Cardiovascular disease is most deadly medical condition in the United States. Medial vascular calcification is a disease that often precedes other more serious cardiovascular diseases that have high mortality. In order to research new therapies for the treatment of medial vascular calcification, an in vitro cell culture model must be developed that mimics the process in vivo. This disease is shown to be an active, cell-mediated process where the vascular smooth muscle cells (VSMCs) in the arteries are differentiating into osteoblast-like cells and depositing hydroxyapatite mineral in the artery walls. By administering inorganic phosphate to cell culture medium, an osteogenic shift can initiated in VSMCs in vitro resulting in calcium deposition and an increase in bone related proteins. We propose to develop and characterize a model for vascular calcification and investigate the effects of magnesium supplementation on in vitro calcification and cellular phosphate uptake.

A leukemia Differentiation Factor (DF), that induced differentiation of rat leukemia MIA C51 cells, was isolated from endotoxin-stimulated rat lung conditioned media.

Disabled-2 (Dab2) is a mitogen-responsive adaptor protein playing a key role in multifaceted cellular functions, such as endocytosis, epithelial-mesenchymal transition (EMT), immune function, stem cell differentiation, oncogenesis, cell signaling, and inflammatory responses. The adenosine deaminase RNA-specific binding protein (ADAR1) is a multifunctional RNA-editing enzyme that can convert adenosine to inosine, which can modulate gene expression and cellular functions in multiple pathways, such as mRNA translation by changing codons and the subsequent protein sequence, pre-mRNA splicing by changing splice site sequences, RNA stability by altering sequence for nuclease recognition, and RNA structure-dependent functions by altering RNA-protein interactions. ADAR1 has displayed a largely pro-tumorigenesis role, especially its immunosuppressive function in cancer cells, which attributes ADAR1 as a potential novel immune checkpoint for cancer treatment. In our lab, we employed an F9 mouse teratocarcinoma stem cell differentiating model and confirmed that Dab2 is an indispensable element for retinoic acid (RA)-induced F9 cell differentiation. Interestingly, our new findings indicated that during the process of RA-induced F9 cell differentiation, both the protein levels of Dab2 and ADAR1 are significantly upregulated, and siRNA-mediated Dab2 silence results in the silence of ADAR1. In addition, results from EMT models and statistical analysis from the human TCGA database further indicated that there is a positive correlation between the expression of Dab2 and ADAR1. Our results imply that Dab2 and ADAR1 may cooperate with each other to modulate cellular functions, which will present a novel mechanism for the mechanistic study of Dab2 in tumorigenesis.

