Medical Biology - Theses
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ItemDistinct precursors of the dendritic cell subtypes( 2006-03)Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response. Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8+ and CD8- subtypes. Each subtype appears to have a specialised role in the various arms of immunity and tolerance. Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes. The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation. Particular was given to DC of the spleen. Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steady-state DC subtypes.
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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.
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ItemThe developmental pathways of splenic dendritic cells( 2011)Dendritic cells (DC) are professional antigen presenting cells, specialised in the activation of naïve T-cells. DC can be further subdivided into circulating plasmacytoid DC (pDC), and conventional DC (cDC), which are functionally distinct subsets. cDC can be further subdivided into peripheral cDC, and lymphoid tissue resident cDC. In this thesis, we focus on the developmental pathways of murine splenic DC – that is, resident cDC and pDC. DC potential has been found in multiple early precursors, and an immediate resident cDC precursor has been described in the spleen. Two precursors have been described in the bone marrow that may form the developmental bridge between the upstream precursors, and the committed pre-cDC. We and others have described common DC progenitors (CDP), which are c-kitintflt3+M-CSFR+, and negative for lineage antigens (lin-). CDP are restricted to differentiation into cDC and pDC. Another precursor, the macrophage-DC progenitor (MDP) has been described as an intermediate stage on the pathways to macrophage and DC development. The definition of a macrophage-DC progenitor includes potential for both macrophages and steady state cDC within single progenitors. In these studies, the ability of a single progenitor to give rise to both macrophages and steady state DC, and thus the existence of a macrophage DC progenitor, has not been established. Here, we have here investigated the developmental potential of the putative MDP populations. Contrary to previous data, we find that the populations defined in the literature as ‘MDP’ are not restricted to the macrophage and DC lineages, but rather retain potential for other haematopoietic lineages. To detect single progenitors with both macrophage and steady state DC potential, we have developed a clonal assay using M-CSF and flt3-ligand, the cytokines that drive macrophage and steady state DC development respectively. We find no evidence of a progenitor with both macrophage and DC potential within the progenitors previously described as ‘MDP’. To determine whether an MDP exists outside of the populations described as such in the literature, we have examined alternative fractions of the bone marrow for candidate ‘MDP’. We find a population within the lin-M-CSFR+CD16/32lowc-kitint/highsca-1-flt3+ BM fraction gives rise to macrophages and steady state cDC on a clonal level. However, this population is not restricted to these lineages. Thus, we find no evidence for a macrophage DC progenitor as a common intermediate on the pathways to macrophage and DC development. In addition, we have investigated the pathways to plasmacytoid DC development. We find that flt3 ligand (FL)-driven differentiation of pDC occurs via multiple developmental pathways. We have described an intermediate precursor on the pathway from development of common lymphoid progenitors to pDC. Furthermore, we have established the validity of using a history of expression of recombinase-activating gene 1 and the presence of D – J rearrangements at the immunoglobulin heavy chain locus as a marker of developmental history. We here demonstrate that the presence of these markers in pDC indicates a developmental history distinct from those pDC that lack these markers. The description of pDC in vivo both with and without a history of RAG1 expression, or with and without D – J rearrangements thus indicates the operation of multiple developmental routes to pDC in vivo. We have also investigated the role of external factors in the pathways of DC development. We find that FL plays an important role in directing multipotent cells into the DC lineage. We further show that the cytokines interleukin-10 and granulocyte-macrophage colony stimulating factor promote DC development. In this thesis, we have clarified the pathways to the development of the resident dendritic cell subsets, and described some of the factors involved in regulating these pathways. The elucidation of the pathways to steady state resident DC development will form the basis for understanding the regulation of these pathways, and the adaptation of these pathways under conditions of infection.