High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: A high fat diet (HFD) reduces beta-cell mass, impairs glucose signalling and is involved in the development of Type 2 diabetes. Malnutrition during gestation is hypothesized to irreversibly damage beta-cell development. The transcription factors Pdx-1 and Pax 4 are involved in islet cell development. Pdx-1 is reported to regulate expression of GLUT-2, glucokinase (GK) and the insulin gene. Aims The aim of this study is to investigate, in the neonatal and weanling rat, the effect of exposure to a HFD in utero and/or lactation on weight, glucose and insulin concentrations, islet cell development, pancreatic transcription factors and glucose sensing genes. Methods Neonatal and weanling rats were exposed to a maternal HFD for defined periods of gestation and/or lactation. After termination, pups were weighed and glucose and insulin concentrations determined. mRNA expression of Pdx-1, Pax 4, GLUT-2 and GK was quantified by LightCycler PCR. Pancreatic sections were immunostained for insulin and glucagon (islet cell development), and for Pdx-1, GLUT-2 and GK (beta-cell function) followed by image analysis. Results: Exposure to an in utero HFD throughout gestation resulted in hyperglycaemic pups with reduced beta-cell volume and number, Pdx-1 and GK immunoreactivity. In contrast the alpha-cell volume, number and size were augmented in neonates exposed to a HFD throughout gestation. Most weanlings were hyperglycaemic and hypoinsulinaemic. In some weanlings, reduced beta-cell number and beta- and alpha-cell size was observed. Pdx-1 mRNA was overexpressed in weanlings exposed to a maternal HFD for the final week of gestation or throughout both gestation and lactation, but reduced in those only exposed throughout lactation. Pax 4 mRNA was reduced in weanlings exposed to a maternal HFD for the first or final week of gestation, throughout gestation or throughout lactation. In most of the weanlings, GLUT-2 mRNA expression was reduced whereas immunoreactivity for GLUT-2 was increased. Both GK mRNA expression and immunoreactivity were reduced in most of the weanlings. Conclusions • Exposure to an in utero HFD throughout gestation induced hyperglycaemia in neonates. The reduced Pdx-1 expression appears to play a role in the compromised beta-cell development, and concomitant with the reduced GK levels, contributes to the hyperglycaemia in these neonates and may make them susceptible to beta-cell failure. • In most weanlings exposed to a HFD in utero and/or during lactation the hyperglycaemia and hypoinsulinaemia suggest compromised beta-cell function. The GK mRNA expression and immunoreactivity were reduced thereby impairing glycolysis which would result in reduced insulin secretion contributing to the hyperglycaemia. Furthermore, beta-cell development is adversely affected by the HFD in some weanlings. This would contribute to reduced beta-cell function and may eventually result in beta-cell failure. GLUT-2 immunoreactivity was increased in some, suggesting a compensatory adaptative mechanism to restore glucose homeostasis. • A maternal HFD has adverse effects both in neonates and weanlings on beta-cell development, transcription factor and glucose sensing gene expression and induced hyperglycaemia and hypoinsulinaemia in some of the offspring. Ways to ameliorate the HFD-induced attenuation of key beta-cell genes to ensure normal beta-cell function are important for future research in Type 2 diabetes. / AFRIKAANSE OPSOMMING: ‘n Hoe vet diet (HVD) verminder beta-sel masse, glukose signale