Thesis--Lund. / Extra t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1991. / Added t.p. with thesis staement inserted.

Thesis (doctoral)--Lund University, 1991. / Added t.p. with thesis staement inserted.

Thesis--Lund. / Extra t.p. with thesis statement inserted.

OBJECTIVE: In today’s world, diabetes has become an ever-growing crisis, with no definitive cure yet found. In a report conducted by the American Diabetes Association in March of 2018, it was noted that 1.5 million Americans are diagnosed with diabetes each year (ADA, 2018). Insulin is both a limited and expensive source, with prices of Lispro, a rapid-acting insulin, costing upwards of $306 per 1000 units, to Glargine, a basal-analog insulin, costing $298 per 1000 units (McEwen et. al, 2017). Because of this, many diabetics are left with no alternatives to properly treat their blood sugars and maintain a healthy HbA1c level, a laboratory measure of glucose bound to hemoglobin that indicates a diabetic’s blood sugar over a two to three-month period (Mayo Clinic, 2018). Even with insulin treatment, diabetics can suffer from microvascular complications ranging from nephropathy, retinopathy, or even death thereafter if not properly cared for (Klein et. al, 2005). In turn, many researchers have delved into analyzing and perfecting a potential treatment procedure known as islet transplantation that can serve to eliminate the necessity of insulin injections and pump devices, and replace the beta cells destroyed by complications from Type I diabetes. Islet transplantation is the process of extracting healthy islets from the pancreas of an organ donor, purifying the islets in cell culture media that works to recover islet cells, known as islet isolation, and injecting the isolated islets into diabetic recipients whose beta cells are nonfunctional (Alejandro et. al, 2018). The goal of this procedure is to restore proper function of endogenous islets in the body, which contain beta cells that work to secrete insulin and better regulate the body’s glucose metabolism. While pancreatic islet transplantation can reverse diabetes, the process is inefficient, with many islets lost to hypoxemia before the islets become vascularized (Kumatzu et. al, 2018). We hypothesize that by prevascularizing islets ex-vivo, and using a gelatinous scaffold seeded with endothelial cells, one can avoid ischemic induced loss. This will ensure that islets are delivered the necessary oxygen and nutrients they need in order to restore endogenous function. By inserting this prevascularized device into the subcutaneous space of C57 BL/6 mice, islets can be surrounded by a vast blood network, allowing them to function similarly to when they are in the pancreas. If completed properly, this could ease the difficulty of diabetics continuously having to self-regulate their blood sugar levels by multiple injections of exogenous insulin each day. METHODS: Prior to implanting a prevascularized device into the mouse model, we isolated and purified healthy islets from the pancreases of C57 B/L 6 mouse donors, using the steps outlined in the Edmonton Protocol (Shapiro et. al, 2006). Each device could house approximately 300-400 islets, so about two to three mouse donors were used per vascularized graft implanted. In conjunction with IVIVA Medical, the functionalized, three-dimensional islet graft was created and contained a perfusable vascular bed to better ensure islet survival and improve integration immediately after implantation. Mice were monitored daily to ensure the graft was stable inside the subcutaneous space and to ensure the mice were not experiencing any adverse reactions from the implant. On specified days post-implantation, the graft was explanted from the mouse, along with the surrounding tissue, to analyze the foreign body reaction experienced from the implantation and whether a vascular network formed. The tissue sample was then sent to the Histopathology Department for further processing and analysis. RESULTS: In all three groups in the study, foreign body reactions were expressed by the recruitment and presence of multiple cell lines, including macrophages, dendritic cells, and B and T lymphocytes. While immune cells proliferated, there were limited endothelial cells and islets present post-implantation, indicating the presence of hypoxemia, poor vascular formation, and a potent inflammatory response, ultimately leading to islet dysfunction. CONCLUSION: While prevascularizing the scaffolds helped them better perfuse while in the subcutaneous space, we found that the inflammatory reaction, coupled with improper islet seeding, did not initially lead to islet graft survival. With modifications, we plan to create a stronger vascular network to surround the islet cells that would ensure their durability and survival in the long-term. In utilizing this data, future research can work to better stabilize islet cells, with the end goal of translating this work into human models in the near future.

No description available.

No description available.

The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. / Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. / The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. / DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. / Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. / This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.

Clinical islet transplantation is a promising treatment for type 1 diabetes. It involves the transplantation of pancreatic islets, isolated from a donor, into the portal vein of a recipient in order to replace his/her dysfunctional islets. Though promising, islet implantation into the liver is greatly hindered by numerous problems, including mechanical stresses, inflammatory responses, exposure to high drug and toxin loads, as well as irretrievability. In order to address these concerns, investigation into alternative implant sites, such as the subcutaneous site, has intensified. Transplantation of islets within these extrahepatic sites is commonly met with three primary obstacles: 1) inadequate spatial distribution of the cells; 2) oxygen deficiency in the local environment; and 3) insufficient vascularization within and around the implant. Thus, the objective of this proposal is to engineer a superior bioartificial pancreas, a device combining novel biomaterials and insulin-secreting cells, by focusing on these critical issues, specifically how to best reduce islet aggregation, as well as increase oxygen delivery, both in the short term and long term. A highly macroporous silicone scaffold will be engineered to distribute the islets three-dimensionally, while not imparting diffusion resistances commonly encountered in microporous materials. Macroporous scaffolds will also permit vascular in-growth. In order to sustain oxygen levels at the moment of device implantation, a novel, oxygen generating disk, which relies on the decomposition of calcium peroxide, will be developed and incorporated alongside the scaffold to deliver short-term supplemental oxygen. Therefore, it is postulated that these bioactive scaffolds, which interact with islets on a spatial, chemical, and biological level, will improve the viability as well as the function of islets, both in vitro and in vivo, as compared to naked islets under extrahepatic conditions.

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting.