Não é claro como o receptor de glicocorticóide (GR) contrarregula a atividade do STAT5 na transição da gestação para a lactação. Dexras1 é uma pequena proteína G ativada por dexametasona (DEX), que regula morfologia celular, crescimento, etc. Neste estudo detectamos a expressão de Dexras1 em células beta de ilhotas pancreáticas. DEX induz a expressão de Dexras1 em células RINm5F, que está aumentada na gestação e diminuída na lactação. A expressão protéica de 11<font face=\"Symbol\">&#946HSD1, enzima ativadora de glicocorticóides (GCs), segue esse perfil. A ligação tanto do GR como do STAT5, analisada por ChIP assay, ao promotor do gene da Dexras1 aumentada por DEX é revertida por prolactina (PRL), e está diminuída e aumentada na gestação e lactação, respectivamente. DEX induz a associação ao GR, fosforilação e translocação nuclear do STAT5b. O silenciamento gênico de Dexras1 promoveu aumento da secreção de insulina, e aumentou os níveis de pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 e PKA. Sendo assim, a regulação de Dexras1 por PRL e GCs contribui para a secreção de insulina característica do periparto. / It is not clear how glucocorticoid receptor (GR) counteracts STAT5 activity during the transition of pregnancy to lactation. Dexras1 is a small G protein activated by dexamethasone (DEX) that controls cell morphology, growth, etc. In the present study we detected Dexras1 expression in pancreatic beta cell. DEX induces Dexras1 expression in RINm5F cells, which is increased in pregnancy and decreased in lactation. The expression of 11<font face=\"Symbol\">&#946HSD1, the glucocorticoids (GCs) activating enzyme, followed Dexras1 profile in pancreatic islet. Both GR and STAT5b bindings to Dexras1 gene promoter, analyzed by ChIP assay, are increased by DEX and PRL counteracts this effect. Both bindings are decreased in pregnancy and increased in lactation. DEX induces STAT5b association to GR, phosphorylation and nuclear translocation. Dexras1 knockdown using small interference RNA (si-RNA) promoted an increase in insulin secretion, as well as increased levels of pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 and PKA. Thus, Dexras1 regulation by PRL and GCs contributes to insulin secretion during peripartum.

Made available in DSpace on 2017-07-21T19:59:57Z (GMT). No. of bitstreams: 1 Nayara Carvalho Leite.pdf: 2701096 bytes, checksum: 1dca8ecc3a7945789cec9125b5a0c33b (MD5) Previous issue date: 2013-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A obesidade é um fator de risco para o desenvolvimento do diabetes tipo 2. O exercício físico reduz o tecido adiposo, modula a secreção e ação da insulina preservando a homeostase glicêmica. A administração de glutamato monossódico (MSG) induz lesões hipotalâmicas que levam a obesidade. O controle da secreção de insulina depende da formação do ATP nas células β pancreáticas, processo acoplado a rotas metabólicas glicolíticas e mitocondriais. O presente estudo investigou o efeito do exercício físico no metabolismo da glicose em ilhotas pancreáticas de ratos MSG-obesos. A obesidade foi induzida pela administração de MSG (4g/Kg). Controles (CON) receberam salina. Aos 21 dias os animais foram separados em 4 grupos CON-SED (sedentários); CON-EXE (exercitados); MSG-SED e MSG-EXE. O exercício consistiu em natação (3x/semana/30min). As ilhotas foram isoladas e incubadas com glicose (16,7 mM) na presença ou ausência dos seguintes bloqueadores do metabolismo da glicose: 1-Ácido Iodoacético (IAA, 1mM), bloqueia a glicólise; 2– Ácido alfa-ciano-4-hidroxicinâmico (-CHC, 1mM), evita o metabolismo do piruvato; 3– Fluoroacetato de Sódio (SF,2 mM) inibe o ciclo do ácido tricarboxílico (AT); 4– Rotenona (ROT, 1M) e 5- Antimicina (ANT, 50nM), inibidores respectivamente dos complexos mitocondriais I e III. A expressão proteica do transportador GLUT2 foi avaliada em ilhotas pancreáticas. Os dados foram avaliados por análises de variância (ANOVA) ou Teste t de Student (p<0,05). Ratos MSG-SED desenvolveram obesidade, resistência à insulina e hipersecreção de insulina em relação aos ratos CON-SED. A natação evitou a hiperinsulinemia, corrigiu a resistência