Zugl.: Giessen, Universiẗat, Diss., 2007.

The gastrointestinal tract comprises a number of digestive organs including the stomach and pancreas. The stomach is involved in the digestion and short term storage of food while the pancreas is a mixed endocrine and exocrine gland which provides the body with hormones and enzymes essential for nutritional utilisation. The pancreas consists of three different cell lineages, acinar, ductal and endocrine cells. The endocrine cells, organised in the islets of Langerhans, are scattered throughout the exocrine parenchyma and regulate blood glucose levels by production of hormones such as glucagon and insulin. The Nkx family of homeodomain proteins controls numerous processes during development. Previous studies have identified two members belonging to the Nkx6 subfamily of Nkx proteins, Nkx6.1 and Nkx6.2. We have described the cloning and embryonic expression pattern of Nkx6.3. All three members of the Nkx6 gene family were shown to be expressed in partially overlapping domains during the development of the gastrointestinal tract and the central nervous system. Nkx6.2 was also identified as a transient marker for pancreatic exocrine cells. Analysing gene expression patterns and morphological features in tissues and organs is often performed by stereologic sampling which is a labour-intensive two dimensional approach that rely on certain assumptions when calculating e.g. β-cell mass and islet number in the pancreas. By combined improvements in immunohistochemical protocols, computational processing and tomographic scanning, we have developed a methodology based on optical projection tomography (OPT) allowing for 3D visualisation and quantification of specifically labelled objects within intact adult mouse organs. In the pancreas, this technique allows for spatial and quantitative measurements of total islet number and β-cell mass. We have further developed a protocol allowing for high resolution regional analyses based on global OPT assessments of the pancreatic constitution. This methodology is likely to facilitate detailed cellular and molecular analysis of user defined regions of interest in the pancreas, at the same time providing information on the overall disease state of the gland. Type 1 diabetes mellitus (T1D) can occur at any age and is characterized by the marked inability of the pancreas to secrete insulin due to an autoimmune destruction of the insulin producing β-cells. Information on the key cellular and molecular events underlying the recruitment of lymphocytes, their infiltration of the islets of Langerhans and consequent β-cell destruction is essential for understanding the pathogenesis of T1D. Using the developed methodology we have recorded the spatial and quantitative distribution of islet β-cells and infiltrating lymphocytes in the non obese diabetic (NOD) mouse model for T1D. This study shows that the smaller islets, which are predominantly organised in the periphery of the organ, are the first to disappear during the progression of T1D. The larger islets appear more resistant and our data suggest that a compensatory proliferative process is going on side by side with the autoimmune-induced β-cell destruction. Further, the formation of structures resembling tertiary lymphoid organs (TLOs) in areas apparently unaffected by insulitis suggests that local factors may provide cues for the homing of these lymphocytes back to the pancreas.