en speel ‘n rol in Tipe 2 diabetes. Die hipothese is dat wanvoeding gedurende swangerskap lei tot onomkeerbare betasel beskadiging. Die transkripsiefaktore Pdx-1 en Pax 4 speel rolle in eilandselontwikkeling. Daar is bewyse dat Pdx-1 die uitdrukking van die GLUT-2, glucokinase (GK) en insulin gene reguleer. Doelstelling: Die doel van hierdie studie is om, in die pasgebore en gespeende rot, die effek van ’n HVD in utero en/of laktasie op gewig, glukose en insulin konsentrasies, eilandselontwikkeling, pankreatiese transksripsiefaktore en op glukosewaarnemingsgene te ondersoek. Metodes: Pasgebore en gespeende rotte is vir bepaalde periodes van gestasie en/of laktasie blootgestel aan ’n HVD van die moeder. Na terminase, is kleinjies geweeg en die glukose- en insulienkinsentrasies bepaal. mRNA uitdrukking van Pdx-1, Pax 4, GLUT-2 en GK is geoes met LightCycler PCR. Snitte van die pankreas is gekleur met insulien en glukagon (eilandsontwikkeling) en vir Pdx-1, GLUT-2 en GK (betaselfunksie) gevolg deur beeldanalise. Resultate: Bloodstelling aan ’n in utero HVD regdeur gestasie het hiperglisemie versoorsaak in pasgebore rotte met verlaagde betasel volume en aantal, Pdx-1 en GK immunoreaktiwiteit. In teenstelling daarmee was die alfasel se volume, aantal en grootte verhoog in pasgebore rotte wat regdeur gestasie aan 'n HVD blootgestel was. Meeste van die gespeende rotte was hiperglisemies en hipoinsulinemies. In sommige gespeende rotte, was daar ’n verlaging van betasel hoeveelheid en grootte en in alfasel groote. Oormatige uitdrukking van Pdx-1 mRNA het plaasgevind in speenlinge wat aan ’n HVD van die moeder vir die laaste week van gestasie of regdeur gestasie en laktasie blootgestel was, maar dit was verlaag in die speenlinge wat net tydens laktasie blootgestel was. Pax 4 mRNA was verlaag in speenlinge wat aan ’n HVD van die moeder blootgestel was vir die eerste of laaste week van gestasie, regdeur gestasie of regdeur laktasie. In meeste van die speenlinge is onder-uitdrukking van GLUT-2 mRNA, maar ’n verhoging van GLUT-2 immunoreaktiwiteit gevind. Beide GK mRNA uitdrukking en immunoreaktiwiteit was laer in meeste van die speenlinge. Gevolgtrekkings: • Blootstelling aan ’n in utero HVD regdeur gestasie lei tot hiperglisemie in pasgebore rotte. Die verlaagde Pdx-1 immunoreaktiwiteit speel klaarblyklik ’n rol by die geaffekteerde betaselontwikkeling. Dit, saam met die verlaagde immunoreaktiwiteit vir GK, kan bydra tot die hiperglisemie in hierdie pasgebore rotte. In die meeste van die speenlinge wat aan ’n HVD blootgestel was, dui die hiperglisemie en hipoinsulinemie op geaffekteerde betaselfunksie. Die GK mRNA uitdrukking en immunoreaktiwiteit is verlaag, wat weer glikolise benadeel, en dit sal lei tot verminderde insulienafskeiding wat bydra tot die hiperglisemie. Betaselontwikkeling word voorts negatief beinvloed deur die HVD, wat blyk uit die verlaagde aantal en grootte van betaselle. Dit sal bydra tot verminderde betaselfunksie. Dit kan uiteindelik tot betaselversaking lei. GLUT-2 immunoreaktiwiteit was verhoog in hierdie speenlinge, wat dui op ’n kompenserende aanpassingsmeganisme om glukose homeostase te herstel. ’n HVD van die moeder het ’n negatiewe uitwerking op betaselontwikkeling, transkripsiefaktor en glukosewaarneming geenuitdrukking in beide die pasgebore en gespeende rotte, en geinduseerde hiperglisemie en hipoinsulinemie in sommige kleintjies. Dis belangrik vir toekomstige Tipe 2 diabetes navorsing dat daar na gekyk moet word om die HVD-geinduseerde verlaging van sleutel betaselgene te verbeter vir optimale betaselfunksie.

