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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Islet pathobiology in congenital hyperinsulinism in infancy

Han, Bing January 2017 (has links)
Congenital Hyperinsulinism of Infancy (CHI) is a potentially lethal condition caused by excessive, unregulated insulin release from pancreatic β-cells. It is a complex clinical condition and the current understanding of this disease is still not completed. In this thesis, we investigated the disease islet pathobiology from 4 main perspectives; Using nucleomegaly as a novel diagnostic marker; Identifying the mosaic of immature delta-cells in atypical CHI (CHI-A); Assessing the insulin secretory profile at the ultrastructural level; Investigating endocrine cell turnover and the driving force/mechanism behind it. By quantifying the enlarged nuclei in the endocrine pancreas of patients with CHI, we discovered that the increased incidence of nucleomegaly is pathognomic for diffuse CHI (CHI-D). This finding potentially set a novel diagnostic hallmark for intraoperative diagnoses. A characteristic of CHI-A is a combination of active and quiescent islets. The maintained expression of NKX2.2 in somatostatin positive cells suggests an immature delta-cells phenotype in quiescent islets and this is potentially contributing to the pathobiology of CHI-A. By examining the insulin secretory profile at the ultrastructural level, as well as investigating the crucial exocytosis-related genes from both RNA and protein levels, our data suggested a greater secretory capacity in β-cells from focal CHI lesion compared to CHI-D. Despite seeing a maintained potential for proliferative (Ki67) in CHI samples, there was no significant increase in apoptosis rates (cleaved caspase-3) and whole cell mass compared to control samples. Alterations in the cellular localisation of cell cycle regulators are a plausible explanation for these abnormal disease dynamics. These data expanded our knowledge on understanding CHI, and provided us new clues for the phenotypical alterations and pathobiological mechanisms in patients with this disease. Meanwhile, they also provided new insights in the future management of CHI.
2

Pancreatic progenitor cell lines derived from patients with congenital hyperinsulinism

Eastwood, Lauren Elizabeth January 2013 (has links)
Islet transplantation has proved to be a useful treatment for Type 1 diabetes mellitus, but inadequate supplies of transplantable donor tissue have intensified the need to find a renewable source of β-cells. Human embryonic stem cells (HESC) are pluripotent and may offer a viable alternative to donor islets, but their targeted differentiation to a more specific β-cell phenotype is proving challenging. One strategy to restore β-cell mass is through activation of progenitor cells present in the pancreas. The aim of this study was to isolate and characterise progenitor cells from pancreatic tissue obtained from patients with Congenital Hyperinsulinism of Infancy (CHI). An enzymatic digestion was used to isolate islets from four patients with CHI and CHI-derived cell lines (NES139, NES140, NES140 and NES144) were subsequently derived that had the potential to proliferate in vitro. The previously-described cell line NES2Y was utlised as a control cell line for comparison. Using RT-PCR, exon array, qPCR, immunocytochemistry and Ca2+ microfluorimetry techniques this thesis examines both the molecular and physiological characteristics of these four CHI-derived cell lines to establish their potential as populations of pancreatic progenitor cells. Genotyping revealed that all of the patients carried mutations in the SUR1 gene, ABCC8. Pancreatic endocrine progenitor markers (e.g. PDX1, SOX9 and HLBX9) as well as islet precursor markers (e.g. NKX2.2, NKX6.1, NEUROD1, PAX6 and FOXA2) were identified and their expression was stable over continuous cell culture. However, each of the cell lines failed to express other markers, specifically NGN3, PAX4, and ISLET1. Cell lines developed from each patient then underwent a fibroblast to epithelial-like morphological transition. High-throughput exon array analysis revealed a significant down regulation of ACTA2, VIM and upregulation of CDH1 (q value < 0.05), a gene expression pattern associated with a mesenchymal-to-epithelial transition. Analysis at the mRNA level identified that CHI-derived cell lines expressed those channels and transporters associated with the β-cell function of glucose-stimulated insulin secretion (GSIS). Yet, when expression of all five endocrine hormones was investigated, mRNA expression was undetectable in three CHI-derived cell lines, except for the expression of insulin in NES143. Protein level assessment, however, failed to detect any expression of insulin. Functional studies examining whole cellular calcium dynamics and those underlying GSIS revealed that, whilst ATP (0.1 mM) and histamine (0.1 mM) readily raised intracellular Ca2+, each of the cell lines failed consistently to respond to tolbutamide (0.1 mM), glucose (20 mM), diazoxide (0.1 mM) and KCl (40 mM), except for NES140 which responded to applications of acetylcholine (0.1 mM). Given the display of cellular plasticity, molecular and physiological characteristics, the data show CHI-derived cell lines mimic pancreatic progenitor cell populations. More importantly, they represent islet precursor cells of the secondary transition phase of pancreatic development. Future studies should concentrate on the inductive potential of these cells to produce mature insulin-secreting β-cells.
3

