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

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

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

The emergence and spread of drug resistance have hindered the campaign for malaria eradication. The development of new drug targets is critical for our anti-malarial arsenal of interventions. Plasmepsins, which are aspartic proteases expressed by malaria parasites, serve important functions for parasite survival. Among the 10 members of this enzyme family, plasmepsin X (PMX) is essential for P. falciparum growth and has been shown to be involved in the egress of merozoites from infected red blood cells and the invasion of merozoites into red blood cells. Several aspartic protease inhibitors have anti-malarial activity on P. falciparum and are proposed to target PfPMX. The aim of this project was to investigate if these compounds affect P. knowlesi growth and whether PMX is a cross-species target for antimalarial development. This work showed that two aspartic protease inhibitors, 49c and 1SR, caused inhibition of P. knowlesi parasite growth. In further studies, live cell imaging demonstrated that these compounds inhibit P. knowlesi parasite growth by blocking parasite egress. Next, the optimal condition for protease activity was characterised after the expression and purification of a functional recombinant P. knowlesi plasmepsin X (rPkPMX). Using a fluorogenic protease assay, both 49c and 1SR were shown to inhibit the activity of rPkPMX. Furthermore, rPkPMX was able to cleave synthetic substrates, which were based on the predicted cleavage sites of PfSUB1, PfRAP1, PfRh2, TgROP1 and TgMIC6 predicted cleavage sites. By screening a panel of aspartic protease inhibitors, the BACE1 inhibitor, LY2886721, was identified as an inhibitor of rPkPMX activity as well as P. knowlesi and P. falciparum parasite growth. Therefore, PMX can be used as a cross-species target for antimalarial drug development.

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

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of the joints, affecting 0.5% to 1% of global population. Current targeted therapies antagonize the debilitating effects of key inflammatory mediators and immune cells. However, few patients achieve complete remission, prompting novel therapeutic approach. Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) was first identified as a haemopoietic growth factor but is now recognised as a proinflammatory cytokine in a number of autoimmune inflammatory diseases, including RA and Multiple Sclerosis (MS). GM-CSF promotes destructive joint inflammation by priming pro-inflammatory phenotypes of myeloid cells, such as neutrophils, monocytes and macrophages. Accordingly, therapies targeting GM-CSF or its receptor are currently under clinical evaluation in RA and MS and seem promising. However, much remains to be explored how GM-CSF is dynamically regulated during arthritis, especially in autoantibody-mediated inflammation as seen in seropositive RA patients. This work aims to characterize the cellular source of GM-CSF during antibody-induced arthritis and evaluate the cell-intrinsic negative regulation of GM-CSF signalling in myeloid cells and arthritis. I utilized the autoantibody-driven, immune complex-mediated serum transfer induced arthritis (STIA) murine model, which mimics the effector phase of seropositive RA patients. Using the novel GM-CSF dual reporter mice, joint-infiltrating Natural Killer (NK) cells were found to be main GM-CSF-producing cells during STIA. By using NK-deficient (Mcl1fl/fl:Ncr1-Cre) mice, NK-depleted (anti-NK1.1 antibody-treated) mice or specifically deleting GM-CSF production by NK cells (Csf2fl/fl:Ncr1-Cre), I was able to show the importance of NK cells and their GM-CSF-producing function in maintaining arthritis (Chapter 3). GM-CSF on myeloid cells induced the Cytokine Inducible SH2-containing (CIS) protein, a member of the suppressor of cytokine signalling (SOCS) protein family. Using Cish-/- mice, I showed that CIS negatively regulated GM-CSF signalling post activation, which is evident in intracellular signalling pathways, effector cell functions and in antibody-induced arthritis (Chapter 4). Taken together, this study provides new insights into the pathogenesis of antibody-driven, GM-CSF-mediated autoimmune inflammation and provide a rationale towards designing novel anti-inflammatory agents such as NK modulator or CIS mimetics for RA.

Plasmodium falciparum resistance to artemisinin-(ART) based combination therapies (ACTs) and other antimalarials poses a major threat to malaria control and elimination. Current efforts are aimed towards identifying potent antimalarials which inhibit multiple stages of the parasite lifecycle or discovering novel drug targets which may help overcome ART-resistance. This work aimed to characterise two aspartyl proteases of P. falciparum which may hold promise as antimalarial targets. One strategy recently proposed to overcome ART-resistance is the synergistic use of a parasite-selective proteasome inhibitor to sensitise ART-resistant parasites to artemisinin. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the P. falciparum ubiquitin-proteasome system (UPS) could yield a combination therapy to tackle ART-resistance. DNA-damage inducible protein 1 (DDI1) is a previously uncharacterised essential aspartyl protease in P. falciparum. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPS in mammalian cells. In other organisms, DDI1 plays a role in shuttling proteins to the proteasome for degradation via its ubiquitin-like domain. We hypothesise PfDDI1 is an active aspartyl protease and plays a role in the parasite’s UPS. To investigate the role of DDI1 in the UPS and parasite survival, we identified a DDI1 orthologue in P. falciparum and characterised this using several strategies. We utilised CRISPR-Cas9 to knock out, tag and inducibly knock down DDI1 across the asexual lifecycle of P. falciparum, and study the effect of this on parasites. Expression of recombinant DDI1 proteins provided insight into the protease activity and substrate repertoire of PfDDI1. Together these studies provide insight into the domain architecture, essentiality and function of PfDDI1 and clues into its potential as an antimalarial target. Development of an antimalarial to block parasite transmission between humans and mosquitos is also a viable strategy to reduce malaria burden. In this study, we also explore a potential transmission-blocking target, Plasmepsin VII (PMVII) and create tools to enable further study of this aspartyl protease in sexually reproductive gametocytes. These tools are vital to determine the function and substrate repertoire of PMVII and elucidate its potential as an antimalarial target.