O Diabetes Mellitus do tipo 1 (DM1) é uma doença metabólica multifatorial caracterizada por hiperglicemia e hipoinsulinemia crônicas, decorrentes da destruição progressiva das células &#946 pelo sistema imunológico. Durante a progressão do DM1, as ilhotas de Langherhans são invadidas por células do sistema imune que secretam citocinas pró-inflamatórias, gerando um quadro denominado insulite. A exposição das células &#946 a essas citocinas leva a ativação de diversas vias de sinalização que aumentam o estresse oxidativo e de retículo endoplasmático, contribuindo para a indução da morte das células &#946. Existem muitos estudos que investigam as vias moleculares que levam à destruição da célula durante o DM1, contudo é necessário um melhor entendimento da regulação e contribuição dessas diferentes vias para o desenvolvimento dessa patologia para que se possa desenhar terapias mais apropriadas para impedir seu desenvolvimento ou até mesmo para se atingir uma cura. Algumas das dificuldades encontradas na aplicação desses estudos estão relacionadas ao fato de que eles são conduzidos em sua maioria em células &#946 ou ilhotas isoladas em cultura. Sabe-se que há uma importante regulação entre os diferentes tipos celulares presentes na ilhota e também entre as células da ilhota e as células adjacentes a ela, o que, sem dúvida, influencia no destino da célula &#946 frente a um ataque autoimune. Este trabalho visou aperfeiçoar o conhecimento acerca do comportamento das células da ilhota pancreática frente ao desenvolvimento do DM1, utilizando técnicas de imunomarcação em cortes histológicos pancreáticos de três modelos animais de DM1. No primeiro modelo: Influência do exercício físico na indução do DM1, mostramos que o exercício físico é capaz de prevenir a destruição das células &#946 e potencialmente estar envolvido na transdiferenciação celular para recuperação de células endócrinas na ilhota desses animais. No segundo modelo: Efeito de NOX1 e NOX2 na viabilidade e função de células &#946 \", observamos que a presença dessas NADPH oxidases parece ter influência na estrutura e provavelmente na viabilidade de células &#946. No terceiro modelo: Papel de HNF4 &#945 na viabilidade e função das células &#946, mostramos que a imunomarcação das ilhotas de animais KO para esse fator de transcrição nas células &#946 é uma ferramenta de extrema importância para esse estudo. O desenvolvimento desse trabalho possibilita que a investigação de diferentes vias envolvidas na destruição das células &#946 seja realizada no ambiente em que essas células se encontram, permitindo avaliar a ativação de vias especificas, como por exemplo ativação de NF-kB e validar os resultados observados em células isoladas. / Type 1 Diabetes Mellitus (DM1) is a multifactorial metabolic disease characterized by chronic hyperglycemia and hypoinsulinemia, which is due to the progressive and specific destruction of &#946 cells by the immune system. During the progression of DM1, the islets of Langherhans are invaded by cells of the immune system that secrete proinflammatory cytokines, in a process called insulitis. Exposure of &#946 cells to these cytokines leads to the activation of several signaling pathways that increase oxidative and endoplasmic reticulum stress, contributing to the -cell death. There are many studies that investigate the molecular pathways that lead to the destruction of the &#946-cell during DM1, but a better understanding of the regulation and contribution of these different pathways to the development of this pathology is necessary in order to design more appropriate therapies to prevent their development or even to achieve a cure. Some of the difficulties encountered in the application of these studies are related to the fact that they are conducted mostly on &#946-cells or isolated islets in cell culture. It is known that there is an important regulation between the different cell types present in the islet and also between the islet cells and the cells adjacent to it, which undoubtedly influences the fate of the &#946-cell against an autoimmune attack. This work aimed to improve the knowledge about the behavior of pancreatic islet cells in the development of DM1, using immunostaining techniques in pancreatic histological sections of three animal models of DM1. In the first model, \"Influence of physical exercise on the induction of DM1\", we showed that physical exercise is able to prevent the destruction of &#946 cells and potentially be involved in cell transdifferentiation for the recovery of endocrine cells in the islet of these animals. In the second model, \"Effect of NOX1 and NOX2 on viability and &#946 cell function\", we observed that the presence of these NADPH oxidases appears to influence the structure and probably the viability of &#946 cells. \"In the third model:\" Role of HNF4&#945 in viability and &#946 cell function, we show that the immunostaining of islets of KO animals for this transcription factor in &#946 cells is a tool of paramount importance for this study. The development of this work enables the investigation to be performed in the environment of these cells, allowing to evaluate the activation of specific pathways and validate the results observed in isolated cells.

Diabetes Mellitus is a group of complex and heterogeneous metabolic disorders characterized by hyperglycemia. Even though the condition has been extensively studied, its causes and complex pathologies are still not fully understood. The occurring damage to the pancreatic islets is strikingly heterogeneous. In type 1 diabetes, the insulin producing beta cells are all destroyed within some islets, and similarly in type 2 diabetes, some islets may be severely affected by amyloid. At the same time other islets, in the near vicinity of the ones that are affected by disease, may appear fully normal in both diseases. Little is known about this heterogeneity in susceptibility to disease between pancreatic islets. This thesis examines the physiological and pathophysiological characteristics of islet subpopulations. Two subpopulations of islets were studied; one constituting highly vascularized islets with superior beta cell functionality, and one of low-oxygenated islets with low metabolic activity. The highly functional islets were found to be more susceptible to cellular stress both in vitro and in vivo, and developed more islet amyloid when metabolically challenged. Highly functional islets preferentially had a direct venous drainage, facilitating the distribution of islet hormones to the peripheral tissues. Further, these islets had an increased capacity for insulin secretion at low glucose levels, a response that was observed abolished in patients with recent onset type 1 diabetes.  The second investigated islet subpopulation, low-oxygenated islets, was found to be an over time stable subpopulation of islets with low vascular density and beta cell proliferation. In summary, two subpopulations of islets can be identified in the pancreas based on dissimilarities in vascular support and blood flow. These subpopulations appear to have different physiological functions of importance for the maintenance of glucose homeostasis. However, they also seem to differ in vulnerability, and a preferential death of the highly functional islets may accelerate the progression of both type 1 and type 2 diabetes.