Medical Biology - Theses
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ItemA CRISPR/Cas9-based investigation of inflammasomes in infectious disease and autoinflammation( 2017)Inflammasomes are a family of innate immune signalling platforms that are activated in response to tissue damage or infection. Inflammasome stimulation results in activation of the inflammatory protease caspase-1, which induces a lytic cell death program known as pyroptosis, and maturation and release of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. The potent inflammatory cascade triggered through activation of the inflammasomes is protective against many bacterial pathogens that either invade host cells or produce toxins that deregulate key homeostatic mechanisms within innate immune cells such as monocytes and macrophages. De-regulation of inflammasome signalling, such as gain-of-function mutations in inflammasome components, can result in autoinflammatory pathology. In order to investigate the function and regulation of inflammasomes, Clustered, Regularly Interspersed, Short, Palindromic Repeats (CRISPR)/Cas9 gene editing technology has been utilised to delete various inflammasome components from human myeloid cell lines or from mice. The alternative inflammatory caspases, caspase-11 in mice and caspases-4 and -5 in humans are activated directly by cytoplasmic lipopolysaccharide (LPS), a key component of the cell wall of gram-negative bacteria. These caspases are able to induce pyroptosis independently of caspase-1, but are only able to trigger IL-1β and IL-18 release in a caspase-1-dependent manner. In this thesis, the roles of caspase-4 and caspase-5 in the response to cytoplasmic lipopolysaccharide (LPS) and invasive gram-negative bacteria have been investigated in a human monocytic cell line. While both caspases responded to infection with live gram-negative bacteria, free LPS that was transfected into the cytoplasm activated only caspase-4. This suggests that caspases-4 and -5 may be activated by distinct stimuli or through different mechanisms. This work also interrogates the role of the inflammasome-forming receptor pyrin, in both autoinflammatory disease and the anti-bacterial immune response. A serine to arginine mutation in pyrin at amino acid position 242 results in a newly described autoinflammatory condition known as Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). A monocytic cell line expressing the S242R mutant of pyrin has been created and it was demonstrated that this mutation results in spontaneous inflammasome activity. Under homeostatic conditions, serine 242 is phosphorylated and interacts with the 14-3-3 family of adapter proteins to keep pyrin inactive. Deletion of specific 14-3-3 isoforms also resulted in spontaneous production of mature IL-1β. Finally, the expression of pyrin in various myeloid compartments and its role in in vivo models of bacterial infection have been investigated using a pyrin-deficient mouse line. Two isoforms of pyrin were detected that were differentially expressed among myeloid populations. Additionally, no role for the pyrin inflammasome was observed in a Dextran Sodium Sulfate (DSS)-induced colitis model, or Citrobacter rodentium, Salmonella Typhimurium or Mycobacterium tuberculosis infection models.
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ItemNeutrophil extracellular trap-associated cell death - role in gout and relationship to alternate forms of cell death( 2017)Cell death has emerged as a critical process in many facets of human disease, ranging from cancer to inflammation and cardiovascular disease. One such modality, Neutrophil Extracellular Trap (NET)-related cell death, or NETosis, is a form of cell death with potential implications in a wide range of human conditions but, at present, understanding of the mechanisms of NETosis and its physiologic and pathological consequences is limited. Finding the key mechanisms underlying NETosis will illuminate roles for NETosis in human diseases and animal models of these conditions, and provide targets for intervention. This thesis examines NETosis in the context of the human inflammatory disease, gout, and explores the relationship between NETosis and the other main types of cell death - necroptosis and apoptosis. First, I developed a novel time-lapse imaging-based quantitative analysis of NETosis. The assay combines multi-well format live-cell microscopy technique with computerized analysis to obtain detailed kinetic information about the two key components of NETosis - cell death and chromatin decondensation. The technique allowed me to demonstrate that different NETosis stimuli, namely phorbol myristate acetate and monosodium urate crystals, exhibit different kinetics of cell death. Exploring the differences further, I found that crystals induce NETosis through a nicotinamide adenine dinucleotide phosphate oxidase-independent pathway that is distinct from PMA-induced NETosis. I also observed that both forms of NETosis depend on neutrophil elastase activity to cause chromatin decondensation, but not cell death. I observed NET-like structures in samples taken from gout patients both during the acute inflammation of a gout attack and from the uninflamed crystal-rich tophus tissue, a feature of chronic gout. The NETs released during MSU-induced NETosis were resistant to serum nucleases relative to PMA-induced NETs, suggesting that these DNA structures may persist within tissues, as seen in the patient samples. This nuclease resistance was at least partially attributable to the presence of increased actin in MSU-induced NETs relative to PMA-induced NETs, as identified using proteomic techniques. Given the presence of NETs in both inflammatory and uninflamed contexts, I demonstrated that the presence of NETs dampens the IL-1β responses of macrophage-like cells and reduces crystal-induced macrophage cell death. I further demonstrated that NETosis is distinct from both apoptosis and necroptosis. Unexpectedly, two MLKL inhibitors did inhibit NETosis, but this is an indirect effect, dependent on accelerating apoptosis. This finding highlighted that over time in culture, neutrophils lose the ability to release NETs when stimulated with PMA and this loss of “NET competence” is mediated by caspase activation. Using the power of a live cell imaging approach to simultaneously quantify cell death and NET release, my studies have advanced our fundamental understanding of the mechanistic differences underlying NETosis induced by different stimuli. Further, by applying this assay, I was able to establish that proteins within the apoptosis and necroptosis pathways that had been previously attributed roles in the NETosis pathway, were in fact dispensable. Consequently, I anticipate this assay and the mechanistic insights it provides will play an important part in advancing our understanding of the NETosis pathway.