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

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

Acute Myeloid Leukaemia (AML) is a vastly heterogeneous blood disorder with a poor prognosis for patients older than 65. Our group has been focused on developing new treatments for AML to replace the standard intensive chemotherapy. Previous data showed that the SMAC-mimetic birinapant in combination with the caspase inhibitor IDN could kill different types of AML both in vitro and in vivo through activation of necroptosis cell death pathway. However, over 50% of the patient samples tested in study showed resistance to necroptosis. This project aims to determine the molecular mechanisms that mediate necroptosis resistance in AML and identify new regulators of necroptotic pathway. The results obtained in this study will expand the knowledge of necroptosis signalling in leukaemia and will contribute to the optimal clinical use of birinapant/IDN drug combination. This project contains 2 parts; (1) We will use human AML cell lines that are resistant to necroptosis to determine the molecular changes involved in cell death resistance. (2) We will use CRISPR/Cas9 knock out screen in human AML cell lines that are sensitive to necroptosis, trying to identify new regulators of TNFR1-induced necroptotic pathway. Together these experimental approaches will allow a better understanding of the regulation of TNF-necroptosis signalling in AML. By overexpression wild-type RIPK3 in the KG-1 cell line, we successfully sensitised KG- 1 cells to necroptosis, which indicates that the KG-1 endogenous RIPK3 is dysfunctional. By cDNA sequence of KG-1 endogenous RIPK3, we detected several mutant base pairs, which may lead to the dysfunction, but this result needs further prove by genome sequence, which is undergoing. By CRISPR knock out screen, we found several targets that may lead to the necroptotic resistant, and MAGE3 is the most research-worthy one. Knockout MAGEB3 in the MV4;11 cell line led to the downregulation of RIPK3 and the necroptotic resistance. However, this result could not be repeated on the U937 cell line, and the mechanism of how MAGEB3 regulates RIPK3 is still unclear. Further research will be done on MAGEB3 to have a better understanding of the role of MAGEB3 in the necroptotic pathway. Together, all these results gave a better understanding of the necroptotic pathway and may contribute to the treatment of AML.

Malaria is a major global health problem and a leading cause of death worldwide. The mechanism behind some parts of the parasite life cycle are still obscure, especially the liver stage which is essential for parasite development and maturation. It is likely that the parasite prevents the host hepatocyte from undergoing cell death during invasion. This is especially relevant for Plasmodium vivax as the hypnozoite can lay dormant in a liver cell for months, even years. We hypothesise that P. vivax encodes proteins to inhibit host cell death in liver. We used the computer algorithm I-TASSER to identify several P. vivax proteins which were predicted to have similar structures to human proteins involved in cell death. We expressed these P. vivax proteins in mammalian cells and performed functional tests to investigate their potential roles experimentally. Identification of P. vivax proteins that influence host cell death would improve our understanding of how P. vivax can survive for prolonged periods in the host cell during liver stage and may accelerate the development of new drugs for malaria liver stage, which is necessary for the ultimate goal of eliminating malaria.

Production of high-quality, specific antibody is essential for lifelong protection against pathogens. Generation of antibodies is the end result of a complex and highly regulated process of B cell development and signalling. Defects anywhere in this process can lead to immune dysregulation, resulting in primary immunodeficiency and/or autoimmunity. This thesis explores a new hypothesis – that the majority of cases of primary immunodeficiency, and likely other complex immune disorders, are caused by the combinatorial effect of multiple small defects in B cell function or ‘health’ that sum to cause disease in patients. Further, that we can measure and model these defects quantitatively in vitro. To test this hypothesis, we focused on Common Variable Immunodeficiency (CVID), a clinically heterogenous primary immunodeficiency, united by antibody deficiency, where most cases are sporadic and, presumably, polygenic. In this thesis, we outline the development of a novel in vitro pipeline and accompanying parametric mathematical modelling tools to identify and measure the functional cause of immunodeficiency in individual patients. These in vitro assays were first developed and calibrated on healthy donors, to measure healthy human B cell responses to T-dependent and T-independent stimulation. These assays measure cell division, death, differentiation, isotype switching and antibody production, to reveal the innate programming of B lymphocytes in response to T-independent and T-dependent stimuli. Here, we observed, for the first time, autonomous programming of the division-burst size (division destiny) in human B cells, a phenomena previously demonstrated in murine lymphocytes. We applied cyton modelling to human lymphocytes for the first time, to explain the parameters underlying the synergy between two signals. Additionally, by testing a number of unrelated healthy donors, we established a healthy donor ‘range’ of B cell responses for comparison to patients. We subsequently applied these assays to a cohort of CVID patients and identified a number of quantitative differences. These included: 1. A striking, severe early survival defect of patient naive B cells; 2. A defect in the ability of patient naive B cells to differentiate to antibody secreting cells and produce switched antibody, and; 3. Reduced proliferation of patient naive B cells in response to T-independent stimulation. We utilised mathematical modelling tools to demonstrate that this reduced proliferation is explained by a combination of multiple small defects in the parameters that control the B cell response (times to divide, times to die etc) that sum together in a non-linear manner to magnify their impact on immune responses. Furthermore, the relative contribution of each parameter to the final immune deficiency was individually determined and found to be unique for each patient. This work significantly improves our understanding of the functional causes of immunodeficiency and offers a clear path toward improved clinical diagnosis and targeted treatment strategies for individual patients.