Browsing by Subject "Pancreas"
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Item Application of NMR in the characterization of existing and development of new methods for pancreas preservation(2012-10) Scott III, William EarlIslet transplantation is an emerging treatment for type-1 diabetes. Pancreas preservation has been identified as an area for improvement since many pancreata are exposed to >6-8 hours of cold ischemia reducing viable islet yields. Traditionally utilized methods of preservation depend on low temperatures and specialized preservation solutions to maintain viability. Oxygenation during preservation has been identified as a potential means to extend preservation and improve isolation outcomes. The two-layer method (TLM) was developed to oxygenate pancreata during preservation. After initial excitement, since it relies on oxygen delivery by surface diffusion alone, and oxygenation of human sized pancreata in this manner is impossible; its use clinically has faded. Persufflation (PSF), gaseous oxygen perfusion, offers an alternate means of actively delivering oxygen to tissue via the vasculature. Due to the inability of small animal models to properly demonstrate oxygen limitations, and the lack of consistent availability of human pancreata a porcine model was developed. In order to aid in the development and proper assessment of new techniques, the use of 31P-NMR spectroscopy to monitor ATP during preservation was investigated. It was able to confirm that while TLM may provide adequate oxygenation for maintenance of ATP levels in smaller pancreata (rat) it indeed is unable to maintain ATP in larger organs such as the pig or human pancreas. In contrast PSF demonstrated the ability to maintain pig or human pancreata for at least 24 hours with only a minimal decay in ATP levels observed. The relevance of these findings was confirmed both histologically as well as by the gold standard, islet isolation. Paired studies demonstrated the ability of PSF to maintain and possibly improve outcomes while extending preservation times to at least 24 hours. The methods developed in this dissertation can be applied to the development and comparison of other novel methods of preservation in the pancreas and other organs.Item Comparison of individually-adjusted heparin versus ultra low-dose aspirin for prevention of thromboemboli in immune-mediated hemolytic anemia in dogs and the safety of ultrasoud-guided fine needle aspiration of the feline pancreas: a case-control Study(2014-06) Crain, Sarah KathleenThe following thesis is a compilation of the clinical studies designed and performed over a 3-year period at the University of Minnesota College of Veterinary Medicine. The studies, which include a prospective, randomized clinical trial and a retrospective case-controlled study, are unrelated to each other in subject matter, but both proved to be challenging in their own way, and had a lot to teach as far as the challenges and benefits unique to each type of clinical trial. The chapters first include a review of canine autoimmune hemolytic anemia, which provided significant insight into the common types of studies performed in veterinary medicine as well as provides background on a complex and interesting disease. The second and third chapters detail the studies performed, as well as discuss the specific challenges, shortcomings, and learning issues presented with each study. Despite careful planning and anticipation of the potential issues that would be encountered, each study was imperfect in its own way. Performing these studies was a very valuable way to learn about the challenges of study design, data collection, and data evaluation.Item Fatty Acid Binding Protein 4 Alters Obesity Associated Cancer Metabolism via Lipid Desaturation and Redox Signaling(2019-05) Wirth, KeithBackground: An association of obesity with cancer incidence and worsened clinical outcomes has been established. Circulating and local levels of fatty acid binding protein 4 (FABP4), a lipid chaperone and adipokine, have been correlated with degree of obesity and metabolic dysfunction, and more recently with breast and pancreas cancer prognosis. FABP4 transcriptional regulation has been linked to cellular redox status, with nuclear factor (erythroid-derived 2)-like 2 (Nrf2) being an upstream regulator of this balance. We hypothesized FABP4 modifies fatty acid saturation indices in cancer, driving an altered redox status, and ultimately inducing tumor proliferation. Methods: Panc1 pancreatic adenocarcinoma and MCF7 breast cancer cells were treated with recombinant FABP4, R126Q (FABP4 point mutant), and HTS01037 (FABP4 inhibitor.) Cell growth and proliferation was assessed. Targeted lipidomic analysis was performed in the same conditions. Whole cell reactive oxygen species (ROS) was quantified via Amplex Red assay. Nrf2 activity was quantified via antioxidant response element luciferase assay, and cell proliferation with chemical inhibitor (brusatol) was assessed. Untargeted gene expression profiles after FABP4 or HTS treatment were studied via RNA sequencing. C57BL/6J FABP4 knockout (AKO) and littermate wild type (WT) mice were injected with Pan02 and E0771 cells, murine pancreas and breast cancer cell lines. Tumor volume and progression was evaluated. Results: Panc1 and MCF7 cells treated with recombinant FABP4 demonstrated increased proliferation relative to control and point mutant protein treatment. This increase was abolished with HTS treatment. Unsaturated/saturated fatty acid ratio was decreased with FABP4 treatment and increased with HTS treatment. ROS levels were decreased and Nrf2 activity was concurrently increased with exposure to exogenous FABP4. Nrf2 gene expression profile was upregulated with FABP4 treatment, independent of ER stress. Tumor progression was significantly decreased in AKO mice. Conclusions: FABP4 induces a shift in the fatty acid saturation index of tumor cells, activating Nrf2 expression and decreasing intracellular ROS, independent of ER stress, allowing for aggressive tumor proliferation.Item Modulation of prostate and pancreatic PhIP-DNA adducts by consumption of cruciferous and apiaceous vegetables in male wistar rats(2013-05) Warnert, Marissa A.PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ) is one of the most abundant heterocylic aromatic amines (HAA), which are dietary procarcinogens in over-cooked meat. Some phase I biotransformation enzymes activate several HAA, including PhIP, while specific phase II enzymes can detoxify PhIP and other HAAs. Cruciferous vegetables induce phase I and phase II enzymes, while apiaceous vegetables inhibit phase I enzymes; but it's unknown if their combined intake acts synergistically on carcinogen metabolism. We evaluated the effects of cruciferous and apiaceous vegetables, and their respective phytochemicals, either alone or combined, on prostate and pancreatic PhIP-DNA adducts in rats. For prostate, PhIP-DNA adducts were reduced by 1-week intake of apiaceous vegetables (by 33%, P < 0.05), PEITC/I3C (by 45%, P < .001), and the combination of PEITC/I3C + furanocoumarins (30% reduction, P < .01). There were no effects in pancreas. Our results suggest fresh vegetables and phytochemicals modulate biotransformation enzymes and may influence cancer risk.Item Reprogramming of different cell types into pancreatic beta cells by using transcription factor genes Pdx1, Ngn3 and MafA(2012-08) Akinci, ErsinIn this dissertation we studied the effect of the pancreatic transcription factor genes Pdx1, Ngn3 and MafA, all of which were cloned into single adenoviral construct. First, we investigated the reprogramming competency to Pdx1, Ngn3 and MafA of different cell types at different developmental stages from mouse and rat. Second, we looked at the effect of these three genes on a pancreatic rat exocrine cell line AR42J-B13 to see if this gene combination provides a true beta cell reprogramming event. Next, we investigated the effect of some small molecules to see if they increased the reprogramming efficiency of this gene combination. Finally we tried to sort the small fraction of insulin-positive cells formed after Pdx1, Ngn3, MafA transduction to enrich the insulin-positive cell population hence getting more accurate data from them.Transduction of different cell types with Ad-PNM showed that the Pdx1, Ngn3 and MafA gene combination is enough to stimulate the expression of Insulin genes with varying intensities depending on cell type. Rat cell lines responded to Ad-PNM in a better way than mouse cell lines by activating more beta cell genes. The number of responding cells to Ad-PNM was also higher for the rat cell lines than the mouse cell lines. Moreover progenitor-like cells as well as the cells developmentally related to beta cells are prone to be reprogrammed into beta-like state. Supporting this final statement, embryonic mouse heptoblasts from Pdx1-GFP transgenic mice gave the most promising result by activating the endogenous Pdx1 as well as many other important beta cell genes upon transduction with Ad-PNM.Further characterization of the effect of Ad-PNM on the most resposive cell type AR42J-B13 cells showed that after Ad-PNM the cells became post-mitotic, began to express Insulin genes together with many other beta cell genes, produced mature insulin hormone, changed the epigenetic state of their chromatin, and rescued diabetic mice if transplanted into the kidney capture. However, some important beta cell genes were not activated thereby making these cells unresponsive to glucose which is an indispensable beta cell quality. It is obvious that even though the changes are dramatic, the combination of Pdx1, Ngn3 and MafA did not provide a true beta cell reprogramming in vitro at least for this cell system. Among some of the small molecules which had been reported to favor beta cell formation, regeneration and/or survival, three of them including DAPT, BIX-01294 and NECA increased the effect of Ad-PNM on mouse hepatocyte-derived small cells. Moreover when all three were used together they showed a better efficiency. Even though there was a significant increase in the number of insulin positive cells, the fraction of the Ad-PNM-responding cells was still low after the small molecules. For that reason the effect of small molecules on reprogramming efficiency of Ad-PNM was compared by counting the fraction of insulin-positive cells out of Ad-PNM-bearing cells between control and experimental cell groups instead of performing qRT-PCR.Of the attempts that we performed to increase the low fraction of insulin-positive cells via different sorting techniques to get more accurate and reliable results from Ad-PNM responding cells, only fluorescence-activated cell sorting of insulin-immunostained cells worked. Even though these cells were enriched through the FACS, because the cells were nonviable not alive we could not perform any downstream application that necessitate living cells. This system allows us to do qRT-PCR only.