the blood affecting the normal development of white blood cells. AML is a heterogeneous disease, with many distinct subtypes defined by a long list of morphological and molecular markers. These markers provide important information as the patient’s prognosis varies dramatically between different subtypes. AML patients harbour a low number of mutations compared to other cancers while the expression of thousands of genes is often dysregulated. This makes RNA sequencing (RNA-Seq) an appealing instrument to track both genetic and transcriptional determinants of response in leukaemia. This thesis tackles relapse in AML by studying a cohort of bulk RNA-Seq samples extracted from Core-Binding Factor (CBF) AML, where patients generally respond well to their initial therapy but the incidence of relapse is above 40\% over five years. Data was collected as part of an Australian clinical trial and serial samples were available to study disease progression. As detecting mutations from RNA is not commonly performed, this thesis describes bioinformatics workflows to detect mutations from related samples in RNA and to track them over time. The workflows were defined as the result of a benchmarking effort using validated mutations from public RNA-Seq data from AML, as well as assessing the sensitivity at different library sizes. This investigation was instrumental in defining a cost-efficient sequencing design for our analysis of the CBF-AML cohort and will be a useful reference for future work in this area. This thesis also introduces new strategies and software to integrate mutation calls from existing bioinformatics software and to aid visualisation of the results. These include new software developed with the statistical software R, to standardise and combine variant calling outputs as well as static and interactive plots to explore changes in serial samples over time. A great challenge in analysing bulk cancer RNA is to estimate the signal of interest while taking into account the large biological heterogeneity induced by tissue composition. This thesis tackles the problem by adopting a statistical methodology to estimate and adjust for the unwanted variability in the data exploiting the gene expression data itself. We found that our results from RNA-seq profiling had large consistency with the published literature on CBF-AML, including the definition of gene expression signatures, gene fusions and recurrently mutated pathways. In addition, this thesis offers a genome-wide perspective of the molecular changes occurring in CBF-AML as the disease progresses, highlighting genomic lesions not routinely screened in clinical diagnosis. This thesis demonstrates the strength and limitations of using only RNA-Seq to study cancer genomes. The methodological approaches developed in this thesis should serve as a way that other researchers can improve the detection, tracking and interpretation of molecular alterations in cancer transcriptomes.

Cytosolic sensor proteins like NLRP1 (NOD-like receptor containing a pyrin domain 1) play a fundamental role in mediating innate immunity. Upon activation they form signalling hubs that recruit the adaptor protein ASC (apoptosisassociated speck-like protein containing a CARD) and procaspase-1 to form an inflammasome. Procaspase-1 is in turn activated and processes the cytokines pro-IL-1b and pro-IL-18 as well as the pore-forming protein GSDMD (Gasdermin D) into their mature forms, resulting in inflammation and pyroptosis. When dysregulated, inflammasomes are often involved in the development of autoinflammatory diseases. Therefore, it is of major interest to understand the molecular mechanisms underlying the regulation of inflammasome sensors. A biochemical approach was taken to investigate the structural basis of inflammasome formation. Producing and characterizing recombinant protein of separate domains of NLRP1 demonstrated that NLRP1 autoinhibition is not mediated by direct intramolecular interaction of the N-terminal PYD with other domains. Additionally, the full-length NLRP1 protein was characterized by biochemical and structural means. Size exclusion chromatography indicated that recombinant NLRP1 forms oligomers in solution. Small-angle X-ray scattering confirmed this observation and further allowed the calculation of a molecular envelope of the NLRP1 oligomer. The oligomeric state of the protein was estimated to be hexameric, based on the particle volume derived from the molecular envelope. Furthermore, a highly sensitive reversed-phase HPLC assay was employed to measure the ATP hydrolysis activity of recombinant full-length NLRP1. In contrast to previous reports, we found that NLRP1 hydrolyses ATP at a low rate. The physiological relevance of this activity was investigated by taking a mutational approach in functional assays in cells measuring inflammasome activation. Substitution of residues identified by computational analysis of the nucleotide binding site suggested that ATP hydrolysis is involved in maintaining NLRP1 in an autoinhibited state. A similar approach was taken to investigate the involvement of direct modifications of the NLRP1 protein in regulating inflammasome activity. Functional effects of a single nucleotide polymorphism, which leads to the amino acid substitution M1184V in the NLRP1 protein and is described to increase autoproteolysis in the NLRP1 FIIND domain, were investigated. The results showed that increased cleavage can amplify or inhibited activation of NLRP1 in the context of different stimuli. Moreover, a potential phosphorylation within the CARD domain was identified as another essential modification regulating the activity of NLRP1. Overall, this work provides new insights into the role of structural mechanisms, ATP as a cofactor and posttranslational modifications in regulating NLRP1 inflammasome activity.

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.