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ItemStructural investigations of pro‑apoptotic Bcl‑2 family proteins( 2017)The Bcl‑2 protein family regulates the intrinsic apoptotic pathway through an intricate network of protein:protein and protein:membrane interactions. The pathway culminates in the permeabilisation of the mitochondrial outer membrane by the pro‑apoptotic effector proteins Bak and Bax, an event that irreversibly commits a cell to death. To facilitate membrane permeabilisation, Bak and Bax undergo a series of conformational changes to convert from inert monomers to membrane‑embedded homodimers that nucleate and propagate apoptotic oligomers. While great strides have been made in structurally characterising these conformational changes, questions remain surrounding homodimer interactions with the membrane, oligomerisation, and membrane pore formation. This thesis addresses these questions by providing structures of lipids bound to Bak BH3:groove core homodimers (Chapter 2). These are the first structures of any Bcl‑2 family protein in complex with lipid. They reveal symmetric binding sites for phospholipid headgroups and acyl chains. In one structure, adjacent Bak homodimers are cross‑linked by the acyl chains of single phospholipids, suggesting homodimer oligomerisation could be mediated by lipid. Bak oligomers could be dissociated with phospholipase A2, supporting a role for lipid in oligomer stability. Collectively, the structures presented here indicate that lipids may play a direct role in Bak oligomerisation. Like Bak, Bax homodimerises and oligomerises on the mitochondrial outer membrane. The original Bax BH3:groove core homodimer structure was solved as a GFP fusion at low resolution. Here, a tetrameric structure consisting of two Bax BH3:groove core homodimers alone was solved at high resolution (Chapter 3), providing details for canonical interactions in atomic detail. A crystal structure of Bax BH3:groove core homodimers containing lipid was also solved, although the structure could not be refined due to severe twinning. This result demonstrates that Bax core domains also associate with lipid, and provides a starting point for crystal optimisation. Pro‑survival Bcl‑2 family proteins antagonise the apoptotic function of Bak and Bax by preventing their activation and sequestering their activated forms. Sequestration of activated Bak and Bax in heterodimeric Mode 2 complexes involves binding of the Bak/Bax BH3 domain to a conserved hydrophobic groove. Beyond this, little is known regarding the topology of these complexes. The pro‑survival protein Bcl‑XL can undergo similar conformational changes to Bak and Bax, but whether it forms BH3:groove heterodimers with Bak/Bax was unknown. Using cysteine cross‑linking on mitochondria, I show that Bcl‑XL can form reciprocal BH3:groove heterodimers with Bax, and possibly Bak (Chapter 4). These results challenge a simplistic view of Mode 2 complexes, implicating more extensive interactions beyond the canonical BH3 in groove interface. Bok is a third potential pro‑apoptotic effector protein that shares sequence similarity with Bak and Bax, but its role in apoptosis remains unresolved. To investigate the structure and function of Bok, I developed a recombinant expression system to produce human, rat, and chicken Bok. The first crystal structure of Bok, from the chicken, reveals the canonical Bcl‑2 family fold, with deviations that may explain its proposed constitutive activity (Chapter 5). The structure paves the way for mutagenesis studies that will further our understanding of this enigmatic protein.
