Medical Biology - Theses
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ItemCytokine signalling in haematopoietic cells( 2018)Cytokine receptor signalling is essential for cell survival, proliferation and subsequent differentiation of haematopoietic stem cells (HSCs). Cytokines control development of haematopoietic progenitors into cells of the myeloid, lymphoid and erythroid lineages by stimulating cell cycle progression, proliferation and differentiation as well as by inhibiting apoptosis. My work focusses on Interleukin-3 (IL-3) and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), two essential cytokines in haematopoiesis. Binding of a cytokine to its specific receptor leads to the activation of multiple kinase signalling pathways, including the JAK/STAT, Ras-MAP kinase (MAPK) and PI3-kinase/AKT pathways. In this signalling network, the IkappaB Kinase (IKK) complex plays an important role as a downstream signalling hub. My research investigates how IL-3 mediated IKK activation promotes the survival of myeloid cells and what role this process may play in the development of related diseases, such as myeloproliferative disorders. Using immortalised growth factor (IL-3 or GM-CSF) dependent myeloid progenitor cells (FDMs), as well as employing various in vivo mouse models of haematopoietic development, I was able to show that 1) IKK is a major signalling hub linking IL-3, TNFR1 and p53 signalling to control the survival in haematopoietic cells by describing for the first time a role for the E3 ubiquitin ligase MDM2 downstream of IL-3- or TNFalpha-mediated IKK2 activation, suggesting crosstalk between the NF-kB signalling and p53 signalling pathways; 2) IKK regulates cellular metabolism through activation of NF-kB- and p53-dependent metabolic target genes by showing that deletion of IKK2 but not IKK1 in hematopoietic cells significantly alters cellular metabolism, impairing oxidative phosphorylation and upregulating glycolysis due to altered expression of p53-dependent metabolic target genes; 3) IKK plays a crucial role during haematopoietic development, regulating myeloid cell proliferation, lineage commitment and survival, showing that deletion of IKK2 but not IKK1 in haematopoietic progenitor cells severely affects haematopoietic development by skewing lineage commitment in vivo, resulting in neutrophilia, elevated circulating interleukin-6 and lethality due to severe gastrointestinal inflammation. The work presented in this thesis provides new important insights into the role of IKK in haematopoietic cells and haematopoietic development and clearly demonstrate that IKK1 and IKK2, the two catalytic subunits of the IKK complex, have distinct functions depending on the context of activation. In the future, this fact could be exploited to develop novel targeted therapies to specifically target a subunit in disease settings such as haematopoietic malignancies where aberrant NF-kappaB activity is frequently observed.
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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.