Medical Biology - Theses
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ItemA genetics-based investigation into the regulation of RIPK1 and caspase-8 during cell death and disease( 2022)Cell death is a fundamental process needed for healthy development, immunity and life. The tight control and regulation of cell death signalling is important for cellular homeostasis, and the de-regulation of cell death is a hallmark of many diseases ranging from infection to cancer. Several regulated cell death (RCD) pathways have been described, with genetically encoded cell death signalling molecules and effectors dictating cellular fate. Some of these, such as necroptosis and pyroptosis, are highly inflammatory and immunomodulatory, while others, such as apoptosis, are generally considered non-inflammatory and tolerogenic. Caspase-8 is a critical cell death protein that also has a pleiotropic role in inflammation. Receptor interacting protein kinase (RIPK)1 liaises with external signals to control the death and inflammatory functions of caspase-8, but major gaps remain in our understanding of how RIPK1 regulates the death and non-death functions of caspase-8. Identifying and characterising the mechanisms that control caspase-8 activity is crucial to understanding how we might best therapeutically target cell death signalling to overcome relevant diseases. This PhD thesis explores the regulation of caspase-8 activity and identifies key upstream checkpoints to therapeutically intersect and modulate caspase-8 activity. Firstly, this thesis genetically delineates a unique caspase-8-dependent cell death triggered by combined signalling of host-derived interferon (IFN)-gamma and pathogen ligands that engage Toll-like receptors (TLRs). Experiments show that caspase-8 cell death signalling is licensed by nitric oxide (NO), which is produced by the inducible nitric oxide synthase (iNOS) protein. Physiologically, both caspase-8 and iNOS contributed to disease severity in a model of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection, suggesting iNOS might licence damaging cell death and inflammation during coronavirus disease of 2019 (COVID-19). Secondly, the physiological role of Mind Bomb-2 (MIB2), a recently described pro-survival protein that prevents caspase-8 activation by RIPK1 in cancer cells, is examined using novel MIB2 gene targeted mice. This thesis reveals the physiological function of MIB2 in vivo and examines the function of MIB2 in both inflammation and cancer disease models to determine whether therapeutics designed to inhibit MIB2 could be used to safely activate caspase-8. These studies find that deficiency or inactivation of MIB2 is well-tolerated in mice and does not impact important biological processes including development, haematopoiesis, viability or fertility. Interestingly, challenging MIB2 knockout mice to drive excessive caspase-8 activity leads to enhanced cell death-induced dermatitis, while inactivation of MIB2 limits tumourigenesis in a model of inflammation-driven colorectal cancer. This thesis provides critical insight into the regulation of caspase-8 and uncovers distinct modes of regulation detailing how elevated NO or, inhibition of MIB2 contribute to excessive cell death and disease. This work aids the design of next generation treatments to overcome cell death resistance and transforms our understanding of how caspase-8 is regulated in inflammation and disease.
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ItemStructural and Functional Investigation of the BCL-2 Family Member, BCL-rambo( 2022)The BCL-2 family proteins are the faces of mitochondrial apoptosis, governing cell fate through interactions that mediate mitochondrial outer membrane permeabilisation. While the molecular intricacies of the pathway have been characterised in great detail, putative BCL-2 proteins of unknown function remain. BCL-rambo (or BCL-2-like protein 13) is one such protein. This enigmatic BCL-2 family member was first discovered in 2001 and 20 years later, a consensus concerning its role has yet to be reached. With reports suggesting a pro-apoptotic or an anti-apoptotic role for BCL-rambo, whilst others implicating a role in mitophagy, the deconvolution of its influence within these established fundamental signalling pathways is of great interest considering their potential for dysregulation in disease. This thesis aimed to characterise the BCL-rambo protein from the ground-up. Harnessing a combination of molecular, structural, and biochemical techniques, this work investigated the structure and function of BCL-rambo in the context of apoptosis and mitophagy. The effect of BCL-rambo expression on apoptosis is examined in Chapter 3, which details attempts to identify binding partners within the BCL-2 family of proteins. Chapter 4 describes the design and production of recombinant BCL-rambo protein used for X-ray crystallisation studies, and reveals the structure of the BCL-rambo BCL-2 homology region. Chapter 5 assesses the role of BCL-rambo in mitophagy and investigates its ability to bind ATG8-family proteins. This chapter also examines how phosphorylation of BCL-rambo affects its affinity for ATG8-family proteins and includes X-ray structures of a phosphorylated BCL-rambo peptide bound with ATG8-family proteins. Together, these studies address the discrepancy regarding the function of BCL-rambo, supporting a role for BCL-rambo as a mitophagy receptor, and elucidates the first experimental three-dimensional structure of BCL-rambo. This work also provides an intriguing example of divergent protein evolution and brings the field a step closer to completing the BCL-2 family portrait.
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ItemManipulating cell death pathways to promote clearance of HIV-1( 2021)HIV is a chronic retroviral infection first recognised in humans 40 years ago. Untreated, it leads to progressive CD4 T cell depletion and death approximately ten years post infection. Combination anti-retroviral therapy (cART) is very effective at controlling active HIV replication. However, it needs to be continued daily for the lifetime of the infected individual, leading to a large personal and societal cost. Although the lifespan of HIV infected individuals has approached that of the general population there continues to be excess morbidity and mortality from malignancies and cardiovascular disease. A cure for HIV has eluded the scientific community so far due to a latent reservoir of the virus existing in a small minority of memory CD4 T cells, which contain HIV DNA integrated into the cellular genome. The HIV DNA integrates can be replication competent or defective. The vast majority are defective but there exists a small pool of these cells that harbour replication competent virus. These latent cells containing integrated HIV DNA downregulate their cell differentiation markers compounding the search for these cells even further. Unfortunately, these cells are unaffected by cART and during cART interruption they can reactivate and infect naive CD4 T cells. Cell death and survival in HIV infection is balanced by host and viral factors. The most well characterised form of cell death in HIV infection is apoptosis, which can occur via both extrinsic and intrinsic pathways and can be triggered by multiple events. Actively infected cells die due to viral cytopathic effects and immune clearance, but central memory CD4 T cells infected with HIV appear to be more resistant to cell death via upregulation of important anti-apoptotic proteins that block the cell death pathways. However, this upregulation can be exploited to drive cells towards death by blocking their action. SMAC mimetics are compounds that drive cell death in extrinsic apoptosis by blocking the action of IAPs. This thesis explores the addition of SMAC mimetics to standard cART therapy with the hypothesis that by targeting these upregulated proteins this can deplete the latent reservoir of HIV infection. For the first time, I show that SMAC mimetics delay the time to viral rebound in HIS HIV mice. I also describe preliminary work targeting both the extrinsic and intrinsic apoptosis pathways.
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ItemCell Death Mechanisms in T cell Differentiation and Homeostasis( 2021)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.
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ItemIdentifying Novel Regulators of Intrinsic Apoptosis( 2021)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.