Role of norepinephrine in glucose homeostasis /

Ste. Marie, Linda, January 2003 (has links)
Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 54-60).
4

Stem cells from patients with congenital hyperinsulinism

Kellaway, Sophie January 2016 (has links)
Diabetes and congenital hyperinsulinism (CHI) are severe diseases affecting the pancreas. Current models for testing drugs to treat these diseases are in vivo in rodents or isolated rodent islets. Differences between the human and rodent pancreas, and ethical issues, mean that in vitro human models are needed. To develop a novel in vitro model for pancreatic diseases, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) were derived from the pancreas of patients with CHI. MSCs from three forms of CHI were phenotypically normal for MSCs, and maintained the CHI-causing mutation. When compared to MSCs from bone marrow, the CHI pancreatic MSCs expressed pancreas-specific gene ISL1 and showed promoter hypomethylation of other pancreatic genes, including PDX1. The CHI pMSCs could be differentiated to cells resembling immature beta-cells, with some beta-cell gene expression (INS, PDX1), but no glucose responsive insulin secretion. CHI associated hypersecretion of insulin was not seen as the ATP-sensitive potassium KATP channels were not being expressed. Addition of the Wnt inhibitor DKK1 markedly enhanced differentiation via induction of neuronal genes. Alongside high insulin secretion, CHI also features increased proliferation. CHI MSCs were also hyperproliferative, and showed alterations to the cell cycle. These changes were related to p27Kip1 localisation, a known affected protein in CHI tissue, and CDK1, a novel regulator for CHI. iPSCs were also derived from focal CHI MSCs and were also phenotypically normal, but did not maintain the pancreatic hypomethylation present in MSCs. The CHI iPSCs were efficiently differentiated to definitive endoderm and PDX1 positive cells. Terminally differentiated iPSCs were endocrine, but were not mature beta-cells. In conclusion, authentic MSCs and iPSCs were derived for the first time from patients with CHI. These stem cells could be differentiated towards beta-cells, but mature glucose responsive beta-cells were not produced. MSC derived beta-like cells secreted insulin but did not have KATP channels, whereas iPSC derived beta-like cells had KATP channel gene expression but not INS. With further optimisation to resolve these, CHI stem cell derived beta-cells may be used for in vitro modelling. Further, the undifferentiated MSCs only show hyperproliferation associated with p27Kip1 and CDK1 and so can be a useful resource for modelling hyperproliferation seen in CHI.
5

Gene expression and cell cycle regulation in human pancreas development and congenital hyperinsulinism

Salisbury, Rachel January 2015 (has links)
The dynamics of β-cell mass are at the focus of an extensive international effort to develop β-cell replacement therapies for type 1 diabetes. During normal fetal development endocrine cells emerge from a pool of PDX1+/SOX9+ multipotent progenitors that transiently express the proendocrine gene NGN3. These cells become hormone-positive and are seen to bud from the ductal structures and aggregate into islet clusters. Congenital hyperinsulinism in its diffuse form (CHI-D) is characterised by an increase in hormone-positive cells associated with ducts and diffuse patterns of insulin expression. CHI-D arises from mutations inactivating the KATP channel and is diagnosed following persistent episodes of hypoglycaemia caused by an inappropriate secretion of insulin. Whilst existing knowledge has focused on the β-cell, we have explored the histology of CHI-D across multiple pancreatic cell lineages. The starting hypothesis considered CHI-D as an over-exuberance of endocrine differentiation with a progenitor population underlying this process. We suggest CHI-D is not simply an excessive proliferation of pre-existing β-cells. Expression of many transcription factors involved in endocrine differentiation were unchanged in CHI-D, NKX2.2 was increased and persisted in δ-cells. The incidence of nucleomegaly was also confirmed in CHI-D samples, predominantly in the β- and δ-cell lineages. Whilst increases in endocrine cell proliferation were subtle, the ductal and acinar cell lineages had significantly elevated proliferation correlating with changes in cell cycle regulation. The expression of NGN3 was profiled in a range of human fetal samples to determine whether a competence window for endocrine differentiation exists during development. Peak expression was observed between 10-17 wpc whilst protein and transcript expression were both reduced by birth and postnatally. Combined with the data in CHI-D and postnatal controls, it is likely that endocrine commitment ceases in human towards the end of gestation and that further increases in β-cell mass rely on proliferation or NGN3-independent pathways. These data provide new clues for the pathological mechanisms of CHI-D and the establishment and maintenance of the β-cell mass in the human pancreas. We have shown an altered potential for cell proliferation in CHI-D in previously unappreciated ways and provide a rationale for studying molecular components of the β-cell to help unlock β-cell proliferation as a therapeutic option in diabetes.
6