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ItemPro-apoptotic therapies for the treatment of Mycobacterium tuberculosis infection( 2017)One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) has killed more than 1 billion people over the past two hundred years, surpassing mortality caused by all other pandemics and epidemics combined. Despite a concerted global effort to reduce transmission, Mtb infects an estimated 10.4 million people and kills 1.4 million people each year. Managing this condition is becoming increasingly challenging because Mtb is fast becoming resistant to all first line antibiotic therapies. Novel interventions beyond iterations on antibiotics are required. Understanding host-Mtb interactions, with a view to targeting host signalling pathways that the organism is reliant upon, is a tenable approach to combatting this deadly disease. Host cells are intolerant of intracellular organisms and consequently Mtb must prevent a cell from dying so that it has time to propagate and disseminate. Exactly how it does so is controversial and poorly understood. In my work, I sought to understand the role of apoptosis in Mtb disease pathogenesis. I dissected the role of the extrinsic apoptotic pathway and associated key molecular components including inhibitor of apoptosis (IAP) proteins. I also examined the role of the intrinsic apoptotic pathway and associated Bcl-2 family of proteins. I found that Mtb infected mouse and human macrophages showed major aberrations in the protein expression levels of IAP and Bcl-2 family molecules such that the stoichiometry of these proteins strongly favoured cell survival. I infected mice that were deficient in the three major mammalian IAPs (cIAP1, cIAP2 andXIAP) and found that in the absence of cIAP1, and to a lesser extent cIAP2, Mtb infected macrophages died and disease pathogenesis was strikingly altered. I then sought to reprogram the extrinsic apoptotic pathway to promote death of Mtb infected cells by using a clinical stage drug inhibitor of cIAPs. I was able to optimise a dosing regimen of the cIAP antagonist, birinapant, that proved efficacious in killing Mtb infected macrophages and in reducing bacterial loads in various strains of mice and in mice engrafted with a human immune system. An examination of the intrinsic cell death pathway also proved very interesting. Again, I used a combination of gene-targeted mice and clinical stage drugs to antagonise the function of several Bcl-2 family pro-survival proteins. Interfering with Bcl-xL function had no effect on Mtb disease pathogenesis whilst antagonising the function of Bcl-2 made the disease worse. Notably, as little as a 50% reduction in Mcl-1 function, examined using Mcl-1+/- mice, produced an improvement in Mtb infection outcomes. Given the success in defining targetable host cell pathways involved in TB pathogenesis, I next investigated if these insights were applicable to latent Mtb infection. A large proportion of people infected with Mtb may not progress to overt disease but remain latently infected and can reactivate disease under certain circumstances. There are well defined indications for treating some people who are latently infected. Treatment of latent infection suffers from the same shortcomings as treatment of active disease. I found that birinapant could be optimised to also impact on latent infection. The significance of my work includes providing valuable insights into how apoptosis plays a critical role in determining Mtb infection and disease outcomes. I believe that my work, for the first time, has identified the key molecular components that regulate cell survival / apoptosis signalling during Mtb infection. The importance and implications of these findings are underscored by my preclinical studies showing that these host cell molecules and pathways can be targeted using clinical stage drugs to promote clearance of Mtb infection and disease.
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ItemInvestigation of cell death pathways in response to TNF and IFNγ( 2017)During my PhD I investigated the regulation of the TNF and IFNγ signalling pathways and their ability to induce cell death. IFNγ is a critical cytokine in the immune response against viral and intracellular bacterial infections. It has also been associated with auto-inflammatory and auto-immune disorders (Pollard et al., 2013; Zhang, 2007), where it was found upregulated together with other pro-inflammatory cytokines like TNF (Ohmori et al., 1997). TNF signalling and the mechanism of cell death induction downstream of the TNF receptor complex has been investigated in detail over the past 4 decades. Although IFNγ was first described 50 years ago, and before TNF, significantly fewer IFNγ signalling components have been discovered compared to the TNF signalling complex. Nevertheless, both cytokines induce equally potent and potentially dangerous systemic responses at low concentrations and must be tightly regulated. I therefore hypothesised that additional IFNγ signalling regulators must exist. In order to discover such novel regulators of the IFNγ signalling pathway I enriched for the IFNγ receptor and identified binding partners using mass spectrometry. Using this approach I identified SPTLC1 and 2, which are two subunits forming the serine palmitoyltransferase, directly interacting with the IFNγ receptor chain 2 (IFNGR2) constitutively. Weak interaction between SPTLC1/2 and IFNGR1, however, was only detected upon IFNGR complex formation induced by IFNγ stimulation suggesting that