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.