à insulina e atenuou o excesso de tecido adiposo em ratos MSG. Ilhotas pancreáticas de ratos MSG-SED apresentam hipertrofia, aumentada expressão do GLUT2, secretando cerca de 25% mais insulina em relação a ilhotas de ratos CON-SED. Em ambos os grupos a natação reduziu em média 25% a secreção de insulina estimulada por glicose. A natação atenuou a hipertrofia dos adipócitos, das ilhotas pancreáticas, bem como corrigiu a expressão do GLUT2 em ratos MSG-obesos. O efeito do bloqueador glicolítico foi mais acentuado em ilhotas de ratos MSG-SED, indicando maior ativação desta via. O bloqueio do complexo I mitocondrial teve efeito similar entre os grupos. Todavia a inibição do complexo III foi menos acentuada em ilhotas de ratos MSG-SED. Os bloqueadores do ciclo AT e transporte do piruvato não inibiram o controle secretor de insulina em ilhotas de todos os grupos; porém o α-CHC exerceu efeito paradoxal em ilhotas de ratos MSG-SED. Alterações da glicólise; do ciclo do AT e do complexo I mitocondrial parecem não estar envolvidas na menor resposta a glicose encontrada em ilhotas de ratos exercitados. Porém, aumento da participação do complexo III mitocondrial foi observado em ilhotas de ambos os grupos exercitados. O tratamento neonatal com MSG induz obesidade, dislipidemia e resistência à insulina, eventos atenuados e/ou revertidos pela natação. A hipersecreção de insulina é corrigida pelo exercício sem alterar a via glicolítica e/ou do ciclo do AT. Todavia, a natação parece elevar a participação do complexo III mitocondrial.

Made available in DSpace on 2017-07-21T19:59:58Z (GMT). No. of bitstreams: 1 Elisangela Gueiber Montes.pdf: 2966733 bytes, checksum: 6b6637d8142736fd55b645149fba4694 (MD5) Previous issue date: 2013-05-29 / Introduction: Adipose tissue excess is associated to the metabolic syndrome (MS), resulting in several physiological changes, such as, glucose intolerance, dyslipidemia, hypertension and insulin resistance, a set of symptoms intimately linked to the development of type II Diabetes Mellitus (DM2). Obese and/or diabetic people also present a low degree of chronic inflammation intertwining the different MS clinical and physiopathological symptoms. Recently, the spleen, one of the main lymphoid organs in our body, has been appointed as the active organ in the obesity inflammatory process. Objective: This study investigated the effects of spleenectomy in SM induced through the neonatal treatment with monosodium glutamate (MSG). Methodology: Wistar male rats received MSG injections (4g/kg) from the first to the fifth day of life, while de control group (CON) received equimolar saline. The animals were weaned on the 21st day and on day 60 half of the animals were submitted to spleenectomy (ESPL) and the spleen was removed. Four experimental groups were formed. CON-NO (not operated); CON-ESPL; MSG-NO; MSG-ESPL. From day 21 to day 90, food and water consumption as well as body weight were evaluated. On day 90, pancreatic islets were isolated using the collagenase technique and incubated in glucose (5,6; 8,3; and 16,7mM) or glucose (11.1 mM) plus L-NAME (2,5; 5,0 and 10,0nM) inhibitor of the nitric oxide producing enzyme (iNOS). The insulin was dosed through radioimmunoassay. Blood was collected and plasma separated for glucose, triglycerides and total cholesterol biochemical dosages. Insulin resistance was evaluated using the insulin tolerance test (1U/Kg). Hemogram automated analysis was also carried out. White and brown fat depots, as well as pancreas and spleen were removed for histological procedures. Data was expressed as mean ± standard error of the mean (sem). Variance analysis (ANOVA), with Bonferroni post-test (p<0,05) were employed. Results and Discussion: Rats MSG-NO presented insulin resistance, increase of about 78% in the insulin, triglycerides and cholesterol levels. Additionally, the content of body fat was around 215% higher in this group in relation to the CON-NO rats, without changing the food control. The size of adipocytes and pancreatic islets was around 58% smaller in this group when compared to CON-NO rats. Pancreatic islets of MSG-NO rats presented insulin