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

Nous avons montré, aussi bien in vitro que in vivo, l’impossibilité de la reprogrammation directe des macrophages en cellules béta, et ce en en utilisant les facteurs publiés dans la littérature actuelle comme étant déterminants et importants pour la différenciation en cellules béta du pancréas. Les macrophages préalablement transduits par des virus, ont été cultivés dans des conditions spécifiques visant à mimer le microenvironnement pancréatique, ou injectés dans le pancréas des souris afin de faciliter la reprogrammation. De plus, l’échec dans la reprogrammation des lignages, la culture des macrophages en présence de composés régulant la différentiation des cellules béta induit la formation d’organoïdes. Ces “clusters” de cellules montrent une forte inhibition du potentiel prolifératif comme a été démontré par l’arrêt du cycle cellulaire. Le criblage de différents composés a permis l’identification de la nicotinamide (vitamine B3) comme étant la molécule responsable de l’inhibition de la progression du cycle cellulaire. Les analyses transcriptomiques ont montré l’augmentation de l’expression des inhibiteurs du cycle cellulaire ainsi que la réduction des niveaux d’expression des régulateurs positifs, confirmant que la nicotinamide régule négativement le cycle cellulaire. De manière intéressante, nous avons montré que ce phénomène est réversible, étant donné que le retrait de la nicotinamide résulte en un réengagement dans le cycle cellulaire et la formation de colonies. Ces résultats indiquent le possible rôle de la nicotinamide dans la régulation du potentiel prolifératif des macrophages Maf-DKO. / We have shown here that direct reprogramming of macrophages into beta cells was not possible, both in vitro and in vivo, using pancreatic fate determinants already reported in current literature. Transduced macrophages were cultured in specific conditions or injected into the murine pancreas, aiming to mimic the pancreatic microenvironment as a reprogramming inducer. Besides this failure in lineage reprogramming, culturing macrophages with compounds regulating beta cell differentiation induced the formation of organoids. These cell clusters showed strong inhibition of the proliferative potential, as demonstrated by the arrest of their cell cycle. Screening of the different compounds, allowed the identification of nicotinamide as the responsible molecule for the inhibition of cell cycle progression. Transcriptomic analysis depicted an increased expression of cell cycle inhibitors along with reduced levels of positive regulators, confirming the nicotinamide-induced negative regulation of cell cycle. Most importantly, we have demonstrated that this phenomenon was a reversible proliferation inhibition, since withdrawal of nicotinamide resulted in cell cycle re-entry and rescue of the colony formation phenotype. Together these results indicate a possible role of nicotinamide (Vitamin B3) in the regulation the Maf-DKO macrophages proliferative ability. Further investigations are needed to assess a role for Nicotinamide in controlling the biology of wild-type macrophages, and determine the molecular pathways controlling this transient phenomenon.

OBJECTIVE: Cardiovascular disease (CVD) remains the leading cause of death globally. Reducing high blood cholesterol, which is a dominant risk factor for CVD events, is an essential goal of medical treatment. Statins are known as first‐choice agents. However, clinical trials report that some statins increased the risk for type 2 diabetes (T2D). Our objective was to investigate the effect of different statins on insulin secretion and content from pancreatic β-cells after chronic and acute exposure and determine the underlying mechanisms. METHODS: The effects of simvastatin, lovastatin and pitavastatin on GSIS and content were studied in clonal pancreatic β-cells (INS-1 832/13) cultured in high glucose (12 mM). Insulin content and secretion were measured after chronic and acute incubation of statins using homogenous time-resolved fluorescence (HTRF) insulin assay kit (Cisbio). Intracellular Ca2+ was measured using fura-2 AM (Invitrogen). RESULTS: Simvastatin (25-200 nM) and lovastatin (50-200 nM) significantly inhibited GSIS and depleted insulin content in a dose-dependent manner after 72-hour exposure. When the secretion level was normalized for content, the inhibitory effect was not observed. Simvastatin (200 nM) also increased the amplitude of intracellular Ca2+ oscillations at low glucose, but this was not reflected in the amplitude of oscillatory insulin release. In contrast, pitavastatin (25-200 nM) did not affect GSIS and only decreased insulin content at the highest dose tested. CONCLUSION: Inhibition of GSIS by simvastatin and lovastatin could be due to depletion of insulin content. Decreased Ca2+ sensitivity may also contribute to inhibition of GSIS by simvastatin. Pitavastatin had less inhibitory effect on GSIS and insulin content as compared to simvastatin and lovastatin indicating that not all lipophilic statins have a detrimental impact on GSIS. We suggest that statins may have differential mechanistic effects on β-cells some of which may contribute to the risk of T2D.