Regulation of host health is intricately coordinated by a diverse interplay of immune cells detecting assaults from pathogens via recognition of pathogen associated molecular patterns (PAMPs) to mount an immune response, as well as detecting damage associated molecular patterns (DAMP) to indicate an area of damage and signal tissue repair. The gastrointestinal tract is a major signaling hub for such immune responses, as intestinal epithelial cells (IECs) compose the epithelial barrier, immune cells surveillance breached barriers to regulate the gut microbiome, and intestinal stem cells (ISCs) proliferate to replenish the IEC pool. One such method for regulating these cellular functions downstream of PAMPs/DAMPs within the gastrointestinal tract is via NF-κB signaling. This cellular signaling pathway is activated by one of two pathways: the well- defined canonical NF-κB pathway and the understudied noncanonical NF-κB pathway. The noncanonical NF-κB pathway is unique as it requires NIK, the NF-κB-inducing Kinase, to further elicit signal transduction of this pathway. Noncanonical NF-κB activation is critical to maintaining gut health, as signaling is regulated at a precise level to ensure a balance of pro-/anti-inflammatory signals to elicit a proper damage response. Any perturbations to NIK-activated signaling significantly predisposes the gastrointestinal niche towards chronic inflammatory conditions of the gastrointestinal tract. In this work, we explore the potential involvement of dysregulated noncanonical NF-κB signaling in inducing chronic inflammatory diseases of the gut, including Eosinophilic Esophagitis (upper GI tract), Celiac Disease/Non-Celiac Gluten Sensitivities (small intestine), Inflammatory Bowel Disease (entire intestine/large intestine), and an inflammatory subtype of colorectal cancer being Colitis-Associated Colorectal Cancer (large intestine). We study this pathway via the use of murine models bearing genetic deletions, cellular models, and the generation of miniature organs (i.e. "organoids") in petri dishes. Further, we assess varying levels of NF-κB signaling through the genetic deletions of NIK and RelA to inhibit noncanonical and canonical NF-κB pathways, respectively. Reciprocally, we also examine overactivated signaling via loss of the negative regulatory NLRs, which are proteins that function to impede NF-κB signaling. Clinical relevancy of this work is evaluated using biopsy samples collected from human patients with active disease states. Culminating our work, we find that noncanonical NF-κB signaling levels is both tissue- and cell-type specific in driving disease formation. Finally, we conclude our findings by suggesting the promise of NIK as a potential candidate for disease biomarkers and a target for future drug development. / Doctor of Philosophy / Redness, swelling, heat, pain, and loss of function – these are the five signs of inflammation. Under normal physiological conditions, inflammation is the body's conserved evolutionary response by serving as the first line of defense against infections propagated by foreign invaders like pathogens (i.e. bacteria, viruses, fungi), while also signaling to the immune system to resolve tissue damage. Therefore, properly maintained pro-inflammatory signaling is critical to ensuring a healthy state. However, an imbalance in pro- and anti-inflammatory signaling elicits a long-term, low-grade form of inflammation termed "chronic inflammation". Unresolved chronic inflammation can persist for several months or even years and further predisposes patients to various chronic inflammatory conditions and even inflammation-induced cancer. The NF-κB cellular signaling mechanism is a central regulator of inflammation and can be activated upon either the canonical NF-κB or noncanonical NF-κB pathways. In comparison to its canonical counterpart, the noncanonical NF-κB is vastly understudied, especially in regards to gastrointestinal health. A unique feature of the noncanonical NF-κB pathway is the required stabilization of the NF-κB-inducing Kinase (NIK) protein, which is required for further propagation of this signaling network. As evidenced by our culmination of works, we reveal that Noncanonical NF-κB signaling is critical to gut health, as it maintains a precise cellular signaling mechanism within the gut tract by properly maintaining pro- and anti-inflammatory signaling. Additional, downstream implications include regulation of cell division and activation of cell death to elicit a proper damage response. Within this dissertation, we evaluate the understudied noncanonical NF-κB pathway in various chronic inflammatory diseases of the gut including Eosinophilic Esophagitis (upper GI tract), Celiac Disease/Non-Celiac Gluten Sensitivities (small intestine), Inflammatory Bowel Disease (entire intestine/large intestine), and an inflammatory subtype of colorectal cancer Colitis-Associated Colorectal Cancer (large intestine). Through the use of murine models bearing deletions of genes related to noncanonical NF-κB signaling (esp. NIK), cell models, and the generation of "mini-organs" organoids from isolated intestinal stem cells, we are able to model the involvement of NIK and noncanonical NF-κB signaling in maintaining gastrointestinal health. Clinical relevancy of these findings was further evaluated by quantifying noncanonical NF-κB signaling levels in human biopsies. Culminating our work, we find noncanonical NF-κB signaling to be context-specific in driving disease formation. Finally, we conclude this work by suggesting the promise of NIK as a potential candidate for disease biomarkers and target for future drug development.

No description available.

