No description available.

No description available.

Diabetes is a complex group of metabolic disorders with genetic, immunological, and environmental etiologies. Decades of diabetes research have elucidated many genetic drivers of normal islet function and dysfunction. Furthermore, genome wide associated studies (GWAS) have discovered that most diabetes susceptibility loci fall outside of coding regions, which suggests a role for noncoding elements in the development of disease. This highlights our incomplete understanding of the islet regulome and suggests the need for detailed functional analyses of noncoding genes to precisely determine their contribution to diabetes susceptibility and disease progression. Transcriptome analyses have revealed that the eukaryotic genome is pervasively transcribed. Strikingly, only a small proportion of the transcriptome is subsequently translated into protein; the majority is made up non-protein coding RNAs (ncRNAs). The most abundant class of these ncRNAs are called long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides that lack protein-coding potential. The establishment of lncRNAs, once dismissed as genomic dark matter, as essential gene regulators in many biological processes has redefined the central role for RNA in cells. While evidence suggests a role for lncRNAs in islets and diabetes, in vivo functional characterization of islet lncRNAs is lacking. For my thesis project, I sought to understand the lncRNA regulatory mechanisms that promote pancreas development and function. We conducted comparative transcriptome analyses between embryonic mouse pancreas and adult mouse islets and identified several pancreatic lncRNAs that lie in close proximity to essential pancreatic transcription factors. One of the candidate lncRNAs, Pax6 Upstream Antisense RNA (Paupar), mapped near Pax6, a gene encoding an essential pancreatic regulatory protein. We demonstrate Paupar is enriched in glucagon-producing alpha cells where it promotes the alternative splicing of Pax6 to an isoform required for activation of essential alpha cell genes. Consistently, deletion of Paupar in mice resulted in dysregulation of Pax6 alpha cell target genes and corresponding alpha cell dysfunction. These findings illustrate a distinct mechanism by which lncRNAs can contribute to cell-specific regulation of broadly expressed transcription factors to coordinate critical functions within a cell.

Metabolic syndrome defines a cluster of related comorbidities including obesity, Type 2 diabetes, fatty liver disease, and cardiovascular diseases. Increasingly prevalent in Western countries, metabolic syndrome diseases are a major focus of efforts to understand the complex genetics that underlie disease risk and severity. Immunoglobulin domain-containing receptor 2 (ILDR2) is an ER transmembrane protein first identified as a candidate genetic modifier of diabetes susceptibility in the context of obesity. Obese, leptin-deficient mice with hypomorphic Ildr2 expression had hypoinsulinemic hyperglycemia with reduced beta cell mass, suggesting that ILDR2 plays a role in maintain beta cell mass and function. Further studies proposed a role for ILDR2 in hepatic lipid metabolism as Ildr2 shRNA-mediated knockdown (KD) caused hepatic steatosis in mice. The goal of this thesis work is to clarify the role of ILDR2 in diabetes and hepatic steatosis in an effort to elucidate the specific mechanism of ILDR2. We developed a conditional Ildr2 knockout (KO) allele, enabling tissue-specific ablation in mice. Liver-specific and hepatocyte-specific KO mice did not develop hepatic steatosis. However, liver-specific KO mice treated with adenoviral Ildr2 shRNA accumulated hepatic triglycerides, suggesting off-target effects of the shRNA. Using RNA sequencing and sequence alignment, several gene candidates for shRNA off-targeting effect were identified. Future studies are proposed to elucidate role(s) of these genes in the previously described phenotype of Ildr2 KD mice. I conclude that Ildr2 ablation may contribute to the development of hepatic steatosis, but does not play a major role in hepatic lipid metabolism. We also developed beta cell-specific (RIP2-cre) and pancreas-specific (Pdx-cre) Ildr2 KO mice and characterized them for diabetic phenotypes. Pancreas-specific KO mice displayed impaired glucose tolerance, reduced insulin secretion and decreased calcium signaling in islets. These results confirm a role for ILDR2 in islet cell function. Experiments performed in RIP2-cre beta cell-specific KO mice were confounded by effects of the Cre construct, prohibiting definitive conclusions about the role of ILDR2 in the beta cell. Additionally, because Ildr2 is expressed at low levels in beta cells, we propose that ILDR2 may function in islet macrophages. Overall, this work defines the metabolic functions of ILDR2, clarifying its role in hepatic lipid metabolism, and confirming its role in islet cell function. In addition, I discuss preliminary evidence suggesting that ILDR2 may function in the brain to regulate body weight and metabolism.

