Thesis--University of Oklahoma. / Includes bibliographical references (leaves 51-55).

Advances in medical treatment are leading away from generalized care towards intelligent systems or devices which can sense and respond to their environment. With these devices, the burden of monitoring and dosing for treatment can be removed from the doctor (or the patient) and be placed on the device itself. Implicit closed-loop control systems will allow the device to respond to its environment and release therapeutic agent in response to a specific stimulus. Environmentally responsive hydrogels show great promise in being incorporated in such an intelligent device, such as pH-responsive hydrogels which can swell and deswell in response to changes in the pH of the media. Thus, pH changes can be exploited for controlled and intelligent drug delivery when used in combination with these pH-responsive hydrogels. In this work, heterogeneous, thermal-redox initiated free-radical polymerizations were developed to synthesize novel pH-responsive hydrogels, microparticles, and nanogels. The specific disease of interest was type I diabetes, which requires daily doses of insulin both at a basal amount and either a postprandial or preprandial bolus in order to maintain blood glucose levels within safe limits. To allow pH-responsive hydrogels to be sensitive to glucose, glucose oxidase was incorporated which oxidizes glucose to gluconic acid. A novel inverse-emulsion polymerization method was developed for the synthesis of poly[2-(diethylaminoethyl methacrylate)-grafted-polyethylene glycol monoethyl ether monomethacrylate] (P(DEAEM-g-PEGMMA)) nanogels (100-400 nm) for intelligent insulin delivery. The new polymerization method allowed the incorporation of hydrophilic components, such as glucose oxidase and catalase, as well as PEG surface tethers of lengths 400 Da up to 2000 Da. Surface tethers successfully decreased the surface charge of the nanogels. Insulin loading and release was determined for microparticles which were able to imbibe substantial amounts of insulin from solution when swollen, entrap the insulin when collapsed, and then release the insulin in response to either a pH or glucose stimulus. / text

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 13:38:08.346

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.

Equine herpesvirus-1 (EHV-1) is one of the major viral pathogens causing respiratory disease, abortion, perinatal mortality and neurologic disease among horses resulting in significant economic losses to the equine industry. The virus can also remain latent in the horses and recrudesce at any time. Type-I interferons (IFNs) act as a first line of defense against many viral infections. In this study we investigated the type-I IFN response against the neuropathogenic T953 strain of EHV-1 in equine endothelial cells (EECs). The results showed that after a transient induction of IFN-β mRNA as well as protein at an early time (3h) post infection (p.i.), T953 strain of EHV-1 suppressed further induction of IFN-β at later times (12h onwards). Studies were done to confirm that the suppression of type-I IFN induction at later time points was not due to the normal IFN-β induction kinetics, it was rather because of the active interference by the virus. Investigation of the mechanisms by which T953 interferes with IFN-β production revealed that the virus degraded the endogenous level of the transcription factor, interferon regulatory factor 3 (IRF-3) and also down-regulated the activation of IRF-3 followed by its accumulation in the nucleus. However, T953 infection caused degradation of nuclear factor κB (NF-κB) inhibitory protein IκBα and also induced p50 subunit to translocate into nucleus from cytoplasm suggesting activation of NF-κB signaling. This also indicated that inhibition in the type-I IFN production was probably not due to the inhibition of NF-κB. The results of these studies also indicated that T953 virus was resistant to the biological effect of the recombinant equine IFN-α in vitro. Investigation of the reason of this resistance showed that T953 virus interfered with the cellular JAK-STAT signaling mechanism by which type-I IFN exerts its antiviral effect. Moreover, the studies revealed that downstream of the JAK-STAT signaling, T953 virus also inhibited the expression of cellular antiviral proteins including interferon stimulated gene 56 (ISG56) and viperin. Altogether, these data indicate that the T953 strain of EHV-1 interfered with the host cell innate immune responses by modulating type-I IFN mediated immune responses at multiple levels in vitro.

Differential item functioning (DIF) occurs when individuals from different groups who have equal levels of a latent trait fail to earn commensurate scores on a testing instrument. Type I error occurs when DIF-detection methods result in unbiased items being excluded from the test while a Type II error occurs when biased items remain on the test after DIF-detection methods have been employed. Both errors create potential issues of injustice amongst examinees and can result in costly and protracted legal action. The purpose of this research was to evaluate two methods for detecting DIF: logistic regression (LR) and Mantel-Haenszel (MH). To accomplish this, meta-analysis was employed to summarize Monte Carlo quantitative studies that used these methods in published and unpublished literature. The criteria employed for comparing these two methods were Type I error rates, the Type I error proportion, which was also the Type I error effect size measure, deviation scores, and power rates. Monte Carlo simulation studies meeting inclusion criteria, with typically 15 Type I error effect sizes per study, were compared to assess how the LR and MH statistical methods function to detect DIF. Studied variables included DIF magnitude, nature of DIF (uniform or non-uniform), number of DIF items, and test length. I found that MH was better at Type I error control while LR was better at controlling Type II error. This study also provides a valuable summary of existing DIF methods and a summary of the types of variables that have been manipulated in DIF simulation studies with LR and MH. Consequently, this meta-analysis can serve as a resource for practitioners to help them choose between LR and MH for DIF detection with regard to Type I and Type II error control, and can provide insight for parameter selection in the design of future Monte Carlo DIF studies.

If auditors question a company’s ability to continue existing, they should issue a going concern opinion in the audit report. Whether or not auditors will issue a going concern opinion depends on auditors’ ability to identify going concern problems, as well as their decision whether or not to issue going concern opinions. In Sweden, the going concern accuracy rate has been low compared to other countries. The aim of this study is therefore to analyse whether it is auditors’ lack of ability to identify going concern problems or their decision not to issue a going concern opinion, or perhaps both, that could explain the relatively low accuracy rate. Interviews with four auditors from the Big Four audit firms and four CFOs from middle sized companies were conducted. The results show that there are factors speaking both for and against auditors’ ability to identify going concern problems, why we cannot say for sure whether auditors’ lack of ability to identify going concern problems could be an explanation to the relatively low accuracy rate. The results do however reveal that auditors actively make decisions not to issue going concern opinions to their clients as much as possible, which could explain the relatively low accuracy rate.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

Bone consists of collagen/hydroxyapatite (HA) composites in which poorly crystalline carbonated calcium phosphate is intercalated within the fibrillar structure. Normal bone mineral is a carbonated-apatite, but there are limited data on the effect of mineral containing carbonate on cell response. Although the exact biological role of silicate in bone formation is unclear, silicate has been identified at trace levels in immature bone and is believed to play a metabolic role in new bone formation. To mimic the inorganic and organic composition of bone we have developed a variety of bone graft substitutes. In the present body of research, we characterized the surface composition of human cortical and trabecular bone. When then characterized the surface compositions of the following potential bone substitutes: carbonated hydroxyapatite (CO₃²-HA), silicated hydroxyapatite (Si-HA), and collagen sponges mineralized with calcium phosphate using the polymer-induced liquid-precursor (PILP) process. In the latter substitutes, the PILP process leads to type I collagen fibrils infiltrated with an amorphous mineral precursor upon which crystallization leads to intrafibrillar HA closely mimicking physiological bone mineral. We then determined the osteoblast-like cell response to each bone substitute to characterize the substrate’s effect on osteoblast differentiation. The observations collectively indicate that cells are sensitive to the formatting of the mineral phase of a bone substitute and that this format can be altered to modulate cell behavior.