A Diabetes mellitus em cães é cada vez mais frequente, decorrente de fatores genéticos e/ou ambientais, como um distúrbio endócrino que, de forma semelhante à que ocorre em humanos, falha no controle adequado de glicose no sangue, desencadeia a hiperglicemia, glicosúria e perda de peso. A terapia celular utilizando as células beta-pancreáticas tem sido alvo de estudos, devido à grande demanda de novos casos de Diabetes mellitus e à falta de órgãos para transplantes em humanos e animais. Acredita-se que a ciência possa responder e inovar em tratamentos, encontrando a possível cura para esta doença complexa. Portanto, o objetivo deste estudo foi obter e caracterizar células derivadas do pâncreas fetal canino de animais com idades compreendidas entre 50 e 60 dias de gestação. As células pancreáticas de fetos caninos apresentam morfologia fibroblastóide e crescimento em monocamada em cultivo, apresentam células pluripotentes e proliferativas, não são tumorigênicas e apresentam expressão de PDX1, um fator de transcrição que tem papel importante na ativação do gene promotor da insulina. O pâncreas possui inervação simpática, observado por fibras nervosas TH+. Histologicamente, o pâncreas fetal canino apresenta ácinos num estágio de organização avançado, com parênquima semelhante ao encontrado no cão adulto. As ilhotas pancreáticas são distribuídas no tecido de maneira irregular, organizando-se em pequenos aglomerados de células por entre os ácinos, especialmente próximas aos vasos sanguíneos. A coloração com Ditizona permitiu inferir a presença de insulina no tecido pancreático, o que foi comprovado mediante técnica de imunofluorescência, além da presença de células que expressam o hormônio somatostatina. Os resultados desta investigação indicam que o pâncreas fetal canino demonstra características favoráveis para ser uma fonte viável de células para estudos aplicados à terapia celular em cães. Outras investigações referentes à comprovação da produção de insulina in vitro por essas células se fazem necessárias / Diabetes mellitus in dogs is increasingly common, due to genetic and/or environmental factors such as an endocrine disorder, similarly to what occurs in humans, failure to adequately control blood glucose triggers hyperglycemia, glycosuria and weight loss. Cell therapy using the pancreatic beta cells has been the subject of studies, due to the great demand of new cases of diabetes mellitus and the lack of organs for transplants in humans and animals. It is believed that science can respond and innovate treatments, finding a possible cure for this disease complex. Therefore, the objective of this study was to obtain and characterize derived cells from canine fetal pancreas, of animals aged between 50 and 60 days of gestation. The pancreatic cells of canine fetuses exhibit fibroblastoid morphology and growth in monolayer culture, exhibit pluripotent and proliferative cells, are not tumorigenic and have PDX1 expression, a transcription factor that plays an important role in the activation of the insulin gene promoter. The pancreas has sympathetic innervation observed by TH+ nerve fibers. Histologically, the fetal pancreatic acini canine presents an advanced stage organization with similar parenchyma that found in adult dog. The pancreatic islets are distributed in the fabric unevenly, organizing themselves into small clusters of cells through the acini, especially close to the blood vessels. Staining with Dithizone allowed inferring the presence of insulin in the tissue, which was confirmed by immunofluorescence, in addition to cells that express somatostatin. The results of this investigation indicate that the canine fetal pancreas shows favorable characteristics to be a feasible source of cells for cell therapy applied to studies in dogs. Further investigation regarding the evidence of in vitro production of insulin by these cells are required

