Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Nuclear Factor kappaB (NF-êB) is a transcription factor whose activation is increased in settings of allergic asthma. At least two parallel NF-êB pathways exist: the classical pathway, which plays a role in inflammation and cell survival, and the alternative pathway, which regulates lymphoid cell development and organogenesis. The classical NF-êB pathway regulates inflammatory responses derived from lung epithelial cells; however, the role of the alternative pathway in lung epithelial cells remains unclear. We demonstrate that both classical and alternative NF-êB are activated in lung epithelial cells in response to multiple pro-inflammatory agonists, and siRNA-mediated knockdown of alternative NF-êB proteins largely attenuates pro-inflammatory cytokine production. Furthermore, simultaneous activation of both pathways leads to cooperative increases in pro-inflammatory responses, indicating a potential role for both classical and alternative NF-êB in the regulation of epithelial-derived pro-inflammatory responses. NF-êB activation in the epithelium modulates allergic inflammation in mouse models of allergic airway disease, however, its role in the context of an allergen relevant to human asthma remains unknown. In order to address the impact of inhibition of NF-êB in the epithelium in vivo, we utilized a House Dust Mite (HDM)-induced model of allergic airway disease. We demonstrate that HDM exposure activates classical and alternative NF-êB in both murine lung epithelium and human bronchial epithelial cells. Furthermore, following exposure to HDM, airway hyperresponsiveness, neutrophilic inflammation, and remodeling are attenuated in transgenic CC10-NF-êBSR (airway epithelial specific inhibitor of classical and alternative NF-êB) mice in comparison to wild type mice. Our data also demonstrate that specific knockdown of the alternative NF-êB protein, RelB, in the lung partially protects against HDM-induced pro-inflammatory responses, indicating that both classical and alternative NF-êB are important in HDM-induced responses. NF-êB proteins are modified by the redox-dependent post-translational modification, S-glutathionylation, under conditions of oxidative stress. S-glutathionylation of IKKâ, an upstream kinase in the NF-êB pathway, is known to decrease its catalytic activity; however, it is unknown how S-glutathionylation of IKKâ occurs. GSTP is an enzyme that catalyzes protein S-glutathionylation under conditions of oxidative stress and has been associated with the development of allergic asthma. We aimed to determine whether GSTP regulates NF-êB signaling, S-glutathionylation of IKKâ, and pro-inflammatory cytokine production. We demonstrate that siRNA-mediated knockdown of GSTP modulates NF-êB activation, NF-êB transcriptional activity, and pro-inflammatory cytokine production in response to LPS, a component of a bacterial cell wall. Furthermore, we demonstrate that GSTP associates with IKKâ in response to agonist stimulation and dampens IKKâ-induced pro-inflammatory cytokine production, surprisingly, independent of its catalytic activity. We also show that GSTP associates with other proteins of the NF-êB pathway, indicating a potential dual mechanism for repression of NF-êB-induced signaling. These studies collectively demonstrate that classical and alternative NF-êB contribute to epithelial-derived inflammatory responses, and GSTP may be a novel target by which NF-êB can be regulated.

The processes of lung ventilation and perfusion, diffusion, and gas transport make up the system of breathing and tissue oxygenation. Here, we present several mathematical formulations of the essential processes that contribute to breathing. These models aid in our understanding and analysis of this complex system and can be used to form treatments for patients on ventilators. With the right analysis and treatment options, patients can be helped and money can be saved. We conclude with the formulation of a mathematical model for the exchange of gasses in the body based on basic reaction kinetics.

With previous indication of the Group VIA phospholipase A2 (iPLA2β) enzyme regulating ER-stress induced apoptosis in β-cells by regulating the anti-apoptotic protein Bcl-xL via alternative splicing, our lab postulated iPLA2β to be utilizing a similar mechanism to regulate apoptosis in mice lung. Our previous lab work has shown implications of lung function compromise in iPLA2β-/- mice, and we speculated the cause to be due altered lung architecture stemming from the attenuation of apoptosis. The western blot analysis in this study suggested that iPLA2β is involved in the regulation of Bcl-xL, but the mRNA ratios of the splice variants suggested that alternative splicing is not the mechanism iPLA2β is utilizing for the regulation in our animal models. Additionally, the observation and assessment of the lung morphology of the iPLA2β-/- and wild type mice suggested that iPLA2β does not play an integral role in lung morphology.

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. While Radiation therapy is effective at killing tumor cells or inhibiting their growth initially, development of resistance to treatments and recurrence of tumors are major issues. One of the major goals of Dr. Gewirtz’s laboratory has been to develop strategies to overcome the resistance and attenuate disease recurrence. One of these attempts involve employing vitamin D and its analogs in combination with radiation therapy. Our proposed studies were based on a previous finding where vitamin D and vitamin D analogs such as EB 1089, were shown to enhance the response to radiation in breast cancer through the promotion of autophagy. We extended these studies to non-small cell lung cancer (NSCLC) and were able to validate that 1,25-D3 (the hormonally active form of vitamin D) and EB 1089 does in fact sensitize A549 and H460 cells and prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack the vitamin D receptor or functional p53, respectively, 1,25-D3 failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when p53 was induced in otherwise p53 null H1299 NSCLC cells. Sensitization by 1,25-D3/ EB 1089 was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D3 or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by the vitamin D receptor, p53 and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D3 and EB 1089 in NSCLC.

Overexpression of oncoprotein MDM2 and mutations of tumor suppressor p53 are frequently observed in human cancers. The NFκB pathway is one of the deregulated pathways in oncogenesis. The overall goal of the project was to study the regulation of NFκB pathway by MDM2 in lung cancer. Our first effort was to determine the frequency of MDM2 overexpression in human lung tumor samples and to identify co-occurring abnormal gene expression by studying the levels of MDM2 and members of NFκB pathway with respect to p53 status. Higher than normal levels of MDM2 were found in approximately 30% of the cancer samples harboring wild-type (WT) and mutant p53. Expression of NFκB2, a mutant p53 inducible gene showed significant statistical correlation with MDM2 in cancer samples that harbored WT p53. A downstream target gene of NFκB2, Bcl2, showed a significant correlation to MDM2 levels, independent of p53 status. Lung cancer samples harboring mutant p53 exhibited elevated levels of NFκB2 though not statistically significant. Our next step was to corroborate findings from lung tumor samples with data from lung cancer cell line harboring WT p53-H460. Consistent with lung tumor samples, ectopic overexpression of MDM2 in H460, showed elevated expression of NFκB2 and Bcl2 with promoter upregulation of NFB2. Silencing of MDM2 proportionally downregulated NFκB2 and Bcl2 in H460 cells. Domain analysis of MDM2 suggested that increase in the NFκB2 promoter activity was not confined to the p53 binding domain of MDM2 suggesting their interaction via p53-dependent and p53-independent mechanisms. A functional cell growth assay showed retarded cell proliferation with downregulation of MDM2. Data from human lung tumor samples and lung cancer cell line suggest that overexpression of MDM2 mediates NFB2 upregulation to confer growth advantage, thus favoring oncogenesis.

Chronic respiratory diseases affects many people worldwide with little known about the mechanisms diving the pathology, making it difficult to find a cure. Improving the understanding of smooth muscle and extracellular matrix (ECM) interaction is key to developing a remedy to this leading cause of death. With currently no relevant or controllable in vivo or in vitro model to investigate diseased and normal interactions of small airway components, the development of a physiologically relevant in vitro model with comparable cell attachment, signaling, and organization is necessary to develop new treatments for airway disease. The goal of this study is to create a mechanically, biologically and structurally relevant in vitro model of small airway smooth muscle tissue. Synthetic Poly-L-Lactic Acid (PLLA) and decellularized pig lung ECM (DPLECM) were electrospun to form nanofibrous mats that can closely mimic natural bronchial tissue. The addition of DPLECM significantly changed the PLLA scaffold mechanically, biologically, and physically to bring it closer to the characteristics of the human lung. DPLECM scaffolds exhibited a significant decrease in the elastic modulus compared with PLLA alone. Histological staining and SDS-PAGE showed that after scaffold fabrication, essential proteins or protein fragments in natural ECM are still present after processing. Human bronchial smooth muscle cells (HBSMCs) seeded onto PLECM scaffolds formed multiple layers of cells compared to scaffolds composed solely of PLLA. Phenotype of smooth muscle is better maintained when DPLECM is incorporated into the scaffold shown by enhanced contractile protein expression and increased collagen production for normal smooth muscle remodeling of the scaffold. In summary, this research demonstrates that a PLLA/DPLECM composite electrospun mat is a promising tool to produce an in vitro model with the potential to uncover unknown characteristics of bronchiole smooth muscle behavior in diseased or normal states.