hypersecretion, followed by alterations in the response to the iNOS blocker. The hematocrit and number of leucocytes was around 50% higher in MSG-NO rats when compared to the CON-NO group. The ESPL abolished hyperinsulinaemia, insulin resistance and reduced the food ingestion in MSG rats. However, ESPL elevated approximately 21% the level of triglycerides and cholesterol in the MSG-ESPL. MSG-ESPL rats presented alteration in the lymphocyte and granulocyte profile after surgery. The ESPL reduced approximately 25% and 63%, respectively, the hypertrophy of adypocites and pancreatic islets in MSG rats, as well as normalized the insulin hypersecretion. Finally, SPL normalized the response to the iNOS blocker. Conclusions: Spleenectomy reduces obesity, hyperinsulinaemia and resistance to insulin in MSG rats, such alterations might be related to reduction in the inflammatory process originated in the spleen. / Introdução: O excesso de tecido adiposo está associado a instalação da síndrome metabólica (SM), reunindo diversas alterações fisiológicas, tais como, intolerância a glicose, dislipidemia, hipertensão e resistência a insulina, um quadro intimamente ligado ao desenvolvimento de Diabetes Mellitus tipo 2 (DM2). Indivíduos obesos e/ou diabéticos também apresentam um baixo grau de inflamação crônica interligando os diferentes sintomas clínicos e fisiopatológicos da SM. Recentemente o baço, um dos principais órgãos linfoides do nosso corpo, tem sido apontado como órgão participante do processo inflamatório na obesidade. Objetivo: O presente trabalho investigou os efeitos da esplenectomia na SM induzida pelo tratamento neonatal com glutamato monossódico (MSG). Metodologia: Ratos Wistar machos receberam durante os 5 primeiros dias de vida injeções de MSG (4g/Kg). Controles (CON) receberam salina equimolar. Os animais foram desmamados aos 21 dias e aos 60 dias metade dos animais foram submetidos a esplenectomia (ESPL) para retirada do baço, formando-se quatro grupos experimentais: CON-NO (não-operado); CON-ESPL; MSG-NO; MSG-ESPL). Dos 21 aos 90 dias foram avaliados consumo alimentar, hídrico e peso corporal. Aos 90 dias ilhotas pancreáticas foram isoladas pela técnica da colagenase e incubadas com glicose (5,6; 8,3 e 16,7mM) ou glicose (11.1mM) mais L-NAME (2,5; 5,0 e 10,0mM) inibidor da enzima produtora de óxido nítrico (iNOS). A insulina foi dosada por radioimunoensaio. O sangue foi coletado e plasma separado para dosagens bioquímicas de glicose, triglicerídeos e colesterol total . A resistência a insulina foi avaliada pelo teste de tolerância a insulina (1U/Kg). Análise automatizada do hemograma também foi realizada. Os depósitos de gordura branco e marrom, bem como, o pâncreas e o baço foram retirados para procedimentos histológicos. Os dados foram expressos como média ± erro padrão da média (epm), Análise de Variância (ANOVA), com pós-teste de Bonferroni (p<0,05) foi usado. Resultados e Discussão: Ratos MSG-NO apresentaram resistência à insulina, aumento de aproximadamente 78% nos níveis de insulina, triglicerídeos e colesterol. Adicionalmente o conteúdo de gordura corporal foi cerca de 215% maior neste grupo em relação aos ratos CON-NO, sem modificar o controle alimentar. O tamanho dos adipócitos e das ilhotas pancreáticas do grupo MSG-NO foi aproximadamente 115% maior, enquanto o número de adipócitos e de ilhotas pancreáticas foi cerca de 58% menor neste grupo comparado aos ratos CON-NO. Ilhotas pancreáticas de ratos MSG-NO apresentam hipersecreção de insulina, acompanhado de alterações na responsividade ao bloqueador da iNOS. O hematócrito e o número de leucócitos foi cerca de 50% maior nos ratos MSG-NO comparados ao grupo CON-NO. A ESPL aboliu a hiperinsulinemia, a resistência a insulina e reduziu a ingestão alimentar nos ratos MSG. Todavia, a ESPL elevou aproximadamente 21% o nível de triglicerídeos e colesterol no grupo MSG-ESPL. Ratos MSG-ESPL apresentaram alteração no perfil de linfócitos e granulócitos após a cirurgia. A ESPL reduziu aproximadamente 25% e 63% respectivamente a hipertrofia dos adipócitos e das ilhotas pancreáticas de ratos MSG, bem como normalizou a hipersecreção de insulina. Finalmente a ESPL normalizou a responsividade ao bloqueador da iNOS. Conclusões: A esplenectomia reduz a obesidade, a hiperinsulinemia e a resistência a insulina em ratos MSG, alterações que podem estar relacionadas a redução do processo inflamatório oriundo do baço. Palavras chave: Baço, Inflamação, Ilhotas