Apart from CFRD, low cognitive skill index (CSI) was also found in CF patients and was attributed the lacking of vitamin E. Since it is known that insulin plays a role in the learning and memory, decreased plasma insulin level in CF patients is an alternative mechanism for impaired cognitive function. Although numerous studies have found that insulin can improve learning and memory, the mechanism of it is not well understood. In this study, we investigated the effect of insulin on the expression of hippocampal early-phase long-term potentiation (E-LTP) in the immature rats. Hippocampal brain slices were acutely prepared from 10-12 days and 2 months old rats and field excitatory postsynaptic potentials (tEPSCs) were recorded from CA1 region by a multi-electrode in vitro recording system. In the control group, the hippocampal slices of neonatal rats showed no increase in the magnitude of fEPSC after conventional high frequency stimulation (HFS). After pretreatment of the slices with 0.08ng/ml insulin for over one hour, there was no significant change in the magnitude of E-LTP. However, when the insulin concentration increased to 0.8ng/ml, a significant increase in the magnitude of E-LTP was observed. On the contrary, any doses of insulin failed to affect the magnitude of E-LTP of mature rats. These results suggested that insulin could dose-dependently facilitate the production of E-LTP in the hippocampus of infant rats. Application of AG-1024, an inhibitor of insulin receptor, largely abolished the insulin-dependent E-LTP in immature rats rather than adult rats, indicating the involvement of insulin signaling pathway in the insulin effect. On the other hand, increasing the concentration of glucose from 11mM to 22 or 33 mM did not facilitate the E-LTP and application of indinavir, a blocker of insulin-sensitive glucose transporter-4, did not inhibit the effect of insulin. Therefore, it is unlikely that the facilitory action of insulin on E-LTP is via an indirect effect on glucose homeostasis or utilization. Pretreatment with the MAPK pathway inhibitor PD98059 blocked insulin-mediated E-LTP facilitation. Furthermore, the tetanic stimulation induced a significant increase in the level of phosphorylated p42MAPK in the insulin-treated hippocampus than that in the control group. In conclusion, our results suggested that insulin could facilitate the production of hippocampal E-LTP in infant rats, which may play an important role in modulating the expression of LTP in the developing brain and perhaps is an underlying mechanism for the improving effect of insulin on learning and memory. Since insulin plays an important role in the developing brain, perhaps the deficiency of insulin effect resulted from CF patients induces the impairment of cognitive function. / Cystic fibrosis (CF), which is caused by the deficiency of cystic fibrosis transmembrne conductance regulator (CFTR), is the most common autosomal recessive systemic disease with an incidence of 1: 2500 in Caucasians. Cystic fibrosis-related diabetes (CFRD), as one of the complications of CF patients, is regarded as one of the leading co-morbidity in CF patients. The mechanism ofCFRD is attributed to the reduced number of islets due to pancreatic fibrosis caused by the loss of CFTR in pancreatic duct. However, the above mechanism failed to explain the dynamics of insulin secretion induced by glucose tolerance test (GTT) in some CF patients and therefore, we were forced to re-consider the mechanism for the pathogenesis of CFRD. Interestingly, the following facts imply that perhaps there is another mechanism for the onset of CFRD: decreased insulin secretion and decreased plasma HCO3 - concentration was observed in the metabolic acidosis disease, plasma HCO3- level increased accompanied by the elevation of plasma insulin after food intake and CFTR accounted for HCO3 - transport in many epithelial cells. These facts promoted us to hypothesize that