Medical Biology - Theses
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ItemSteady-state and emergency dendritic cell development at a clonal level( 2019)Recent clonal fate and single cell RNA-sequencing studies demonstrate that significant lineage imprinting is already in place within individual haematopoietic stem and progenitor cells (HSPCs). Dendritic cells (DCs) represent one such branch of haematopoiesis and are responsible for pathogen-sensing and activation of the adaptive immune response. At the population level, all three major DC subtypes including type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs), can be generated from a restricted common DC progenitor (CDP) population downstream of HSPCs. However, recent clonal evidence has suggested earlier subtype-specific imprinting within the CDP and even early HSPC populations. Therefore, the current hierarchical model of haematopoiesis is insufficient to explain the complexity and dynamics of DC development. The aim of this thesis was to investigate the development of DCs at the single cell level. One caveat of most prior single cell lineage tracing studies was that clonal fate was only measured at a single time point. Therefore, questions remain as to whether the fate bias observed at a snapshot in time is consistent with earlier or later times. Here, using cellular barcoding, I develop an experimental and computational framework to allow robust periodical examination of lineage outputs of thousands of transient clones during DC development in vitro. I reveal that single HSPC clones are largely programmed regarding the types of DCs to make (fate), the number of DCs to produce (size), and when DC generation occurs (timing). Together, I define these unique properties as a clone’s cellular trajectory. Importantly, I demonstrate that a large proportion of early HSPCs are already committed towards either cDC or pDC generation, even when clonal output is measured over time. This finding is consistent with and further complements the most recent evidence of DC subtype imprinting during early haematopoiesis. Exogenous administration of Flt3 ligand (FL) is known to preferentially induce ‘emergency’ DC development, and is shown to provide promising therapeutic benefits in various conditions such as infection and cancer. However, how FL signals regulate cell proliferation and differentiation during early DC development is largely unknown. In this thesis, I investigate the clonal aetiology of this process. Using cellular barcoding, I demonstrate that emergency DC generation is predominantly driven by increased expansion of pre-existing HSPC clones that are already primed with DC potential. Consistently, enhanced cell cycle activity is found to be prominent within most early HSPCs after short exposure to FL. In particular, using a single cell multi-omics profiling approach, I identify key cellular and molecular events within a unique group of early HSPCs that are most responsive to FL stimulation, which include increased cell division, maintenance of hyper-proliferative potential and establishment of a DC lineage program. Collectively, the findings presented in this thesis provide new insights into the control and regulation of DC fate within individual HSPCs during steady-state and emergency haematopoiesis, with important implications regarding the maintenance or manipulation of DC generation in health and disease.
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ItemScreening for breath: identifying Aurkb as a novel regulator of lung development( 2019)The development of the lung is a highly regulated and complex process that is not fully characterised. Although there have been many studies into the development of the lung many of the mechanisms regulating lung organogenesis are still unclear. In recent years, the importance of epigenetic regulators in embryogenesis has been established but epigenetic control of lung morphogenesis is largely underexplored. I have developed a novel in vitro assay to grow embryonic lung stem cells. These cells, called pneumospheres express the early lung progenitor factor Sox9 and recapitulate the E11.5 lung throughout their time in culture. Pneumospheres can be genetically and chemically manipulated to assess the role of signalling pathways and genes-of-interest on lung progenitor cell self-renewal or differentiation. Using pneumospheres I performed a shRNA knockdown screen, targeting 130 genes involved in epigenetic regulation. Nineteen genes were identified in the screen and validated using in vitro and ex vivo culture systems to determine their role lung stem cells and branching morphogenesis. These experiments identified Aurora kinase B (Aurkb) as an interesting candidate gene. Aurkb exerts a dual role as a regulator of cell cycle and epigenetic control through phosphorylation of histones. Disruption of Aurkb either by short-hairpin RNA or by chemical inhibition in vitro abrogates growth of lung epithelial progenitor cells and causes defects in cell cycle, leading to an accumulation of cells in G2/M of the cell cycle. Conditional deletion of Aurkb in the embryonic lung, leads to a complete lack of lung tissue at birth and severe epithelial growth retardation can be seen as early as midgestation. Understanding the regulation of lung development will provide a better understanding of the lung organogenesis and how disruptions in normal biology can cause early lung diseases such as bronchopulmonary dysplasia or can have an impact on lung disorders later in life such as COPD or lung cancer.