Appendices data can be found at: A: http://dx.doi.org/10.15142/T35P49 B: http://dx.doi.org/10.15142/T3201B C: http://dx.doi.org/10.15142/T3X59V D: http://dx.doi.org/10.15142/T3SG6K F: http://dx.doi.org/10.15142/T3NP4N

Cystic Fibrosis (CF) is the most common hereditary genetic disorder among Caucasians. Pseudomonas aeruginosa is a major cause of morbidity in cystic fibrosis patients. Chronic infection with P. aeruginosa eventually occurs and is associated with a switch to biofilm formation of the bacteria. The symptoms and pathology of acute and chronic P. aeruginosa infections differ greatly. The first line of defense within the lung is the physical barrier of the lung epithelia. The examination of established biofilm interactions with lung epithelia is difficult. Here, I use the Calgary Biofilm Device co-culture system to conduct the concurrent analysis of established biofilms and planktonic bacteria with A549 lung cells. Comparison of P. aeruginosa biofilm and planktonic bacteria’s effects on A549 lung cells showed that planktonic bacteria caused more A549 cell rounding and death, while biofilm stimulated more IL-8 release by epithelial cells. Biofilm was shown to secrete significantly more Pseudomonal Elastase than planktonic, causing A549 morphological changes and loss of tight junctions. The antimicrobial peptide LL-37 was shown to differentially affect biofilm and planktonic bacteria. LL-37 caused a decrease in twitching of planktonic bacteria and exposure to LL-37 for 48 hours resulted in a decrease in elastase secretion likely due to down-regulated type 2 secretion. When established biofilms were compared with newly adherent biofilms, young biofilms were shown to have characteristics similar to both planktonic bacteria and mature biofilms. From this data we can follow the pattern of bacterial virulence as P. aeruginosa transitions from the planktonic mode of growth to the eventual mature biofilm that is associated with chronic infection. In conclusion, this study provides the foundation for a co-culture system that can be used to study the host-pathogen interactions of mammalian epithelia with established P. aeruginosa biofilms. The future adaptations of this model will better represent the in vivo characteristics of chronic lung infection to delineate ongoing virulence mechanisms of the bacteria causing host cell stimulation and damage. / May 2016

<p>The small GTPases <italic>HRAS, <italic>NRAS and <italic>KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in <italic>RAS, with 98% of those occurring in the <italic>KRAS isoform. While there have been many advances in the understanding of <italic>KRAS&ndash;driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of <italic>Kras&ndash;driven tumorigenesis in the lung:</p><p>(<italic>I) Despite nearly identical protein sequences, the three <italic>RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, <italic>KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to <italic>HRAS and <italic>NRAS. To determine the consequences of rare codon bias during <italic>de <italic>novo tumorigenesis, we created a knock-in <italic>Kras<super>ex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the <italic>Kras<super>ex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non&ndash;oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in <italic>Kras tumorigenesis.</p><p>(<italic>II) Lung cancer patients exhale higher levels of NO and <italic>iNOS<super>-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes <italic>Kras&ndash;driven lung adenocarcinoma, and NOS inhibition may decrease <italic>Kras&ndash;driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L&ndash;NAME in a genetically engineered mouse model of <italic>Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic <italic>Kras<super>G12D and dominant-negative <italic>Trp53<super>R172H in lung epithelial cells. L&ndash;NAME treatment was provided in the water and continued until survival endpoints. In this model, L&ndash;NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.</p> / Dissertation