the loss of HCO3--induced insulin secretion resulting from the deficiency of CFTR is an alternative mechanism for the onset of CFRD. Our results showed that HCO3- could induce insulin secretion of isolated islets from rats. Ca2+ imaging revealed that HCO3- dose-dependently induced an increase in intracellular Ca2+ ([Ca2+] i) in RIN-5F cells, an insulin-secreting cell line. Removal of extracellular Ca2+ or addition of nifedipine, the blocker of L-type Ca 2+ channel, decreased the effect of HCO3- significantly, indicating the activation of L-type Ca2+ channel during HCO3- stimulation. The inhibitory effect of BaCl2 implied the involvement of K+ channel. The results that HCO3--induced increase in [Ca 2+]i was reduced by PKA inhibitor and sAC blocker demonstrated that the pathway of sAC-cAMP-PKA-ATP-sentitive K+ channel (K ATP channel) was responsible for the effect of HCO3 -. The reduction of extracellular Cl- or the inhibitor of anion exchanger (AE) inhibited the [Ca2+]i increase induced by HCO3- significantly but the omission of external Na+ failed. The facts that CFTR blocker decreased the effect of HCO3- markedly and the expression of CFTR in RIN-5F cells revealed by western blotting suggested the CFTR-mediated HCO3- transport. These results suggested that HCO 3- could induce insulin secretion in a CFTR-dependent manner, which provided a new insight into the understanding of pathogenesis of CFRD and paved the way for the therapy of CFRD. / Zhao, Wenchao. / "November 2010." / Advisers: Chang Chan; Wing Ho Yung. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 115-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Another growth factor candidate is a recently recognized bioactive peptide, islet-neogenesis associated protein (INGAP). A master pancreatic transcription factor, pancreatic duodenal homeobox-1 (Pdx-1), was overexpressed in PSCs by the adenovirus-mediated transfer method in the present study. With the infection of adenovirus expressing Pdx-1, several beta-cell developmental genes, including Isl-1, Beta2, Nkx2.2, Nkx6.1 and the endogenous Pdx-1 were found to be upregulated temporally in our PSCs-derived ICCs. Meanwhile, previous study has shown that Pdx-1/INGAP-positive cells represent a new stem cell subpopulation during early stage of pancreatic development. We thus explore whether any functional integration of Pdx-1 and INGAP in the growth and functional maturation of PSCs. In order to achieve this proposition, the effects of over-expressing PSCs with the Pdx-1 adenovirus in conjunction with the treatment of INGAP were then investigated. Interestingly, differentiation of the PSC-derived ICCs was not further enhanced by the synergistic treatment of Pdx-1 and INGAP when compared to those ICCs infected with adenovirus expressing Pdx-1 alone, as revealed by the endogenous Pdx-1 and insulin gene expression and their C-peptide content. These data might provide some clues to the intricate interaction between Pdx-1 and INGAP in regulating the ICC and/or the pancreatic endocrine differentiation. (Abstract shortened by UMI.) / Due to the scarcity of fetal pancreas for generating functional insulin-secreting cell clusters for sufficient islet transplantation, we targeted for searching pancreatic stem/progenitor cells. Putative PSCs can be aggregated and differentiated into islet-like cell clusters (ICCs) when exposed to serum-free medium containing various conventional growth factors, including HGF, GLP-1, betacellulin and nicotinamide. / Fetal pancreatic tissue consisting of immature progenitor cells serves as a potential source of stem cells as they possess a higher replicative capacity and longevity than their adult counterparts. / Two novel candidates and a key pancreatic transcription factor on the PSC/ICC proliferation and differentiation were investigated in the present study. One of them is a ubiquitously expressed multi-PDZ-domain protein, PDZ-domain-containing 2 (PDZD2), which was previously found to express in the mouse beta cells and exhibit mitogenic effects in beta cell line. Results showed that PDZD2 was detected in high levels in both human fetal pancreas and in PSCs. Results indicate the potential involvement of PDZD2 in regulating PSCs proliferation and differentiation and pancreatic development. / Suen Po Man, Ada. / "July 2007." / Adviser: P.S. Leung. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0051. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 194-214). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Ma, Man Ting. / "July 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 186-207). / Abstracts in English and Chinese. / Abstract --- p.I / List of Publications --- p.VI / Acknowledgements --- p.VIII / Table of Contents --- p.X / List of Figures --- p.XV / List of Tables --- p.XVIII / List of Abbreviations --- p.XIX / Chapter CHAPTER1 --- INTRODUCTION / Chapter 1.1 --- The Pancreas --- p.2 / Chapter 1.1.1 --- Structure of pancreas --- p.2 / Chapter 1.1.2 --- Structure and function of exocrine pancreas --- p.6 / Chapter 1.1.3 --- Structure and function of endocrine pancreas --- p.9 / Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.9 / Chapter 1.1.3.2 --- Glucose-stimulated insulin secretion from islets --- p.12 / Chapter 1.2 --- Type 1 Diabetes Mellitus (T1DM) --- p.14 / Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus --- p.14 / Chapter 1.2.2 --- Autoimmunity in T1DM --- p.17 / Chapter 1.2.3 --- Management ofTlDM --- p.20 / Chapter 1.2.3.1 --- Insulin replacement --- p.20 / Chapter 1.2.3.2 --- Pancreas and islet transplantation --- p.21 / Chapter 1.2.3.3 --- Stem-cell-based transplantation --- p.22 / Chapter 1.3 --- The Adaptive Immune System --- p.26 / Chapter 1.3.1 --- T-lymphocytes --- p.26 / Chapter 1.3.2 --- B-lymphocytes --- p.29 / Chapter 1.3.3 --- Major histocompatibility complex (MHC) --- p.30 / Chapter 1.3.3.1 --- Classification of MHC molecules --- p.30 / Chapter 1.3.3.2 --- Structure of MHC class I and II molecules --- p.32 / Chapter 1.3.3.3 --- Function and regulation of MHC molecules --- p.34 / Chapter 1.3.4 --- HLA-G and its immuno-modulatory properties --- p.36 / Chapter 1.4 --- Transplantation Rejection --- p.40 / Chapter 1.4.1 --- Mechanisms involved in transplantation rejection --- p.40 / Chapter 1.4.2 --- Immunobiology of rejection --- p.41 / Chapter 1.4.2.1 --- Direct allorecognition pathway --- p.42 / Chapter 1.4.2.2 --- Indirect allorecognition pathway --- p.43 / Chapter 1.4.2.3 --- Semi-direct allorecognition pathway --- p.43 / Chapter 1.4.3 --- Xenotransplantation --- p.46 / Chapter 1.5 --- Cytokines and Immunity --- p.48 / Chapter 1.5.1 --- Interferons --- p.48 / Chapter 1.5.1.1 --- Interferon-γ and its immune regulation --- p.49 / Chapter 1.5.1.2 --- Effect and kinetics of interferon-γ on MHC molecules expression --- p.53 / Chapter 1.5.1.3 --- Regulation of interferon-γ production --- p.56 / Chapter 1.5.2 --- Interlukins --- p.58 / Chapter 1.5.2.1 --- IL-10 and its immune regulation --- p.58 / Chapter 1.5.2.2 --- IL-10 and HLA-G --- p.59 / Chapter 1.6 --- Stem Cells and their Immunogenicity --- p.62 / Chapter 1.6.1 --- Embroynic stem cells --- p.62 / Chapter 1.6.2 --- Mesenchymal stem cells --- p.64 / Chapter 1.6.3 --- Neural stem cells --- p.68 / Chapter 1.6.4 --- Fetal stem cells --- p.69 / Chapter 1.6.5 --- Potential immuno-study in human fetal pancreatic stem cells --- p.70 / Chapter 1.7 --- Aims and Objectives of study --- p.72 / Chapter CHAPTER2 --- MATERIALS AND METHODS / Chapter 2.1 --- Isolation of Pancreatic Progenitors (PPCs) from Human Fetal Pancreas and Induction of Islet-like Cell Cluster (ICCs) Differentiation --- p.75 / Chapter 2.1.1 --- Tissue procurement --- p.75 / Chapter 2.1.2 --- Tissue processing and PPCs culture --- p.75 / Chapter 2.1.3 --- In vitro differentiation of PPCs into ICCs --- p.78 / Chapter 2.1.4 --- Interferon-γ and