Hiperinsulinismo congênito em crianças brasileiras: histopatologia, proliferação das células do pâncreas e genética dos canais K+ / ATP / Congenital hyperinsulinismin in brazilian neonates: histopathology, cells proliferation and KATP channels genes

Lovisolo, Silvana Maria 06 April 2009 (has links)
O hiperinsulinismo congênito (CHI) é um distúrbio do pâncreas endócrino, mais freqüentemente causado por alterações dos canais de membrana KATP das células , resultando em secreção inapropriada de insulina e hipoglicemia severa e persistente nos recém-nascidos, que leva ao óbito ou a seqüelas neurológicas graves, se não diagnosticado a tempo. O diagnóstico depende da análise dos dados clínicos, laboratoriais, morfológicos e genético-moleculares (50% apresentam mutações dos canais KATP). As duas formas histopatológicas descritas requerem cirurgias radicalmente opostas: pancreatectomia quase-total (95-98%) na forma difusa que acomete todo o pâncreas, ou apenas exerese do foco adenomatoso de células , medindo em média 4,5 mm, na forma focal, e portanto a sua distinção é essencial durante o exame intra-operatório de congelação ou através de [18F]-L-Dopa PET-CT. Dez pacientes com CHI difuso e um com CHI focal, submetidos a pancreatectomia, foram analisados em relação a parâmetros clínicos, histopatológicos, de proliferação das células (IHQ de dupla marcação Ki-67 / insulina) e quanto à presença de mutações nos genes das únicas duas proteínas (SUR 1 e Kir 6.2) que formam os canais KATP, e comparados a 19 pâncreas controles normais da mesma faixa etária. Pacientes e controles foram estratificados em 3 meses e > 3 meses de idade. Nucleomegalia, ausente nos controles, foi observada apenas na forma difusa. Os critérios histológicos de maturação normalmente mais freqüentes nos controles 3 meses, foram freqüentemente observados nos recém-nascidos com CHI difuso > 3 meses, sugerindo um retardo na maturação do pâncreas endócrino destes pacientes. O índice de proliferação das células (Ki-67- LI), muito elevado nos focos adenomatosos da forma focal, foi útil na distinção destes focos dos agregados frouxos de ilhotas, histologicamente muito semelhantes, observados em dois casos difusos e um controle, que apresentam níveis de Ki-67-LI cerca de 10 vezes menor. Na forma difusa o Ki-67-LI também foi estatisticamente mais alto do que nos controles. Este é o primeiro estudo de pacientes com CHI no Brasil, e embora existam diferenças epidemiológicas entre os países relacionadas à determinação genética do CHI, não foram constatadas mutações ou novos polimorfismos nos exons 33-37 do gene ABCC8 (SUR 1) de 10/10 pacientes ou no único exon do gene KCNJ11 (Kir 6.2) de 4/10 pacientes / Congenital hyperinsulinism (CHI) is a rare pancreatic endocrine cell disease which most severe cases are found to be, at least in half of patients, associated with genetic defects in the -cell KATP channels. The aim of this study was to evaluate eleven Brazilian patients diagnosed, by standard criteria, as CHI non responsive to clinical therapy, and submitted to pancreatectomy, regarding: histology, -cell proliferation (IHC Ki-67 / insulin) and -cell KATP channels genes mutations in blood samples. For comparison of histology and -cell proliferation, 19 pancreatic control samples were included. According histology, ten patients were classified as diffuse and one as focal form. Nucleomegaly and -cells with abundant cytoplasm were absent in controls, and observed only in the group of diffuse CHI patients. Ki- 67-LI was useful to differentiate the adenomatous areas of the focal form CHI neonate from loose clusters of islets found in two diffuse form and one control samples. Proliferation was much higher in the focal CHI adenomatous areas, but diffuse CHI patients also have statistically higher Ki-67-LI than controls. This is the first genetic study of CHI patients in Brazil, and no mutations or new polymorphisms were found in the ABCC8 gene (SUR 1) (exons 33-37) or in the only exon of KCNJ11 gene (Kir 6.2) in 4/4 patients evaluated. On the other hand, enhanced -cell proliferation seems to be a constant feature in these patients both in diffuse and focal forms
7