Langerhan’s cells (LCs) are the immune sentinels of the skin, sampling the cutaneous microenvironment and presenting captured antigen to T cells. A sheet-like proliferation of LCs is termed Langerhan’s cell histiocytosis (LCH), an enigmatic and poorly understood disorder with a widely varied clinical spectrum and disease course. In non-pulmonary LCH all cases reported to date have been monoclonal. Clonality argues for LCH as a neoplastic rather than reactive disorder. After initial investigation of the limitations of formalin fixed paraffin embedded tissues for downstream analysis, lesions of LCH were collected from 4 sites across Scotland. To further define the spectrum of LCH, clonality was assessed using an X inactivation assay based on the polymorphous region of the Human Androgen Receptor. To improve understanding of the assay, a study on post-mortem material was undertaken. This demonstrated a unique insight into patterns of X inactivation across different tissues of the same individual and highlighted potential pitfalls in interpretation. An important question was whether lesions of LCH associated with haematopoietic neoplasms were polyclonal or monoclonal proliferations? For the first time, associations of LCH with B-cutaneous lymphoid hyperplasia (B-CLH), lymphomatoid papulosis (LyP) and mycosis fungoides (MF) are reported. In two female cases, the LCs were polyclonal providing some reassurance that such lesions are reactive in nature and should not be regarded as potential second neoplasms. In a more expanded study a wide variety of primary LCH lesions were assessed for clonality. Significant limitations were posed by the quality of the material available; in 2 cases the lesions were found to be polyclonal. This is the first time such a result has been reported. Monoclonality was identified in 2 other cases including one of pulmonary LCH. The findings reported herein suggest that clonality and hence neoplasia cannot be assumed in all cases of primary non-pulmonary LCH. The possible functions of LCs in cutaneous lymphoma were explored. In T-cell lymphoma 2 cases reported here suggest a role for LCs in disease progression. In contrast, LCs play no significant part in the development or progression of cutaneous B-cell proliferations although other types of dendritic cells probably have an important role. By studying proliferations of LCs in a variety of settings, this work has extended knowledge of the spectrum of LCH. Displaying similar histopathological appearances, lesions of LCH may be best defined by clonality as well as cytokine expression and level of maturation. In future, such markers may be employed as prognostic indicators allowing individualised and targeted management.

The majority of human papillomavirus (HPV) types cause cutaneous and mucosal disease. Persistent infection with high-risk HPV types is the primary risk factor for the development of cervical cancer. The ability of the virus to persist is contributed to by numerous immune evasion mechanisms. We previously demonstrated that the HPV type 16 (HPV16) E6 protein, down-regulates epithelial (E)-cadherin expression and that the associated Langerhans cells (LC) depletion may contribute to impaired immune recognition by the host. The aims of this study were firstly to establish if E6 down-regulation of E-cadherin is conserved amongst all HPV types, secondly to determine if the reduced E-cadherin expression correlates with reduced LC density in HPV-infected tissues, thirdly, to identify a region of E6 responsible in E-cadherin regulation and fourthly to establish if down-regulation of cell surface E-cadherin also occurs in another DNA tumour virus, adenovirus (Ad). E6 protein from a range of HPV types representing the α, β and γ genera was expressed in HCT116 cells and the effect on cell surface E-cadherin expression was measured by flow cytometry. In addition, a series of tissues infected with HPV types representative of HPV of α, β, [nu] and γ genera were stained to confirm E-cadherin regulation in vivo and to determine the functional significance of E-cadherin expression in relation to LC localisation. In order to identify the region of the E6 protein that was important for E-cadherin regulation, a series of HPV16 E6 mutants were tested for their ability to regulate E-cadherin. Finally, the effects of Ad on cell surface E-cadherin were examined by measuring E-cadherin expression in Ad infected HCT116 cells. E6 down-regulation of E-cadherin was conserved in α, [nu] and γ genera but was lost in β-HPV types, correlating with the ability of the virus to persist. In vivo analysis of patient tissues confirmed this pattern of E-cadherin regulation by E6 types and showed a direct association between loss of E-cadherin and LC depletion, suggesting that E-cadherin regulation by E6 is the cause of depletion of LC in infected tissue. Mutational analysis of HPV16 E6 led to the identification of a putative E-cadherin regulatory region with a conserved motif, H/L/V-[phi]-X-X-X-X-R. A potential mechanism used by E6 to regulate cell surface E-cadherin involved down-regulation of p21[waf1/cip1] (p21) via a p53-independent pathway. Finally, study of Ad showed a similar ability of the virus to regulate E-cadherin, indicating conservation in another DNA tumour virus. This research shows that E-cadherin regulation by E6 is directly associated with LC depletion and viral persistence. The data presented here suggest that LC depletion by HPV is widely conserved in HPV types that cause persistent disease. E-cadherin regulation contributes to this effect through a specific regulatory region of the protein and manipulation of levels of cellular p21. These data may provide a foundation for the development of therapeutics for HPV that aim to overcome immune evasion by the virus.

No description available.

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.