Myogenic differentiation of muscle cells in culture is characterized by changes in morphology and in pattern of gene expression. When the myoblasts in culture are induced to diffrentiate either by cell to cell contact or by serum-starvation, a vast array of muscle tissue-specific genes including α-actin, are activated and is accompanied by a concomitant down-regulation of non-muscle genes such as, β- and γ-actins, tubulins etc.. The coordinate activation of muscle-specific genes is suggested to be mediated by cis-acting regulatory sequences in the muscle gene-promoters and muscle-tissue-specific DNA-binding proteins belonging to the MyoD class of regulators. But the mechanism behind the repression of non-muscle gene expression during differentiation has not yet been well understood. To date, no consensus has been achieved on the mechanism governing the down-regulation of β- actin gene, and no information is available on the regulation of -y-actin gene during muscle differentiation. The results from the present study showed that during differentiation of BC^3H1 cells the β- and γ-actins genes were down-regulated to ≈25% of their initial levels in undifferentiated cells. Measurement of half-life during differentiation indicated that the half-lives of both β- and γ-actins decreased to ≈25% of their original levels in myoblasts. These results suggest that changes in mRNA stability play an important role in the down-regulation of non-muscle actin genes. Second messengers and oncogenes are known to block the differentiation program of muscle cells. In the present study cAMP and ElA were observed to inhibit the down-regulation of β- and γ-actin genes in BC^3H1 cells. In both cAMP-and E1A- treated cells the β- and γ-actin mRNAs were found to have a higher half-life than the untreated differentiated BC^3H1 cells. This observation also suggests that mRNA stability might play an important role in the regulation of β- and γ-actin gene expression. The muscle-specific α-actin is activated by cell-cell contact and serum-starvation. Results in the present study suggested that cAMP was able to inhibit the activation of α-actin expression mediated by serum-starvation while it had no significant effect on the signal mediated by cell-cell contact. It is hypothesized that the two signals mediating a-actin activation might follow different intracellular signalling pathways. The effects of cAMP and E1A on the expression of muscle-specific and non-muscle actins could be a direct primary event or might be an indirect secondary event, mediated by other intracellular factors such as myogenin. The results showed that cAMP did not block the transcription of the myogenin while secondary evidences suggested that cAMP might negatively-regulate myogenin at a point downstream of transcription. E1A was observed to block the expression of myogenin gene suggesting that E1A might be mediating its effect through myogenin. Because the muscle-specific(α) and non-muscle(β- and γ-) isoforms were expressed both in the presence and in the absence of myogenin, myogenin's role in the regulation of actin genes is unclear. / Thesis / Master of Science (MS)

This dissertation comprises three essays on theoretical and empirical issues in industrial organization. Chapter 1 outlines the issues explored in the subsequent chapters and briefly describes their conclusions. Chapter 2 explores how product differentiation impacts the incentive compatibility condition for firms to sustain implicit collusion in games of repeated interaction where, in contrast to previous studies, I focus on a market which is simultaneously vertically and horizontally differentiated. To achieve this objective, vertical differentiation is incorporated into an otherwise standard Hotelling framework. The ensuing mixed model of differentiation shows how the interrelationships between both forms of differentiation impact the incentives to collude, and is more general since it replicates previous findings throughout the literature. In Chapter 3, a multiproduct oligopoly model admitting product differentiation and a discrete choice demand model are proposed and estimated to determine if patterns of anti-competitiveness exist across distinct segments of the European car market. This chapter focuses on the evolution of price competition at a finer level than has been studied with a view to empirically challenge the notion that the European car market is wholly anti-competitive. Empirical results show that firm conduct varies due to the intensity of within-segment competition among rival firms. There is evidence of softer competition in the larger, mid- to full-sized segments and more aggressive competition in the smaller, entry-level subcompact segment. Chapter 4 represents a formal extension of the analysis in Chapter 3. In this chapter I examine the competitive structure of the U.S. automobile market using proprietary data comprising actual dealer-level transaction prices of several models of cars and light trucks sold in the domestic U.S. market between 2004 and 2007. The chapter is the first such study to employ consumer end-prices for automobiles in a structural New Empirical Industrial Organization (NEIO) framework. Empirical results reveal that there is more aggressive pricing in the light truck segments comprising minivans/SUVs and pickups, Bertrand pricing in the smaller, entry-level car segments, and softer competition in the full-size car segment. There is also a strong preference for domestically produced light trucks although consumers generally prefer to drive fuel efficient vehicles. / Ph. D.

This dissertation consists of three essays in empirical industrial organization with applications in U.S. airline and automobile industries. Chapter 1 motivates the aim of this dissertation with a brief summary of the main goals and findings of the subsequent chapters. The main focus of this dissertation is to higlight the changing environments in the U.S. airline and automobile industries in recent years and investigate their implications for the nature of industry competitiveness. Following the recession of 2000 and post 9/11 events, the U.S. airline industry has undergone major restructuring which has defined the way airlines compete today. Chapter 2 of this dissertation explores the impact of the presence of Low Cost Carriers (LCCs) on consumer welfare in this newly restructured market environment. Previous studies on LCC competition have not addressed the welfare issue and have only been limited to impact of LCC entry on average airfare. Departing from previous literature, this question is posed using a discrete choice model of demand for differentiated products. In chapter 3 we use a structural oligopoly model for differentiated products similar to chapter 2 to unveil the nature of conduct that exists in markets with endpoints which qualify as hubs of legacy carriers. In contrast to previous literature on airline hub market conduct, this chapter investigates the nature of conduct that exists in markets defined exclusively by network carrier hubs as a whole group incorporating product differentiation in the model framework. Finally chapter 4 uses the same methodological framework outlined in chapter 3 to explore the importance of frequent incidence of manufacturer incentives in shaping market conduct in the automobile industry. Unlike past literature on automobile market conduct, this is achieved using proprietary dealer level average transaction price data obtained from J.D. Power and Associates (JDPA) with a focus on the Big Three automakers. Specifically we use the widely successful Employee Discount Pricing (EDP) promotional program of 2005, the first of its kind, as a backdrop to identify changes in the nature of short run conduct among the Big Three that might be signalled by such promotional programs. / Ph. D.

The objective of this study was to assess the potential role of extracellular, cytosolic, and membrane cholesterol in the proliferation and differentiation of bovine myoblasts. In the first experiment, myoblasts isolated from Angus or Angus crossbred steers were cultured with 2% lipoprotein deficient fetal calf serum (LPDS) or normal fetal calf serum. Culturing with LPDS did not alter the cytosolic or membrane cholesterol content, or myoblast differentiation, but inhibited myoblast proliferation, compared to culturing with normal fetal calf serum. In the second experiment, myoblasts were cultured with or without lovastatin, a selective inhibitor of cholesterol synthesis. Culturing with 5 μM lovastatin did not affect medium concentration of cholesterol, but reduced cytosolic and membrane cholesterol contents, compared to culturing with vehicle control. Culturing with 5 μM lovastatin inhibited both myoblast proliferation and differentiation. In the third experiment, myoblasts were cultured with or without methyl-βcyclodextrin (MβCD), a chemical that depletes cholesterol from cell membranes. Treating myoblasts with 10 mM MβCD for 30 minutes reduced membrane and cytosolic cholesterol contents while increasing medium cholesterol concentration. Treating with MβCD inhibited both myoblast proliferation and differentiation compared to treating with vehicle control. Overall, this study showed that lovastatin- or MβCD-induced reductions in cytosolic and membrane cholesterol contents were associated with reduced proliferation and differentiation in bovine myoblasts. These associations suggest that cytosolic cholesterol, membrane cholesterol, or both may play a role in bovine myoblast proliferation and differentiation. / Master of Science / The objective of this study was to assess the potential role of extracellular, cytosolic, and membrane cholesterol in the proliferation and differentiation of bovine myoblasts. In the first experiment, myoblasts isolated from Angus or Angus crossbred steers were cultured with 2% lipoprotein deficient fetal calf serum (LPDS) or normal fetal calf serum. Culturing with LPDS did not alter the cytosolic or membrane cholesterol content, or myoblast differentiation, but inhibited myoblast proliferation, compared to culturing with normal fetal calf serum. In the second experiment, myoblasts were cultured with or without lovastatin, a selective inhibitor of cholesterol synthesis. Culturing with 5 μM lovastatin did not affect medium concentration of cholesterol, but reduced cytosolic and membrane cholesterol contents, compared to culturing with vehicle control. Culturing with 5 μM lovastatin inhibited both myoblast proliferation and differentiation. In the third experiment, myoblasts were cultured with or without methyl-β-cyclodextrin (MβCD), a chemical that depletes cholesterol from cell membranes. Treating myoblasts with 10 mM MβCD for 30 minutes reduced membrane and cytosolic cholesterol contents while increasing medium cholesterol concentration. Treating with MβCD inhibited both myoblast proliferation and differentiation compared to treating with vehicle control. Overall, this study showed that lovastatin- or MβCD-induced reductions in cytosolic and membrane cholesterol contents were associated with reduced proliferation and differentiation in bovine myoblasts. These associations suggest that cytosolic cholesterol, membrane cholesterol, or both may play a role in bovine myoblast proliferation and differentiation.

In this study, ADP-ribosylation reactions in the murine myelomonocytic cell line WEHI-3BD+ were investigated.