O diabetes mellitus tipo 1 é uma doença autoimune causada pela destruição das células β produtoras de insulina, presentes nas ilhotas pancreáticas, por células autorreativas do sistema imune. A opção de tratamento mais utilizada são injeções diárias de insulina exógena, o que configura um tratamento não curativo. Para alcançar a independência de insulina, alternativas como o transplante de ilhotas vêm sendo estudadas. Entretanto, a disponibilidade de pâncreas de doadores cadavéricos para o isolamento destas ilhotas é pequena e os métodos de isolamento, pouco eficazes, sendo necessários de 2 a 4 doadores para atingir o número adequado de ilhotas. Além disso, o transplante apresenta problemas relacionados à enxertia, devidos principalmente à baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Desta forma, estudos explorando alternativas que aumentem a sobrevivência e a funcionalidade dos transplantes e diminuam o número de ilhotas exigido por receptor fazem-se muito necessários. As células-tronco mesenquimais apresentam propriedades interessantes para aplicação em terapia celular. Entre elas, destaca-se o efeito parácrino, que exerce diversas funções benéficas, como o aumento da vascularização, nos locais onde estas células estão presentes. Sendo assim, este trabalho explorou o co-transplante de ilhotas pancreáticas com células-tronco mesenquimais derivadas de tecido adiposo, para o tratamento do diabetes mellitus em modelo murino. Os resultados mostraram que a presença destas células no grupo que recebeu o co-transplante não aumentou a taxa de cura, em relação ao grupo que recebeu somente ilhotas. No entanto, o fenômeno de reversão do diabetes foi antecipado no grupo co-transplantado, o que sugere um possível efeito angiogênico das células-tronco adiposo-derivadas presentes neste grupo. Desta forma, conclui-se que estas células podem exercer atividades benéficas, quando co-transplantadas com ilhotas pancreáticas, para o tratamento do diabetes. / Type 1 diabetes mellitus is an autoimmune disease caused by destruction of insulin-producing β cells, present in pancreatic islets, by auto-reactive cells of the immune system. The most widely used treatment option are daily injections of insulin, which configures a non-curative treatment. To achieve insulin independence, alternatives such as islet transplantation have been studied. However, the availability of pancreas from cadaveric donors for the isolation of these islets is poor and the methods for isolation, ineffective, requiring 2 to 4 donors to achieve the appropriate number of islets. In addition, transplantation presents problems related to engraftment, mainly due to poor vascularization, which leads to β cell death in the first days after transplantation. Thus, studies exploring alternatives that increase the survival and function of transplants and reduce the number of islets required by the recipient are very necessary. Mesenchymal stem cells have interesting properties for application in cell therapy. Among them is the paracrine effect, which has several beneficial functions, such as promoting vascularization in the tissues where these cells are present. Thus, the present study explored the co-transplantation of pancreatic islets with mesenchymal stem cells derived from adipose tissue for the treatment of diabetes mellitus in mice. The results showed that the presence of these cells in the group that received co-transplantation did not increase the cure rate, compared to the group that received islets alone. However, the phenomenon of diabetes reversion was anticipated in co-transplanted animals, which suggests a possible angiogenic effect of adipose-derived stem cells present in this group. Thus, we conclude that these cells may exert beneficial functions when co-transplanted with pancreatic islets for the treatment of diabetes.

