Phosphorylation is a reversible post-translational modification which participates in nearly all human signalling events. The role of phosphorylation in biology has been studied for more than 70 years, yet only a tiny fraction of substrates and their corresponding function have been characterised. Over the last 20 years, phosphoproteomics has developed and now established itself as the primary tool to investigate biological signalling events. Phosphoproteomics enables the global unbiased quantitative detection of more than 10,000 phosphorylated sites on a routine basis. However, despite this sensitivity, phosphoproteomic experiments have primarily been performed in immortalized cell lines and tissue samples containing multiple cell types. Moreover, complex biological information is often distilled and collated into single pathways that attempt to capture general signalling cascades. These generalizations through simplified pathway analysis risks losing valuable insights into biological system and does not make full use of the exquisite sensitivity that phosphoproteomic workflows provide. Additionally, cellular responses to ligands can be radically different between different cell types and cellular states, thus, the generalization of pathways combined with most experiments being performed in immortalized cell lines significantly reduces the power of phosphorylation analysis in human systems. The use of cell lines and mixed tissues has been necessary due to the large protein input requirements in phosphoproteomic experiments. Historically, the protein input requirements have been more than 1mg of protein. However, recent developments decreased the input amount required to 0.2mg of protein per sample. Despite this large improvement, the protein input is still highly prohibitive for most primary cell experiments from mice and men. Primary cells are derived from limited sources and expansion ex vivo is highly limited for most primary cell populations. Based on the need for primary cell compatible phosphoproteomic methods, I present the development of an ultrasensitive phosphoproteomic method which is 4-fold more sensitive than current published methods. Moreover, I present an experimentally derived framework to assist in the design of limited input phosphoproteomic experiments. The framework assists in the selection of timepoints, through comprehensive mapping of TNF induced signalling. Furthermore, the choice of protein input is recommended through an investigation of stimulation induced signalling at different inputs. The experiment is based off three donors and investigates how much material is required to identify donor specific differences in signalling. Lastly, we explore the recovery of phosphorylated peptides from formaldehyde crosslinked cells, which will enable the sorting of cell populations post stimulation. This improved workflow is demonstrated on a murine model of TNF induced T cell proliferation using T cells derived from the spleen that are stimulated ex vivo. However, as ex vivo expansion is not possible in this model, we performed the experiment using 12ug of protein input per sample. Even at these incredibly low levels of input, I determine that TNFR2 stimulation may regulate the metabolism of T cells towards a more sustainable energetic environment favouring memory and regulatory T cells rather than an effector phenotype.

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.