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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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ItemThe role of BOK in apoptosis and development( 2013)The intrinsic (also known as the mitochondrial or BCL-2 regulated) pathway of apoptosis is regulated by members of the BCL-2 family, which can be divided into two main groups depending on whether they posses a prosurvival or pro-death function. BAX and BAK are two key players that are directly involved in the execution of apoptosis by mediating mitochondrial outer membrane permeabilisation (MOMP), which unleashes the cascade of caspases that lead to cellular demolition. The essential overlapping roles of BAX and BAK in the intrinsic apoptotic pathway is demonstrated by the fact that many cell types deficient for both proteins are highly resistant to a broad range of cytotoxic stimuli. In addition, mice lacking both proapoptotic proteins (Bax-/-Bak-/-) displayed several developmental abnormalities (such as persistence of interdigitating cells and excess neurons in the brain), and the few survivors that lived to early adulthood all developed severe lymphadenopathy and autoimmune diseases. However, perhaps surprisingly, some organs that are thought to require apoptosis for proper morphogenesis still developed normally in these Bax-/-Bak-/- animals. This indicates the existence of BAX/BAK-independent apoptotic or other cell death processes for the removal of superfluous cells. BOK, a BAX/BAK-related protein, may play a role in cell death signaling during embryogenesis either on its own, or in a manner overlapping with BAX or/and BAK. This thesis describes the first functional analysis of BOK in embryonic development and apoptosis in a physiological context through the study of BOK knockout mice. Since many BCL-2 family members exert their effects in the hematopoietic system, the Bok-/- animals were examined with particular emphasis on their lymphoid organs. Western blot and quantitative PCR analysis demonstrated that BOK is most prominently expressed in the brain and reproductive tissues. However, BOK-deficient animals remain fertile, displayed no obvious abnormalities, and failed to develop reproducible age-related diseases up to at least one year of age. In response to diverse cytotoxic insults, leukocytes from Bok-/- mice underwent apoptosis at a normal rate. These findings demonstrate that BOK is dispensable for embryonic development, fertility, and diseases-free survival of mice. To investigate whether BOK functions in a manner overlapping with BAX or BAK, Bok-/-Bak-/- and Bok-/-Bax-/- doubly deficient mice were generated. These animals also appeared largely normal, and the additional loss of BOK on top of BAX did not exacerbate or rescue infertility in BAX-deficient males. Surprisingly, aged Bok-/-Bax-/- females were found to accumulate abnormally increased follicles at various developmental stages in their ovaries, suggesting that BOK may function together with BAX to regulate follicle atresia. In the final part of this study, Bok-/-Bax-/-Bak-/- triple knockout mice were generated to investigate whether functional overlap exists between all three proteins. This also tested for the presence of alternative cell death mechanisms in the absence of all proapoptotic multi BH-domain BCL-2 family members (i.e. BOK, BAX and BAK). Although the majority of Bok-/-Bax-/-Bak-/- mice died perinatally, a very small number still managed to survive to weaning, indicating the existence of other (perhaps still uncharacterized) processes that regulate tissue morphogenesis through cell killing or cell loss (e.g. shedding of cells into luminal structures) during certain aspects of murine development. In addition, BOK/BAX/BAK triple knockout embryos, unlike those doubly deficient for BAX and BAK (Bax-/-Bak-/-), were already present at a significantly lower than expected Mendelian frequency just before birth (embryonic day E19), implying that BOK may be required, along with BAX and BAK for normal embryogenesis. Finally, the consequences of the combined loss of all three proteins in the hematopoietic system were examined with the use of chimeric mice. Such analyses demonstrated that BAX and BAK alone are sufficient for the development and homeostasis of hematopoietic cells, whereas BOK is dispensable in these processes.