Zhao Hailu. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 192-210). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and 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

Ng, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 179-204). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.IV / Publications --- p.VII / Acknowledgements --- p.VIII / Table of contents --- p.IX / List of figures --- p.XV / List of tables --- p.XVII / List of abbreviations --- p.XVIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The Pancreas --- p.2 / Chapter 1.1.1 --- Anatomy of Pancreas --- p.2 / Chapter 1.1.2 --- The Exocrine Pancreas --- p.4 / Chapter 1.1.3 --- The Endocrine Pancreas --- p.5 / Chapter 1.1.3.1 --- Structure of Islets --- p.5 / Chapter 1.1.3.2 --- "Functions of α-, β-, y-, ð-, Σ-and PP-cells in Islets" --- p.7 / Chapter 1.1.4 --- Overview of Pancreas Development --- p.9 / Chapter 1.1.4.1 --- Organ Morphology --- p.10 / Chapter 1.1.4.2 --- Cyto-differentiation --- p.12 / Chapter 1.1.4.3 --- Control by Transcriptional Factors --- p.14 / Chapter 1.1.5 --- Postnatal Pancreas Development and Regeneration --- p.18 / Chapter 1.1.5.1 --- Proliferation of Pre-existing β-cells --- p.19 / Chapter 1.1.5.2 --- Neogenesis from Precursor Cells --- p.20 / Chapter 1.1.5.3 --- Transdifferentiation of other Cells --- p.20 / Chapter 1.2 --- Diabetes Mellitus --- p.22 / Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus and Current Treatments --- p.24 / Chapter 1.2.1.1 --- Type I Diabetes Mellitus --- p.24 / Chapter 1.2.1.2 --- Type II Diabetes Mellitus --- p.25 / Chapter 1.2.1.3 --- Gestational Diabetes --- p.27 / Chapter 1.2.1.4 --- Secondary Diabetes --- p.28 / Chapter 1.3 --- Stem Cell therapy --- p.29 / Chapter 1.3.1 --- Stem Cell --- p.29 / Chapter 1.3.1.1 --- Mesenchymal Stem Sell --- p.31 / Chapter 1.3.1.2 --- Embryonic Stem Cell --- p.35 / Chapter 1.3.1.3 --- Induced Pluripotent Stem Cell --- p.36 / Chapter 1.3.2 --- Islets Engineering --- p.37 / Chapter 1.3.2.1 --- Genetic Modification --- p.37 / Chapter 1.3.2.2 --- Directed Differentiation --- p.38 / Chapter 1.3.2.3 --- Microenvironment --- p.38 / Chapter 1.3.2.4 --- In vivo Regeneration --- p.39 / Chapter 1.3.2.5 --- Cell Fusions --- p.40 / Chapter 1.3.2.6 --- Combinatory Treatments --- p.40 / Chapter 1.4 --- The Vitamin A & Vitamin D System --- p.42 / Chapter 1.4.1 --- The Vitamin A --- p.42 / Chapter 1.4.2 --- Vitamin A Metabolism --- p.44 / Chapter 1.4.3 --- Roles of vitamin A in Pancreatic Development --- p.46 / Chapter 1.4.4 --- The Vitamin D --- p.48 / Chapter 1.4.5 --- Vitamin D Metabolism --- p.49 / Chapter 1.4.6 --- Metabolic Functions of Vitamin D in Islets --- p.51 / Chapter 1.4.7 --- Cod Liver Oil --- p.53 / Chapter 1.4.8 --- Interactions between Vitamin A and Vitamin D --- p.53 / Chapter 1.5 --- The Relations of Liver and Pancreas Development --- p.55 / Chapter 1.5.1 --- Endoderm Induction for Hepatic and Pancreatic Differentiation of ESCs --- p.55 / Chapter 1.5.2 --- Bipotential Precursor Population within Embryonic Endoderm --- p.56 / Chapter 1.5.3 --- Pancreatic Islets Promote Mature Liver Hepatocytes Proliferation --- p.57 / Chapter 1.5.4 --- Transdifferentiation --- p.57 / Chapter 1.5.5 --- Transplantation in Liver Niche Promotes Maturation of Insulin-Producing Cells --- p.60 / Chapter 1.5.6 --- Neuronal Relay from the Liver to Pancreatic --- p.61 / Chapter 1.5.7 --- Development of Islets in the Nile Tilapia --- p.62 / Chapter 1.6 --- The Insulin-like Growth Factor-I (IGF1) --- p.64 / Chapter 