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<p>The lung is a unique organ, taking a key part physiology but also playing a vital role in host defense. The lung has therefore developed a complex innate immune system that includes ciliated mucus producing cells that trap and remove larger particles in the larger airways and a specialized set of immune cells in the smaller airways. In addition, a variety of host proteins play a vital role in the immune response in the lung. The structural protein caveolin-1 (cav-1) is known to play a role in the uptake of pathogens and controls a variety of signaling pathways, although less is known about its functions in the lung. Surfactant protein A (SP-A) is a secreted protein that is vital in the innate immune response by interacting with microbes and immune cells. The goal of this work was to further elucidate the specific mechanisms by which cav-1 and SP-A affect the host responses, including inflammation and airway hyperreactivity (AHR), to pathogens and particulates. Using a mouse model of environmental lung injury I show that cav-1 is vital in the host response to inhaled lipopolysaccharide (LPS). Although the cav-1 deficient mice had greater lung inflammatory indices compared to wild-type mice, they exhibited reduced AHR following LPS exposure. The uncoupling of these two parameters led me to investigate the role of cav-1 in the contraction of airway smooth muscle and production of nitric oxide, both of which are known to regulate AHR. The bronchi of cav-1 deficient mice contract less than those from wild-type mice although their structure and receptor independent responses were not altered. The absence of cav-1 also resulted in increased nitric oxide levels in the lavage fluid and increased inducible nitric oxide synthase expression in the lung tissue. Administration of the potent and specific inhibitor 1400W increased AHR to levels comparable to wild-type mice. Following intranasal infection with Mycoplasma pneumoniae mast cell numbers increase in the lungs of mice and AHR is dramatically attenuated in SP-A<super>-/-</super> mice when mast cells are absent. Using SP-A<super>-/-</super> Kit<super>W-sh/W-sh</super> mice engrafted with TNF-alpha<super>-/-</super> or TNF receptor (TNF-R<super>-/-</super>) mast cells, I find that TNF-alpha activation of mast cells via the TNF-R and not mast cell derived TNF-alpha, leads to augmented AHR during Mp infection. Additionally, Mp infected SP-A<super>-/-</super>Kit<super>W-sh/W-sh</super> mice engrafted with TNF-alpha <super>-/-</super> or TNF-R<super>-/-</super> mast cells have decreased mucus production compared to those engrafted with wild-type mast cells, while burden was unaffected. Together, these data help to further elucidate the role of cav-1 and SP-A in innate immunity and may lead to the development of more effective human therapies.</p> / Dissertation

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<p>Improvements in genomic technology, both in the increased speed and reduced cost of sequencing, have expanded the appreciation of the abundance of human genetic variation. However the sheer amount of variation, as well as the varying type and genomic content of variation, poses a challenge in understanding the clinical consequence of a single mutation. This work uses several methodologies to interpret the observed variation in the human genome, and presents novel strategies for the prediction of allele pathogenicity.</p><p>Using the zebrafish model system as an in vivo assay of allele function, we identified a novel driver of Bardet-Biedl Syndrome (BBS) in CEP76. A combination of targeted sequencing of 785 cilia-associated genes in a cohort of BBS patients and subsequent in vivo functional assays recapitulating the human phenotype gave strong evidence for the role of CEP76 mutations in the pathology of an affected family. This portion of the work demonstrated the necessity of functional testing in validating disease-associated mutations, and added to the catalogue of known BBS disease genes.</p><p>Further study into the role of copy-number variations (CNVs) in a cohort of BBS patients showed the significant contribution of CNVs to disease pathology. Using high-density array comparative genomic hybridization (aCGH) we were able to identify pathogenic CNVs as small as several hundred bp. Dissection of constituent gene and in vivo experiments investigating epistatic interactions between affected genes allowed for an appreciation of several paradigms by which CNVs can contribute to disease. This study revealed that the contribution of CNVs to disease in BBS patients is much higher than previously expected, and demonstrated the necessity of consideration of CNV contribution in future (and retrospective) investigations of human genetic disease.</p><p>Finally, we used a combination of comparative genomics and in vivo complementation assays to identify second-site compensatory modification of pathogenic alleles. These pathogenic alleles, which are found compensated in other species (termed compensated pathogenic deviations [CPDs]), represent a significant fraction (from 3 – 10%) of human disease-associated alleles. In silico pathogenicity prediction algorithms, a valuable method of allele prioritization, often misrepresent these alleles as benign, leading to omission of possibly informative variants in studies of human genetic disease. We created a mathematical model that was able to predict CPDs and putative compensatory sites, and functionally showed in vivo that second-site mutation can mitigate the pathogenicity of disease alleles. Additionally, we made publically available an in silico module for the prediction of CPDs and modifier sites.</p><p>These studies have advanced the ability to interpret the pathogenicity of multiple types of human variation, as well as made available tools for others to do so as well.</p> / Dissertation