IL-10 treatment --- p.80 / Chapter 2.2 --- Cell culture of human placental Choriocarcinoma JEG-3 Cell Line --- p.81 / Chapter 2.3 --- RNA Expression Detection --- p.82 / Chapter 2.3.1 --- RNA isolation --- p.82 / Chapter 2.3.2 --- Reverse transcriptase (RT) --- p.83 / Chapter 2.3.3 --- Design of primers for Polymerase Chain Reaction (PCR) and Real-time PCR --- p.84 / Chapter 2.3.4 --- PCR --- p.86 / Chapter 2.3.5 --- Real-time PCR analysis --- p.88 / Chapter 2.3.6 --- Calculation using the comparative CT method --- p.90 / Chapter 2.4 --- Flow Cytometry --- p.91 / Chapter 2.5 --- Western Blotting Analysis --- p.93 / Chapter 2.5.1 --- Protein extraction and quantification --- p.93 / Chapter 2.5.2 --- Western blotting --- p.93 / Chapter 2.6 --- Mixed Lymphocyte Reaction (MLR) --- p.95 / Chapter 2.6.1 --- Isolation of peripheral blood mononuclear cells (PBMCs) --- p.95 / Chapter 2.6.2 --- PPC-PBMCs MLR --- p.98 / Chapter 2.6.3 --- ICC-PBMCs MLR --- p.98 / Chapter 2.6.4 --- Proliferation assay --- p.99 / Chapter 2.7 --- ICC Transplantation --- p.101 / Chapter 2.7.1 --- Streptozotocin-induced diabetic animals for transplantation --- p.101 / Chapter 2.7.2 --- Procedures of ICCs transplantation --- p.102 / Chapter 2.8 --- Histological Analysis of ICC Graft --- p.105 / Chapter 2.8.1 --- H&E staining --- p.105 / Chapter 2.8.2 --- DAB staining --- p.106 / Chapter 2.8.3 --- Immunofluorescence staining --- p.107 / Chapter 2.9 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.109 / Chapter 2.10 --- Statistical Data Analysis --- p.110 / Chapter CHAPTER3 --- RESULTS / Chapter 3.1 --- Immuno-characterization of PPCs and ICCs --- p.112 / Chapter 3.2 --- Effect of cytokines on immune-properties of PPCs and ICCs --- p.115 / Chapter 3.2.1 --- Effect of lFN-γ on MHC-I expression in PPCs --- p.115 / Chapter 3.2.2 --- Effect of lFN-γ and IL-10 on HLA-G expression in PPCs and ICCs --- p.119 / Chapter 3.2.3 --- Effect of IFN-γ on B7H4 expression in PPCs --- p.123 / Chapter 3.3 --- Comparison of immune-properties of PPCs and ICCs from 1st and 2nd trimester --- p.125 / Chapter 3.3.1 --- Differential expression of MHC molecules in PPCs --- p.125 / Chapter 3.3.2 --- Different immune-related gene expression in PPCs and ICCs --- p.128 / Chapter 3.3.3 --- Comparison of IFN-γ activated MHC molecules expression in PPCs/ICCs --- p.134 / Chapter 3.3.4 --- Comparison of other IFN-γ activated genes expression in PPCs --- p.139 / Chapter 3.4 --- Mixed lymphocyte reaction of PPCs from 1st and 2nd trimester --- p.143 / Chapter 3.4.1 --- Effect of PPCs on proliferation of PBMC --- p.143 / Chapter 3.4.2 --- Effect of ICCs on proliferation of PBMC --- p.145 / Chapter 3.4.3 --- Effect of PPCs on cytokine production in PBMC --- p.149 / Chapter 3.5 --- Xenotransplantation of ICCs into diabetic mouse model --- p.152 / Chapter 3.5.1 --- Blood glucose level of diabetic mice after transplantation --- p.152 / Chapter 3.5.2 --- Histological evaluation of transplanted ICCs grafts --- p.154 / Chapter 3.5.3 --- Infiltration of CD45 into transplanted grafts of 1st and 2nd trimester --- p.158 / Chapter CHAPTER4 --- DISCUSSION / Chapter 4.1 --- Expression of selected immuno-regulated genes in PPCs and ICCs --- p.163 / Chapter 4.2 --- Effect of IFN-g and IL-10 on expression of immuno-regulated genes in PPCs and ICCs --- p.166 / Chapter 4.3 --- In vitro studies on immunogenicity of PPCs and ICCs from first and second trimester --- p.171 / Chapter 4.3.1 --- Immune-related genes expression --- p.171 / Chapter 4.3.2 --- IFN-γ activated gene expression --- p.173 / Chapter 4.3.3 --- Mixed lymphocyte reaction --- p.175 / Chapter 4.3.4 --- Cytokine production of PBMC in MLR --- p.179 / Chapter 4.4 --- In vivo Xenotransplantation of ICCs into diabetic mouse model --- p.181 / Chapter 4.5 --- Conclusion --- p.187 / Chapter 4.6 --- Further studies --- p.188 / Chapter CHAPTER5 --- BIBLIOGRAPHY / Bibliography by Alphabetical Order --- p.189