1.6.1 --- IGF1 System --- p.64 / Chapter 1.6.2 --- IGF 1 Regulation --- p.65 / Chapter 1.6.3 --- Roles of IGF 1 in Pancreatic Development and Regeneration --- p.68 / Chapter 1.7 --- Aims and Objectives of Study --- p.70 / Chapter Chapter 2 --- General Materials and Methods / Chapter 2.1 --- Pancreatic progenitor cells (PPCs) and liver stromal cells (LSCs) isolation and cell culture --- p.72 / Chapter 2.1.1 --- Tissue procurement --- p.72 / Chapter 2.1.2 --- PPC and LSC culture --- p.72 / Chapter 2.1.3 --- "Treatments of vitamin A, vitamin D and IGF 1" --- p.76 / Chapter 2.1.4 --- "Cell culture of Caco-2, HepG2 and DU-145" --- p.76 / Chapter 2.2 --- Induction of Islet-like Cell Clusters (ICCs) Differentiation --- p.77 / Chapter 2.2.1 --- In vitro Directed Differentiation --- p.77 / Chapter 2.2.2 --- In vitro LSC Microenvironment --- p.77 / Chapter 2.3 --- RNA Expression Detection --- p.79 / Chapter 2.3.1 --- RNA isolation --- p.79 / Chapter 2.3.2 --- Reverse Transcription --- p.79 / Chapter 2.3.3 --- Polymerase Chain Reaction (PCR) --- p.80 / Chapter 2.3.4 --- Realtime PCR --- p.81 / Chapter 2.4 --- Immunocytochemistry --- p.83 / Chapter 2.5 --- Western Blotting --- p.85 / Chapter 2.5.1 --- Protein extraction and quantification --- p.85 / Chapter 2.5.2 --- Western Blotting --- p.85 / Chapter 2.6 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.87 / Chapter 2.6.1 --- Detection of cell viability --- p.87 / Chapter 2.6.2 --- Detection of cell proliferation --- p.87 / Chapter 2.6.3 --- Measurement of Cell death --- p.88 / Chapter 2.6.4 --- Measurement of IGF 1 level in condition medium --- p.89 / Chapter 2.6.5 --- Measurement of glucose induced insulin secretion --- p.90 / Chapter 2.7 --- Regeneration model --- p.92 / Chapter 2.7.1 --- Regeneration model in neonatal-STZ rat --- p.92 / Chapter 2.7.2 --- Change in IGF1 expression in pancreas and liver --- p.92 / Chapter 2.8 --- Statistical Data Analysis --- p.93 / Chapter Chapter 3 --- Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas. (Stem Cell Rev. 2011;7:53-63) / Chapter 3.1 --- Abstract --- p.95 / Chapter 3.2 --- Introduction --- p.97 / Chapter 3.3 --- Materials and Methods --- p.101 / Chapter 3.3.1 --- Fetal Tissue Procurement --- p.101 / Chapter 3.3.2 --- Culture of Pancreatic Progenitor Cells --- p.101 / Chapter 3.3.3 --- RNA Expression Analysis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.102 / Chapter 3.3.4 --- Western Blot Analysis --- p.103 / Chapter 3.3.5 --- Immunocytochemstry --- p.105 / Chapter 3.3.6 --- PPC Proliferation Assays --- p.106 / Chapter 3.3.7 --- PPC Cell Death Assays --- p.107 / Chapter 3.3.8 --- Statistical Data Analysis --- p.108 / Chapter 3.4 --- Results --- p.110 / Chapter 3.4.1 --- "Expression and Localization of RAR, VDR and RXR, CYP26 and CYP24 in PPCs" --- p.110 / Chapter 3.4.2 --- Incubation of PPC with atRA Enhances PPC Viability due to Increased Proliferation and Anti-apoptosis --- p.111 / Chapter 3.4.3 --- Incubation of PPCs with Calcitriol Enhances Viability due to Increased Proliferation --- p.111 / Chapter 3.4.4 --- Both atRA and Calcitriol Induce Up-regulation of both the RAR and the VDR but not the RXR --- p.112 / Chapter 3.4.5 --- Combination Treatment with atRA and Calcitriol on Cell Viability and NGN3 Expression --- p.112 / Chapter 3.5 --- Discussion --- p.114 / Chapter Chapter 4 --- Human fetal liver stromal cell co-culture enhances the growth and differentiation of pancreatic progenitor cells into islet-like cell clusters (In submission to Gastroenterology) / Chapter 