Breathing is essential for human life, yet tens of millions of people in the U.S. alone suffer from lung diseases. With each breath, lungs are exposed to the external environment. Inhaled air first passes through the trachea, bronchi, and finally the bronchioles before it reaches the alveoli where gases are exchanged. A barrier of epithelial cells protects the airways. In addition, epithelial glands also secrete protein-rich fluids onto the airway surfaces to help maintain sterility. Injury, disease, or other factors can damage these cells, and regiospecific stem cells (SCs) can divide to replace them. However, many important details about lung SCs are still unknown. For example, what processes control SC division? How do region-specific SCs differ from one another? And how does disease or injury impact SC biology? We found that some processes that regulate lung development also control adult SC division following injury. We show that SCs from airway glands give rise to surface epithelial cell types and glandular cell types. In contrast, surface SCs only generated surface cell types. Finally, we identify a type of cell in the glands that can regenerate surface cell types after severe injury. These studies provide new insights into the neighborhoods in which SCs reside in the large airways and processes that control their contribution to airway repair following injury. Overall, this research provides important new insights into adult SC biology and conditions affecting lung health.

The immune systems of cnidaria are important to study for two reasons: to gain a better understanding of the evolution of immune responses, and to provide a basis to partially redress the precipitous world-wide die-offs of reef corals, some of which have been attributed to diseases and stress. Many immune responses share ancient evolutionary origins and are common across many taxa. Using Swiftia exserta, an azooxanthellate ahermatypic local octocoral, as a proxy model organism to study aspects of innate immunity in corals and cnidaria allows us to address both of the reasons listed above while not using endangered species. Utilizing a coral that does not contain symbiotic dinoflagellates (zooxanthellae) simplifies the system by restricting the source of proteins to a single genome. The lack of zooxanthellae in Swiftia exserta also allows the animal’s simple adaptation to lab settings. This study of the innate immune system of an octocoral demonstrates: 1) a novel understanding of the microanatomy of octocoral tissues; 2) that Swiftia exserta has at least two cell types that function as constitutive immunocytes; and 3) the presence of two potent antibacterial peptides, one with a mass between 4694 and 4696 Daltons. My report on the microanatomy of the coenenchyme, the tissue between polyps, advances the understanding of octocoral anatomy by systematically comparing histology sections with electron micrographs. Applying various techniques of enzyme histochemistry, coupled with cryo-preservation, to the coenenchyme I have identified at least two populations of constitutive immunocytes in Swiftia exserta. Two antibacterial proteins are identified by protein purification and antimicrobial testing techniques. The more active protein is partially characterized with modern hyphenated mass-spectrometry techniques, and can be the focus of future study.

<p>Ageing is associated with a progressive decline in cognitive and physical function, as well as neurodegeneration. The DNA damage theory of ageing postulates that phenotypes associated with chronological ageing result from a time dependent accumulation of DNA damage caused by endogenously generated reactive oxygen species (ROS). In this work, we have used a host cell reactivation (HCR) technique to examine base excision repair (BER), the major pathway for removal of ROS generated damage, in fibroblasts from normal individuals and from patients with Cockayne syndrome (CS). The HCR assay utilizes an adenovirus encoded β-galactosidase (β-gal) reporter gene treated with methylene blue plus visible light (MB+VL) to measure BER of 7,8-dihydro-8-oxoguanine (8-oxoG). The results presented here demonstrate that host cell repair mechanisms remove MB+VL generated 8-oxoG from viral DNA and that reactivation of gene expression correlates with cellular repair capacity and requires CSA and CSB. Using the HCR assay, we demonstrate that culturing of primary human fibroblasts in media containing low levels of MB increases BER, suggesting increased DNA repair capacity may play a role in the therapeutic application of MB in Alzheimer’s disease treatment. We also demonstrate that BER decreases <em>in vitro </em>with increasing number of cell divisions, and that HCR of the damaged reporter gene is lower in fibroblasts from older donors. Using a second β-gal reporter gene assay, the enhanced expression assay, we were unable to show a relationship between the degree of decreased BER in CS and severity of clinical phenotype. However, we identified an interaction between CSB and the telomere protein TRF2. Overexpression of TRF2 leads to decreased nucleotide excision repair of UVC induced damage in a CSB dependent manner. We also demonstrate defective telomeres in the absence of functional CSB. The data presented in this work provide additional support for the DNA damage theory of ageing.</p> / Doctor of Philosophy (PhD)