IFNGR1 interacts with SPTLC1/2 indirectly via IFNGR2. SPTLC2 deficient single cell mouse dermal fibroblast showed either normal or increased phosphorylation of STAT1 upon IFNγ stimulation and lack of SPTLC2 had no impact on transcription of classical IFN target genes. Secondly, I investigated the mechanism of cell death induced by IFN in combination with Smac-mimetics, a group of small molecule inhibitors of the inhibitor of apoptosis proteins (IAPs), which have been heavily investigated in context of TNF signalling. Previous studies revealed that inhibition of IAPs renders cells sensitive to TNF induced cell death, which is primarily apoptosis mediated by caspase-8. However, inhibition of caspase-8 by caspase inhibitors triggers an alternative cell death pathway called necroptosis. Here I found that the combination of IFN/Smac-mimetic had a similar impact on survival and, more precisely, induced RIPK3 dependent caspase-8 mediated apoptosis in mouse dermal fibroblasts. Surprisingly, IFN/Smac-mimetic induced killing in HT29 cells was not blocked by deleting caspase-8 and effectors of the necroptotic pathway like RIPK3 and MLKL. In contrast, deficiency of RIPK1 largely protected cells from IFN induced cell death, indicating that a novel form of RIPK1 dependent cell death was being induced. In trying to discover the mechanism we observed that caspase-10 was significantly upregulated by IFN and activated by IFN/Smac-mimetic treatment. HT29 cells deficient for caspase-10, caspase-8 and either MLKL or RIPK3 were completely resistant to IFN/Smac-mimetic revealing an important role for caspase-10 in IFN/Smac-mimetic induced killing. Thirdly I focused on the activation and function of MLKL, the most downstream member of the necroptotic pathway known. Necroptosis has been best studied downstream of the TNF signalling complex, upon IAP and caspase inhibition. We and others propose a model where phosphorylation of the MLKL pseudokinase domain by RIPK3 triggers a molecular switch, leading to exposure of MLKL’s N-terminal four-helix bundle domain, its oligomerisation, membrane translocation, and ultimately cell death. We additionally identified novel phosphorylation sites S158, S228, S248. By mutating these sites and overexpressing phosphomimetic and -ablating MLKL mutants in Mlkl-/- or Ripk3-/-/Mlkl-/- deficient murine fibroblasts I demonstrated that these sites influence MLKL activity and discovered a potential inhibitory effect of RIPK3 on cell death induced by MLKL. Finally, I examined the evolutionarily conservation of the necroptosis inducing activity of MLKL by analysing the function of MLKL orthologs. While the intrinsic ability to lyse membranes, which was tested in liposome assays, is highly conserved, several MLKL orthologs including human MLKL failed to induce cell death when expressed in murine fibroblasts. This suggests the presence of additional poorly conserved, species-specific factors that inhibit or activate MLKL.
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ItemStructural transitions during cell death: bak activation and oligomerisation( 2015)Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This thesis investigates structural transitions occurring to Bak during apoptosis. I present crystal structures of a Bak core/latch dimer and demonstrate the dissociation of the core and latch domains upon Bak activation. I provide the first high-resolution details for the core domain dimer, a subunit upon which the larger Bak oligomer builds. Cellular assays, guided by the presented crystal structures, confirm the physiological relevance of these key events in the intrinsic apoptotic pathway (Chapter 2). I also describe the first crystal structures of Bak in complex with the BH3-domain of Bim (Chapter 3). These studies complement previous work performed on Bax and support an analogous mechanism of activation and oligomerisation. Certain detergents have been reported to activate Bak in vitro. Here I demonstrate that some detergents can oligomerise Bak and/or promote hetero-complexes between Bak and the pro-survival protein Mcl-1. I describe the production of homo-oligomeric and hetero-oligomeric complexes of Bak, which may be amenable to structural studies (Chapter 4). The literature on apoptosis assumes that mouse and human Bak are analogous in structure and therefore function. Here I report structural differences between Bak homologs from these two species (Chapter 5). These differences exist at the site of ligand binding, yet it remains unclear whether they result in functional variations. These data may aid in the development of novel agonists and antagonists of Bak, and could prove fundamental to designing murine-based pre-clinical trials. The BH3-only protein from the Schistosoma mansoni worm has been identified as a potential direct activator of Bak and Bax. This direct activator BH3-only protein is unique as it only binds to one of the pro-survival proteins (Mcl-1). Here I describe the crystal structure of the Schistosoma BH3-domain (sBH3) in complex with Bax. Liposome release assays demonstrate that sBH3 can directly activate Bak and Bax and induce the formation of membrane permeabilising oligomers (Chapter 6). These studies support current models for the activation and oligomerisation of Bax. Defining the structural characteristics of the intrinsic apoptotic pathway provides novel opportunities for drug design. Organic agonists of Bak may prove useful for the treatment of cancers, while antagonists could provide therapy for diseases characterised by excessive cell death.
