Application of novel cellular biology techniques to understanding the physiology and pathophysiology of the developing lung

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Johar, Dina
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Events at the level of cellular physiology are important to both pulmonary vascular development and in the pathophysiological processes that lead to pulmonary diseases of the newborn. Cell remodelling throughout development is a disorder of abnormal lung growth due to tissue hypoplasia or pulmonary artery hyperplasia. My thesis addresses two diseases of the lung: congenital diaphragmatic hernia (CDH) and persistent pulmonary hypertension of the newborn (PPHN). Nitrofen, a teratogen, induces CHD in rats and human, however, the mechanism is not fully known and there is no specific gene mutation associated with nitrofen-induced CDH. PPHN is a respiratory failure during circulatory transition because the lung fails to replace fluids and blood with inspired gases. As a result, the pulmonary artery pressure remains high due to hypertensive remodeling where a higher than normal Filamentous:Globular (F:G-actin) ratio increases stiffness, resulting in a thicker vessel diameter. The overall hypothesis that was examined was that methods such as Next Generation Sequencing (NGS) and Laser Scanning Cytometry (LSC) can provide novel insights to encompass both embryonic lung toxicity and the effect of unabated stress on development and on pathophysiological lung conditions postnatally. The primary objective was to evaluate the utility of two new methods to investigate CDH and PPHN. The research undertaken was able to identify changes in miRNA levels in the nitrofen rat model of CDH using NGS. A total of 186 known mRNA and 100 miRNAs were diferentially expressed in nitrofen-induced hypoplastic lungs. Sixty-four rat miRNAs homologous to known human miRNAs were identified. A subset of these genes may promote lung hypoplasia in rats and/or humans. Potential miRNA pathways relevant to nitrofen-induced lung hypoplasia include PI3K, TGF-β, Wnt and cell cycle kinases. Also, the research demonstrated that LSC can be used to image and quantify cytoskeletal alterations in the form of the F:G-actin ratio at the cellular level. Furthermore, the observations obtained with the LSC in vitro are amenable to validation in any in vivo model of interest. The research undertaken supports the proposal that NGS and LSC can open new avenues for research of CDH and PPHN.
MicroRNA, congenital diaphragmatic hernia, nitrofen, lung hypoplasia, mechanical stretch, newborn, normoxia, F:G actin, hypoxia, vascular remodeling, persistent pulmonary hypertension, cell cycle, apoptosis, Hoechst, laser scanning cytometry, phantom