Medical Biology - Theses
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ItemThe role of microRNAs miR-155 and miR-211 in myeloid malignancies( 2016)MicroRNAs are a class of non-coding RNA molecules that post-transcriptionally regulate several critical cellular processes including cell survival, proliferation and differentiation. A subset of microRNAs has well-established roles in cancer development, and microRNAs are emerging as important biomarkers of disease and potential therapeutic targets. This thesis focuses on the role of two microRNAs, miR-155 and miR-211 in myeloid malignancies, particularly in AML. miR-155 is a well-recognized oncomiR in B cell lymphoma. The role of miR-155 in AML, however, is rather enigmatic. Contrasting data suggest miR-155 can block differentiation and promote proliferation, and so function like an oncogene, or it may repress proliferation, induce apoptosis and promote differentiation, functions more closely aligned with tumour suppressor function. To establish if miR-155 is an oncogene or tumour suppressor in AML, the effects of enforced miR-155 expression were studied in murine models of AML, in vitro and in vivo. I show that enforced expression of miR-155 to the highest levels, suppresses colony formation and cell proliferation of AML cells in vitro, and is selected against in tumours in vitro and in vivo. Over time, however, AML cells are selected for intermediate levels of miR-155 expression, which is associated with restored colony formation and increased tumour burden in vivo. Additionally, I show that the differential effects of intermediate and high miR-155 expression levels in AML result from targeting contrasting transcriptional networks in AML. Importantly, I show that the dose-dependent associations of miR-155 expression and phenotype extends to pediatric AML, where the elevated miR-155 levels expressed in patients correlates to the intermediate and not the high miR-155 expression levels, detected in mouse tumours. miR-211 is a microRNA of which very little is known, and is primarily associated with invasion and metastasis in melanoma. In a screen of 700 microRNAs in an inducible model of myeloid differentiation, miR-211 was one of the most highly differentially expressed microRNAs, suggesting that it may have a role in the regulation of myeloid differentiation and consequently in myeloid leukemia. In this thesis, using a system of enforced miR-211 expression in hematopoietic reconstitution experiments, I describe for the first time a novel role for miR-211 as an oncogene in AML. I show that miR-211 drives a partially penetrant myeloid disease characterized by an expansion of CD11b/Gr-1 myeloid cells, anemia, thrombocytopenia and infiltration of peripheral hematopoietic tissues, phenotypically resembling myeloid leukemia. Additionally, I show that miR-211 expression is elevated in a subset of pediatric AML patients, and present data describing the molecular actions of miR-211 in the disease.
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ItemThe control of normal and leukaemic human cells by the colony stimulating factors( 1986)To examine the control of human haemopoietic cells by the haemopoietic hormones (the Colony Stimulating Factors, CSF’s) both purified factors and purified cell populations are required. Studies were initially performed to characterize the action of purified murine and purified and partially-purified human CSF’s on human marrow cells. Of the purified murine CSF’s, G-CSF was active on human bone marrow cells. The murine molecule Eosinophil Differentiation Factor (EDF) was shown to an Eosinophil-CSF with activity on murine and human cells. Recombinant human GM-CSF (rHGM-CSF) was shown to possess all the biological activities of the partially purified native molecule CSF-α. Both the glycosylated and non-glycosylated preparations of rHGM-CSF showed equivalent biological activity in vitro. Subsequent studies were performed to purify and characterize a population of normal committed-granulocyte progenitor cells and the action of CSF’s on these cells was examined. Normal human promyelocytes and myelocytes were obtained using the monoclonal antibody WEM-G11 and the fluorescence activated cell sorter. These cells demonstrated transient, CSF-stimulated proliferation in vitro and generated neutrophilic clones of less than 40 cells in size (clusters). These cells were stimulated to proliferate by human CSF-α, CSF-β and murine G-CSF. Clone transfer experiments documented the ability of clones initiated by one CSF (α or β) to proliferate when transferred to cultures stimulated by the other CSF. The cross-species activity of human CSF-β and murine G-CSF, and the ability of CSF-β to compete with radio-iodinated murine G-CSF for binding-sites on murine (and human) cells suggested that CSF-β was the human equivalent of murine G-CSF. A comparative study of leukaemic promyelocytes demonstrated that fractionated promyelocytes-myelocytes from patients with chronic myeloid leukaemia also showed transient clonal proliferation in vitro and responded to CSF-α, CSF-β and murine G-CSF. Promyelocytes from the blood of these patients generated clones of only two cells in CSF-unstimulated cultures. This behaviour was mimicked when normal promyelocytes-myelocytes were pulse-stimulated by CSF for 45 min. Leukaemic cells from patients with acute myeloid leukaemia also demonstrated CSF-stimulated proliferation in vitro and were responsive to CSF-α, CSF-β, rHGM-CSF and murine G-CSF. There was however considerable heterogeneity in the CSF-responsiveness of these cells. Differentiation-induction by CSF in leukaemic cells was examined using the human leukaemic cell line HL60. When stimulated by CSF, these cells showed increased expression of myeloid surface antigens (as monitored by three monoclonal antibodies) and decreased numbers of clonogenic cells. In some experiments the number of clonogenic cells was reduced to zero. In an attempt to establish a sensitive micro-assay for CSF, two target cell populations were examined. Normal promyelocytes-myelocytes displayed CSF-stimulated proliferation in a micro-assay system but this was not associated with a heightened sensitivity to CSF. The survival of mature human neutrophils and eosinophils in vitro was however shown to be enhanced by CSF’s in a “lineage-specific” manner and this assay was between 10^2-10^3 times more sensitive to CSF than agar cultures. These studies demonstrated that the Colony Stimulating Factors enhanced survival, stimulated proliferation and stimulated differentiation-commitment of normal and leukaemic human cells.