Theses (Ph. D.)--Marshall University, 2004. / Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

Orientadores: Antonio Carlos Boschiero, Kléber Luiz de Araújo e Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T22:13:57Z (GMT). No. of bitstreams: 1 Paula_FlaviaMariaMourade_D.pdf: 1277288 bytes, checksum: fbf2861e21e4c2c3095813e5df9456e2 (MD5) Previous issue date: 2013 / Resumo: Durante a instalação do diabetes tipo 1 as células beta pancreáticas são alvos do ataque pelo sistema de defesa do organismo. A morte das células beta, em geral por apoptose, é provocada por contato direto com células ativadas do sistema imune e por mediadores inflamatórios tais como: citocinas pró-inflamatórias, quemocinas e radicais livres. As citocinas pró-inflamatórias, como IL1-beta, TNF-alpha e IFN-gamma, produzem grande quantidade de ROS e RNS no interior das células beta e estas, por sua vez, possuem uma baixa defesa anti-oxidante enzimática, principalmente ao que se refere às enzimas que degradam H2O2, como catalase e glutationa peroxidase. Tal combinação resulta no surgimento de estresse oxidativo e morte celular. Adicionalmente, outro sistema de peroxidases, as PRDXs, também atuam na proteção das células beta contra o estresse oxidativo. Neste sentido, o estudo sobre a modulação de PRDXs por agentes inflamatórios é de grande valia, à medida que se tenta descobrir novas vias intracelulares desencadeadas pelas citocinas e alternativas para suprir a vulnerabilidade das células beta pancreáticas ao estresse oxidativo. Para isso utilizamos linhagem de células beta produtoras de insulina RINm5F. Estas células foram expostas às citocinas pró-inflamatórias IL1-beta, TNF-alpha e IFN-gamma e à anti-inflamatória IL-4 e a expressão das PRDXs foi analizada. Nossos resultados demonstram que IFN-gamma e TNF-alpha reduzem a expressão da PRDX6. Quando separadas, essas citocinas alteram somente a expressão protéica, através da ativação de sistemas de proteólise, especialmente de calpaínas e ubiquitina-proteassomo, e via ativação da JNK/c-Jun. A pré-incubação das células RINm5F com a citocina antiinflamatória IL4, bloqueia os efeitos do TNF-alpha ou IFN-gamma sobre a expressão da PRDX6. Em conjunto, IFN-gamma e TNF-alpha reduzem tanto a expressão gênica quanto protéica da PRDX6. As alterações transcricionais ocorrem, provavelmente, por ação sinérgica de mais de uma via intracelular, neste caso, NFkB (ativado pelo TNF-alpha) e STAT1 (ativado pelo IFN-gamma), sendo necessária a participação dessas duas vias para a modulação gênica da PRDX6. A deleção dessa enzima aumenta a suceptibilidade das células RINm5F aos efeitos deletérios de IFN-gamma, TNF-alpha e H2O2, sugerindo função importante da PRDX6 na proteção das células beta ao estresse oxidativo / Abstract: Peroxiredoxins are a family of six antioxidant enzymes (PRDX1-6), and may be an alternative system for the pancreatic beta cells to cope with oxidative stress. This study investigated whether the main diabetogenic pro-inflammatory cytokines or the antiinflammatory cytokine IL-4 modulate PRDXs levels and putative intracellular pathways important for this process in the insulin-producing RINm5F cells. RINm5F cells expressed significant amounts of PRDX1, PRDX3 and PRDX6 enzymes. Only PRDX6 was modulated by cytokines, showing both mRNA and protein down-regulation following incubation of RINm5F cells with TNF-alpha and IFN-gamma but not with IL-1beta. Separately IFN-gamma or TNF-alpha decreased PRDX6 protein but not mRNA levels. The blockage of the JNK signalling and of the calpains and proteasome proteolysis systems restored PRDX6 protein levels. IL-4 alone did not modulate PRDXs levels. However, pre/co-incubation with IL-4 substantially prevented the decrease in PRDX6 induced by proinflammatory cytokines. Knockdown of PRDX6 increased susceptibility of RINm5F cells to the deleterious effects of pro-inflammatory cytokines and to oxidative stress. These results show that, from the PRDXs highly expressed in RINm5F cells, only PRDX6 is modulated by the diabetogenic cytokines IFN-gamma and TNF-alpha. This PRDX6 downregulation depends on the Calpain and proteasome systems and JNK signalling. PRDX6 is an important enzyme for protection against oxidative stress and the interaction between pro- and anti-inflammatory cytokines might be important to determine the antioxidant capacity of the cells / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular

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

Pancreas is known to be an organ producing a variety of exocrine and endocrine substances, where also insulin belongs. This hormone is produced in the body almost solely by specialized β-cells of the Langerhans islets and is stored here in secretory granules. As the β-cells contain large number of these vesicles, an organism can quickly respond to the glucose stimulation. Completely processed insulin is formed in the secretory granules probably as a hexamer, where six insulin molecules are coordinated along two zinc bivalent cations. Appropriate β-cell response to higher glucose level and following insulin secretion is one of the key processes that regulate metabolism in the body. In order to study insulin production, its effects or secretion, permanent pancreatic cell lines are often used as biological models, out of primary cells from islets of Langerhans. This diploma thesis is focused on two permanent cell lines INS-1E and BRIN-BD11. We searched for the ability of the cells to produce insulin, if the hormone is fully processed, as well as zinc content, which could have a great influence on insulin's processing. Using different methods we compared these two cell lines with cells from the Langerhans islets. We succeeded in isolation of secretory granules from all three cell types and we plan to...

