Medical Biology - Theses
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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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ItemMultiple endocrine neoplasia type 2B: modelling the disease in human cells and avian embryos( 2017)Paediatric cancer initiation is difficult to study because the early stages are often prenatal. Patient cells at the time of detection already have complex genetic (including epigenetic) changes, and the cancer cell population is heterogeneous. One way to model cancer initiation at the organism level is to engineer the specific initiating mutation into the appropriate cell lineage of an experimental animal embryo. For the human cell context, an ideal cell model would start with the normal human cell of origin and create candidate initiating mutations. Multiple Endocrine Neoplasia type 2B (MEN2B) is an autosomal dominant complex oncologic disease of the neural crest (NC) cell lineage, a so-called neurocristopathy. It presents with i) multiple mucosal ganglioneuromas including hyperplasia of the enteric (gut) nervous system leading to gastrointestinal disorders, ii) pheochromocytoma, with sympathoadrenal (SA) hyperplasia and catecholamine disturbance, and iii) medullary thyroid carcinoma with C-cell hyperplasia, elevated calcitonin and calcium metabolism disturbance. In addition, patients have marfanoid facial features. MEN2B results from de novo germline gain-of-function mutations in the gene RET, most often M918T. RET codes for the signalling receptor for the growth factor ligand GDNF, hence in MEN2B cells, RET signalling is divorced from GDNF availability. MEN2B is rare but it is often misdiagnosed especially early in life. This is due to the nature and diversity of the lineages affected; SA and enteric NC-lineage cells and thyroid C-cells, the latter being of foregut endodermal entero-endocrine lineage. We were the first to successfully use CRISPR/Cas9 to mutate the developing chicken embryo in vivo, showing phenotypic abnormality. This included creating a single point mutation by homology directed repair in vivo in NC cells at the avian MEN2B homologous site (M910T). For the human cell context, we combined the CRISPR/Cas9 technology and knowledge of embryo development and cell differentiation to create MEN2B M918T cells using the human embryonic stem cell (hESC) lines H9, HES3 and MEL2. We modified a hESC differentiation protocol to produce enteric NC-like cells, showing in vitro that these cells upregulated key NC and enteric genes. Functionally we also showed higher proliferation and greater axon production in the MEN2B mutant cells: this is consistent with the ganglioneuroma phenotype. In addition, we developed a new differentiation protocol to produce human SA progenitors and medullary chromaffin-like cells, as marked by expression of key catecholamine genes TH and PNMT, and expression of adrenaline and noradrenaline by HPLC. These cells are affected in MEN2B patient pheochromocytoma. We then developed a novel differentiation protocol for thyroid C-cell-like cells from hESCs via Definitive Endodermal Cells. These cells produce Calcitonin and we validated their functionality by ELISA assay and compared the MEN2B clones with the control hESCs. These cells are affected in MEN2B patients resulting in medullary thyroid carcinoma.