4.1 --- Abstract --- p.128 / Chapter 4.2 --- Introduction --- p.129 / Chapter 4.3 --- Materials and Methods --- p.133 / Chapter 4.3.1 --- Use of human and animal tissues --- p.133 / Chapter 4.3.2 --- "Cell preparation, characterizations and Differentiation" --- p.133 / Chapter 4.3.3 --- Examination of PPC growth and ICC differentiation and functions with LSC co-culture --- p.133 / Chapter 4.3.3 --- Identification of growth factors and investigation of their effects --- p.134 / Chapter 4.3.4 --- Statistical Analysis --- p.135 / Chapter 4.4 --- Results --- p.136 / Chapter 4.4.1 --- "Isolation, Culture and Characterizations of LSCs" --- p.136 / Chapter 4.4.2 --- Establishment of LSC co-culture system --- p.136 / Chapter 4.4.3 --- LSC co-culture enhances PPC-derived ICC differentiation --- p.137 / Chapter 4.4.4 --- Differential expression of mRNA for cytokines and growth factors between 1st and 2nd trimester LSCs --- p.138 / Chapter 4.4.5 --- Characterization of IGF 1 receptors in PPCs and the effects of exogenous IGF1 on PPC growth and ICC differentiation --- p.139 / Chapter 4.4.6 --- Neutralizing antibodies against IGF1R inhibit ICC differentiation --- p.140 / Chapter 4.5 --- Discussion --- p.142 / Chapter 4.6 --- Supplementary Materials and Methods --- p.147 / Chapter 4.6.1 --- Cell Preparation and culture --- p.147 / Chapter 4.6.2 --- In Vitro ICC differentiation --- p.148 / Chapter 4.6.3 --- RNA expression analysis --- p.149 / Chapter 4.6.4 --- Immunocytochemistry --- p.149 / Chapter 4.6.5 --- PPC viability and cell count assays --- p.150 / Chapter 4.6.6 --- IGF1 and insulin ELISA --- p.151 / Chapter 4.6.7 --- Western blotting analysis --- p.152 / Chapter 4.6.8 --- Neonatal streptozotocin regeneration model --- p.153 / Chapter Chapter 5 --- General Discussion and Future Studies / Chapter 5.1 --- General Discussion --- p.165 / Chapter 5.1.1 --- Proliferative effects and enhance expression of NGN3 by vitamin A and vitamin D on PPC --- p.166 / Chapter 5.1.2 --- Induction of PPC derived ICCs by LSCs --- p.169 / Chapter 5.1.3 --- Potential effects of liver stroma derived IGF1 on PPC derived ICCs differentiation --- p.172 / Chapter 5.1.4 --- Significance of islet engineering in the management of diabetes --- p.174 / Chapter 5.1.5 --- Conclusions --- p.176 / Chapter 5.2 --- Future Studies --- p.177 / Chapter Chapter 6 --- Reference / Reference --- p.180

O tamanduá-bandeira Myrmecophaga tridactyla é uma espécie considerada &ldquo;vulnerável&rdquo; no Brasil, por estar ameaçado de extinção em algumas regiões do país. O presente projeto teve por objetivo identificar e caracterizar as estruturas macro e microscópicas do pâncreas nessa espécie. Para tanto, foram dissecados 16 pâncreas de tamanduás-bandeira provenientes do Hospital Veterinário &ldquo;Dr. Halim Atique&rdquo; do Centro Universitário de Rio Preto (UNIRP). As amostras coletadas, foram provenientes de casos de animais atendidos no referido Hospital e que vieram a óbito. O pâncreas situava-se no antímero esquerdo do corpo do animal, apresentava coloração pálida, corpo central e superfície lobulada. Acompanhava a curvatura ventricular maior do estomago aderindo-se na porção inicial do duodeno. Relaciona-se crâniodorsalmente com o baço e ventrículo gástrico, e caudoventralmente com a cápsula fibrosa renal (que aloja o rim esquerdo) e intestinos. Estruturalmente, o órgão demonstrou duas partes distintas: a primeira delas com características exócrinas, composta por ácinos pancreáticos e a segunda endócrina, formada pelas ilhotas