Introduction : L’efficacité de la greffe d’îlots n’est plus à démontrer mais elle reste l’objet de recherches pour améliorer la qualité et la survie des îlots greffés souvent fragilisés par la destruction enzymatique de leur microenvironnement lors de la procédure d’isolement. Dans ce contexte, les cellules stromales mésenchymateuses (CSM) d’origine pancréatique représentent un outil intéressant par leurs propriétés d’immunomodulation et par leur capacité de sécrétion de facteurs du microenvironnement. L’objectif de ce travail est d’évaluer l’effet des cellules stromales pancréatiques humaines sur les cellules β humaines.Méthodes : Des îlots humains purifiés ont été maintenus en culture pendant plusieurs jours. Les cellules adhérentes se formant en périphérie de l’îlot ont été sélectionnées et immortalisées. Ces nouvelles cellules « human islet-derived stromal cells » (hISC) ont ensuite été caractérisées pour déterminer leur profil mésenchymateux Nous avons ensemencé des cellules β humaines (lignée EndoC-βH1 ou cellules primaires) sur du milieu conditionné de hISC (hISC-CM) utilisé comme support de culture. L’adhérence, la survie, la prolifération, l’insulinosécrétion des cellules β cultivées sur le hISC-CM ont été mesurées et comparées à un support contrôle : la poly-L-lysine.Résultats : Les hISC présentent un profil phénotypique et transcriptomique très proche des CSM issues de la moelle osseuse. D’un point de vue fonctionnel, les hISC présentent une capacité d’immunomodulation. Elles expriment et sécrètent des protéines matricielles connues pour être présentes autour et à l’intérieur des îlots humains tels que les collagènes de type I, IV et VI, la laminine et la fibronectine. Au contact du hISC-CM les cellules EndoC-βH sur adhèrent st s’étalent. Le hISC-CM augmente l’expression du marqueur de prolifération PCNA et améliore la survie et la fonction des cellules EndoC-βH1. D’un point de vue mécanistique, l’interaction cellules β/hISC-CM active la phosphorylation de FAK (focal adhesion kinase) et ERK (extracellular signal-regulated kinases). A l’interface de cette interaction, la sous-unité β1 de l’intégrine est impliquée dans les effets observés du hISC-CM sur l’adhérence et la fonction des cellules β.Conclusion : Nos travaux démontrent l’intérêt prometteur des hISC en tant que cellules de soutien des cellules β humaines par la sécrétion de protéines matricielles pancréatiques. Ces résultats ouvrent de nouvelles perspectives pour le maintien des îlots en culture et leur conditionnement dans un microenvironnement plus physiologique permettant ainsi de préserver leur qualité fonctionnelle lors de la greffe. / Introduction : The efficacy of islet transplantation is well established. However, the procedure still needs improvements in the quality of grafted islets, often weakened by the loss of their surrounding tissue during the isolation process. In this respect, mesenchymal stromal cells (MSC) represent an interesting tool as they have immunomodulatory and anti-inflammatory properties and are known to secrete proteins involved in creating a favorable microenvironment. This work aims to investigate the effect of human pancreatic stromal cells on human β-cells.Methods : We characterized the mesenchymal profile of cells, previously immortalized in our lab from human islets of Langerhans adherent cells, hereafter named hISC (human islet-derived stromal cells). We seeded human β-cells (EndoC-βH1 cell line or primary β-cells) on hISC-conditioned medium (hISC-CM) used as coating of Petri dishes. We assessed spreading, survival, proliferation and glucose-induced insulin secretion of β-cells cultured on hISC-CM as compared to poly-L-lysine coating.Results : Phenotypic and transcriptomic profiles of hISC are close to bone-marrow MSC. The hISC have an immunomodulation capacity. They express and secrete extracellular matrix proteins known to be present around and within human islet such as types I, IV and VI collagens, laminin and fibronectin. EndoC-βH1 seeded on hISC-CM adhere and spread on cell culture surface. We show that hISC-CM has positive effects on EndocC-βH1 proliferation, survival and glucose-induced insulin secretion, as compared to poly-L-lysine. From mechanistic point of view, hISC-CM induces FAK (focal adhesion kinase) and ERK (extracellular signal-regulated kinases) phosphorylations. The β1-integrin subunit is involved in both adhesion and increased insulin secretion of β cells induced through hISC-CM.Conclusion : Our work demonstrates a promising interest of hISC as support cells for human β-cells by scaffolding factors secretion. It opens new perspectives for conditioning human β-cells in a more physiological microenvironment to preserve their functional quality before and after transplantation.

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes.

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic beta cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than beta cell death, suggesting loss of beta cell identity. We undertook this study to examine whether viral infection could induce human b cell dedifferentiation. Using the functional human b cell line EndoC-bH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in beta cell–specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-kB pathway and also in a paracrine non–cell-autonomous fashion through the secretion of IFN-a. Lastly, we identified SOX9 targets in human b cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human beta cell dedifferentiation.

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