Hiperinsulinismo congênito em crianças brasileiras: histopatologia, proliferação das células do pâncreas e genética dos canais K+ / ATP / Congenital hyperinsulinismin in brazilian neonates: histopathology, cells proliferation and KATP channels genes

Silvana Maria Lovisolo 06 April 2009 (has links)
O hiperinsulinismo congênito (CHI) é um distúrbio do pâncreas endócrino, mais freqüentemente causado por alterações dos canais de membrana KATP das células , resultando em secreção inapropriada de insulina e hipoglicemia severa e persistente nos recém-nascidos, que leva ao óbito ou a seqüelas neurológicas graves, se não diagnosticado a tempo. O diagnóstico depende da análise dos dados clínicos, laboratoriais, morfológicos e genético-moleculares (50% apresentam mutações dos canais KATP). As duas formas histopatológicas descritas requerem cirurgias radicalmente opostas: pancreatectomia quase-total (95-98%) na forma difusa que acomete todo o pâncreas, ou apenas exerese do foco adenomatoso de células , medindo em média 4,5 mm, na forma focal, e portanto a sua distinção é essencial durante o exame intra-operatório de congelação ou através de [18F]-L-Dopa PET-CT. Dez pacientes com CHI difuso e um com CHI focal, submetidos a pancreatectomia, foram analisados em relação a parâmetros clínicos, histopatológicos, de proliferação das células (IHQ de dupla marcação Ki-67 / insulina) e quanto à presença de mutações nos genes das únicas duas proteínas (SUR 1 e Kir 6.2) que formam os canais KATP, e comparados a 19 pâncreas controles normais da mesma faixa etária. Pacientes e controles foram estratificados em 3 meses e > 3 meses de idade. Nucleomegalia, ausente nos controles, foi observada apenas na forma difusa. Os critérios histológicos de maturação normalmente mais freqüentes nos controles 3 meses, foram freqüentemente observados nos recém-nascidos com CHI difuso > 3 meses, sugerindo um retardo na maturação do pâncreas endócrino destes pacientes. O índice de proliferação das células (Ki-67- LI), muito elevado nos focos adenomatosos da forma focal, foi útil na distinção destes focos dos agregados frouxos de ilhotas, histologicamente muito semelhantes, observados em dois casos difusos e um controle, que apresentam níveis de Ki-67-LI cerca de 10 vezes menor. Na forma difusa o Ki-67-LI também foi estatisticamente mais alto do que nos controles. Este é o primeiro estudo de pacientes com CHI no Brasil, e embora existam diferenças epidemiológicas entre os países relacionadas à determinação genética do CHI, não foram constatadas mutações ou novos polimorfismos nos exons 33-37 do gene ABCC8 (SUR 1) de 10/10 pacientes ou no único exon do gene KCNJ11 (Kir 6.2) de 4/10 pacientes / Congenital hyperinsulinism (CHI) is a rare pancreatic endocrine cell disease which most severe cases are found to be, at least in half of patients, associated with genetic defects in the -cell KATP channels. The aim of this study was to evaluate eleven Brazilian patients diagnosed, by standard criteria, as CHI non responsive to clinical therapy, and submitted to pancreatectomy, regarding: histology, -cell proliferation (IHC Ki-67 / insulin) and -cell KATP channels genes mutations in blood samples. For comparison of histology and -cell proliferation, 19 pancreatic control samples were included. According histology, ten patients were classified as diffuse and one as focal form. Nucleomegaly and -cells with abundant cytoplasm were absent in controls, and observed only in the group of diffuse CHI patients. Ki- 67-LI was useful to differentiate the adenomatous areas of the focal form CHI neonate from loose clusters of islets found in two diffuse form and one control samples. Proliferation was much higher in the focal CHI adenomatous areas, but diffuse CHI patients also have statistically higher Ki-67-LI than controls. This is the first genetic study of CHI patients in Brazil, and no mutations or new polymorphisms were found in the ABCC8 gene (SUR 1) (exons 33-37) or in the only exon of KCNJ11 gene (Kir 6.2) in 4/4 patients evaluated. On the other hand, enhanced -cell proliferation seems to be a constant feature in these patients both in diffuse and focal forms

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