pancreáticas encontradas nas regiões media, caudoventral e lobar esquerda. A analise ultraestrutural permitiu identificar nas células centro-acinosas do pâncreas vesículas com grânulos de zimogênio, mitocôndrias, Aparelho de Golgi e retículo endoplasmático rugoso / The giant anteater Myrmecophaga tridactyla is a species considered &#39;vulnerable&#39; in Brazil since it is threatened in some Brazilian regions. This study aimed to identify and characterize morphological structures of the pancreas in this species. For this, 16 anteaters pancreas from the Veterinary Hospital &quot;Dr. Halim Atique at University Center of Rio Preto (UNIRP), were dissected. All samples were from animals treated at the hospital which died of natural causes. The pancreas was located in the left antimere of the animal&rsquo;s body, being lobulated and having a pale color and central body. It followed the greater curvature of the stomach, adhering on the initial portion of the duodenum. It was craniodorsally related to the spleen and gizzard, and caudoventrally to the renal fibrous capsule (which houses the left kidney) and intestines. Structurally, the organ had two distinct parts: an exocrine, composed of pancreatic acini; and and endocrine, formed by pancreatic islets found in the medial, caudoventral and left lobar regions. The ultrastructural analysis allowed identifying the central-acinar pancreatic cells with vesicles zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum

A NADPH oxidase participa da secreção de insulina estimulada pela glicose e da produção de superóxido nas ilhotas pancreáticas. Nesse estudo, avaliamos o efeito do ácido palmítico na produção de superóxido e secreção de insulina por ilhotas pancreáticas de ratas e linhagem INS-1E. O palmitato aumentou a produção de superóxido por mecanismo dependente da ativação da PKC e da NADPH oxidase e da oxidação do ácido graxo. O ácido graxo causou a translocação da p47PHOX para a membrana plasmática, processo indicativo da ativação do complexo enzimático. A exposição de ilhotas ao palmitato causou o aumento do conteúdo proteico da p47PHOX e do RNA mensageiro da p22PHOX, gp91PHOX, p47PHOX, pró-insulina e do GPR40. A estimulação da secreção de insulina pelo ácido graxo na presença de alta glicose foi reduzida através do inibidor da NADPH oxidase e também pela inibição da expressão do GPR40 por RNA de interferência. A atividade da NADPH oxidase e a sinalização via GPR40 são mecanismos envolvidos no controle da secreção de insulina estimulada pelo palmitato. / The NADPH oxidase complex is involved in the glucose-stimulated insulin secretion and the superoxide production in pancreatic islets. In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and INS-1E cells. Palmitate increased superoxide production in a process dependent on the activation of PKC, NADPH oxidase and fatty acid oxidation. In fact, palmitate caused p47PHOX translocation to the plasma membrane. Exposure to palmitate for 1 h up-regulated the protein content of p47PHOX and the mRNA levels of p22PHOX, gp91PHOX, p47PHOX, proinsulin and the GPR40. Fatty acid stimulation of insulin secretion in the presence of a high glucose concentration was reduced by the inhibition of NADPH oxidase activity and by the inhibition of GPR40 expression by a small interference RNA. In conclusion, NADPH oxidase is an important source of palmitate-induced superoxide production in beta cells. The NADPH oxidase activity and GPR40 signaling are involved in the control of insulin secretion by palmitate.