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ItemStudies of the role of Mcl-1 in haemopoiesis and leukaemia( 2015)Cell death by apoptosis plays a critical role during embryonic development and in maintaining tissue homeostasis. Consequently, defective apoptosis can lead to degenerative diseases, autoimmunity and tumour development. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by pro- and anti-apoptotic members of the Bcl-2 family of proteins. The principal focus of my studies is Mcl-1, an inhibitor of the intrinsic apoptosis pathway. Mcl-1 is overexpressed in a variety of cancers, including acute myeloid leukaemia (AML) where high levels of Mcl-1 are associated with poor prognosis and drug resistance. Using mouse genetic models, I have investigated the consequences of overexpression of Mcl-1 for haemopoiesis and autoimmunity (Part I) and for the development and treatment of AML (Part II). I. To determine the impact of simultaneously inhibiting the intrinsic apoptosis pathway via overexpression of Mcl-1 and the extrinsic apoptosis pathway via a non-functional Fas receptor, mcl-1 transgenic mice were crossed with faslpr/lpr mice. The combined mutations had little impact on myelopoiesis apart from an increase in macrophages, mainly in the spleen. All major lymphoid subsets were elevated, however, including the “unusual” T cells characteristic of faslpr/lpr mice. Furthermore, the onset of autoimmune disease was markedly accelerated. Thus, consistent with other genetic studies, the intrinsic and extrinsic apoptosis pathways synergise to control autoimmunity. II. To determine the impact of Mcl-1 in AML, I used a mouse model induced by retroviral expression of MLL-AF9, the fusion oncoprotein created by the t(9;11) translocation often found in childhood and treatment-induced adult AML. Overexpression of Mcl-1 or its pro-survival relative, BCL-2, increased the leukaemic burden in the spleen and blood of sick mice although it did not accelerate morbidity. AMLs overexpressing Mcl-1 or BCL-2 tended to have a higher proportion of mature cells compared to ‘wild type’ MLL-AF9 leukaemias. Unlike ‘wild type’ MLL-AF9 leukaemias, which were readily transplantable in non-irradiated recipients, most MLL-AF9 leukaemias overexpressing Mcl-1 and many overexpressing BCL-2 would only transplant if injected into lightly-irradiated recipients. Possible reasons for this unexpected result are discussed. In vitro experiments using short-term lines derived from primary tumours demonstrated that overexpression of Mcl-1 or BCL-2 in MLL-AF9 tumours increased resistance to standard drugs used to treat AML in the clinic. However, even those overexpressing Mcl-1 or BCL-2 were sensitive to the proteasome inhibitor, bortezomib, and to various CDK inhibitors as single agents. The addition of the BH3-mimetic ABT-737 enhanced the response of MLL-AF9 AMLs of all genotypes to standard therapeutics. In contrast, when added to bortezomib or CDK inhibitors, ABT-737 only enhanced the sensitivity of the AMLs that overexpressed BCL-2. Future studies will compare the efficacy of these drug regimens in vivo in transplanted syngeneic immuno-competent mice.
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ItemBIM is critical for DNA damage-induced apoptosis and enforces tumour suppression( 2013)Apoptosis is a highly regulated, ordered form of cell death that is critical for a wide variety of physiological processes. Loss of key apoptotic regulators or other defects in apoptosis can result in either excessive or insufficient removal of cells leading to severe consequences, such as embryonic lethality, autoimmunity or cancer. The BCL-2 family of proteins are key regulators of apoptosis that control the ‘point of no return’ integrating diverse upstream signalling pathways to determine whether a cell will live or die under conditions of stress. Due to their ability to regulate cell survival, abnormalities in the expression of the BCL-2 family members are frequently observed in human cancer. Furthermore due to their ability to initiate apoptosis signalling, the BH3-only subfamily of the BCL-2 protein family are required for mediating tumour cell killing following treatment with many chemotherapeutic agents. Accordingly, mutation or loss of the BH3-only proteins, or their upstream regulators, is associated with chemoresistance and poor treatment response. The tumour suppressor p53 is a critical direct transcriptional activator of the genes encoding the BH3-only proteins PUMA and NOXA. Mutations in p53, with consequent loss of its transcriptional activity, constitute the most frequent abnormality in human cancer and are associated with poor response to anti-cancer therapeutics, particularly those that cause DNA damage. Since p53 functions upstream of the BH3-only proteins in apoptosis signalling, therapies that act independently of p53 to induce the expression of BH3-only proteins are likely to be more efficacious for the treatment of patients that harbour p53-deficient tumours. In this thesis I describe the characterisation of the mechanisms by which DNA damage can induce apoptosis in the absence of p53 function, focussing on those that are relevant to the treatment of p53-deficient tumours. To do this I generated a panel of p53-deficient thymic lymphoma-derived cell lines and determined their ability to undergo apoptosis in response to DNA damaging agents that induce different types of DNA lesions, such as γ-irradiation, etoposide and cisplatin. I have examined changes in protein expression of members of the BCL-2 family following treatment with these DNA damaging agents and identified those members of the BH3-only sub-group that are up-regulated. By generating lymphoma-derived cell lines from mice that lack both p53 and select members of the BH3-only proteins, I have been able to determine which are essential for the induction of DNA damage-induced apoptosis in the absence of p53. Through this approach I have identified novel mediators of DNA damage induced-apoptosis both in lymphoma cell lines and also in primary non-transformed cells deficient for p53. I then sought to determine whether this newly identified pathway plays a critical role in the elimination of pre-leukaemic cells that have sustained physiological DNA damage in vivo (e.g. due to replication induced stress or the stress elicited by oncogene activation). To address this cohorts of mice deficient for p53 plus candidate transducers of this pathway were aged and the lymphoma incidence was compared to those lacking p53 alone. The additional loss of this novel DNA damage induced pathway provoked a pronounced acceleration in lymphoma onset in the p53-deficient mice and also resulted in a more aggressive tumour phenotype. The importance of this tumour suppressor pathway was further characterised by ageing p53-heterozygous mice deficient for pathway members and comparing their tumour incidence to that of mice lacking only a single allele of p53; revealing a profound acceleration of tumour development in this context. BIM has been shown to be a critical tumour suppressor in the Eμ-Myc mouse model of lymphoma and recent evidence has shown that the pro-survival BCL-2 family member BCL-XL is critical to promote survival of cells undergoing neoplastic transformation in this model. To gain further insight into the processes that govern cell survival during neoplastic transformation, I performed experiments to determine whether concomitant loss of BIM would be sufficient to abrogate the delay in lymphomagenesis observed in the absence of BCL-XL. Loss of a single allele of Bim was sufficient to revert this delay and loss of both alleles of Bim provoked a further acceleration in lymphoma onset. Thus through my investigations in vitro into the manner in which apoptosis can be induced in cells lacking p53 and my studies utilising various mouse models of lymphoma development I have identified a novel apoptotic pathway important for the elimination of in p53-deficient pre-leukaemic cells and malignant lymphoma cells. These data provide new mechanistic insights into DNA damage induced apoptosis in vitro and for tumour suppression in vivo.
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ItemFunctional characterisation of Caspase-9 in haematopoiesis( 2012)Caspases are a family of cysteine-aspartic proteases that play essential roles in programmed cell death (apoptosis), programmed necrosis (necroptosis), and inflammation. This work aims to clarify additional reported functions of caspases, and to enhance our understanding of the functional roles of caspases in the blood (haematopoietic system). By genetically dissecting the apoptotic pathway, I show that caspase activation is not required for megakaryocytes to form platelets from their cytoplasm. Rather the opposite is true, apoptotic caspase activation must be restrained for megakaryocytes to survive and produce platelets. In addition, platelets are fully functional without the initiator Caspase-9. Caspase-9-deficient platelets maintain blood clotting (hemostasis), and are capable of facilitating thrombin generation via the exposure of membrane phospholipid phosphatidylserine – supporting the notion that platelet apoptosis and platelet activation are biochemically distinct processes. Herein, I also show that the Bcl-2 regulated caspase cascade is critical for haematopoietic stem cell maintenance. A novel relationship between apoptotic caspase activation and type-1 interferon production – a cytokine known to regulate ‘stem-ness’ – is established. Together, this research refines previously described biological functions for caspases, and provides new insight into the role of caspases in cell death and the physiological consequence of their genetic or pharmacological inhibition.