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Diagnostic contribution to the evaluation of intervention models in the treatment of type 1 diabetes
Zacharovová, Klára ; Saudek, František (advisor) ; Plecitá, Lydie (referee) ; Mandys, Václav (referee)
During treatment of diabetes mellitus by immunointervention or transplantation, it is necessary to monitor the markers of immune destruction or rejection of surviving insulin producing cells. An aim of this thesis is to improve the possibilities of following autoimmunity and to detect the survival of transplanted pancreatic islet in vivo. Partial aims included vitality testing of isolated islets for transplantation by measurement of respiration activity, observing the process of in vitro labeling of isolated islets with superparamagnetic iron oxide (SPIO) contrast agent for subsequent magnetic resonance imaging (MRI) of islets and observing SPIO particles transport after transplantation. We also studied a new dual paramagnetic contrast agent combined with fluorescein intended for identification of the MRI contrast agent in samples for histology. Further, we assessed autoimmune reaction by evaluation of cytokine response to specific stimulation with auto-antigens. We tried to affect beta-cells destruction by polyclonal anti- thymocyte antibodies in a mouse experimental model. A new method of the islet respiration measurement correlated with other methods of islet quality testing and it was suggested as a diagnostic test before clinical transplantation. Results obtained studying the intercellular...
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The role of NADPH oxidase in architecture and function of β cells and Langerhans Islets
Tučková, Štěpánka ; Plecitá, Lydie (advisor) ; Bardová, Kristina (referee)
Local production of reactive oxygen species (ROS) and changes in the redox environment influence the metabolism and function of β cells of the Langerhans islets (LO). Changing the ratio between NAD(P)H / NAD(P)+ redox partners significantly affects sensitive proteins and ROS production. ROS are able to reversibly modify some amino acid residues (eg Cys, Met) of antioxidant enzymes and their interaction partners. Such a signaling cascade allows the transmission of a signal over longer distances and can also interfere with the influence of gene expression. The unique enzyme NADPH oxidase 4 (NOX4) is present on membranes within β cells and constitutively produces H2O2 depending on the presence of NAD(P)H. After glucose stimulation, both NAD(P)H and Nox4 mRNA levels increase. As previously observed in our laboratory, C57BL/6J mice with a specific Nox4 deletion in β cells have a disrupted biphasic insulin release and exhibit insulin resistance in fat and muscle tissue. We found that the absence of NOX4 in C57BL/6J mice affects LO architecture. Wildtype (WT) mice on a normal, predominantly carbohydrate diet (ND) have the majority of small LO with an area of up to 5 000 μm2 (measured on histological sections). High-fat diet (HFD) feeding of WT for 8 weeks leads to the development of diabetic phenotype and...
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Molecular mechanism of insulin release
Tučková, Štěpánka ; Plecitá, Lydie (advisor) ; Bardová, Kristina (referee)
Insulin reduces glucose levels in blood and has impact on metabolism, cell- cycle and proliferation of target body cells. Its secretion from pancreatic β cells is induced right after food consumption, utilization in digestion system and nutrient elevation in blood circulation. Under physiological conditions molecular mechanisms include simultaneous effect of glucose, fatty acids, amino acids, ions, reactive oxygen species and neural and humoral system. The primary inducer is glucose, which is necessary also for insulin gene expression. Glucose modulates the fate of other nutrients, which enhance glucose stimulated insulin secretion. In general, glucose decreases fatty acid beta oxidation and increases the level of effector lipid intermediates in cytoplasm. The key processes within insulin release pathways are the Krebs cycle, pyruvate cycling and glycerole-3-phosphate/free fatty acids cycle. Moreover, endocrine signaling molecules, hormones and nervous system regulate insulin secretion through their receptors on β cell membranes. Disruption of regulatory mechanisms and insulin sensitivity of peripheral tissues may lead to development of diabetes mellitus.
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Diagnostic contribution to the evaluation of intervention models in the treatment of type 1 diabetes
Zacharovová, Klára ; Saudek, František (advisor) ; Plecitá, Lydie (referee) ; Mandys, Václav (referee)
During treatment of diabetes mellitus by immunointervention or transplantation, it is necessary to monitor the markers of immune destruction or rejection of surviving insulin producing cells. An aim of this thesis is to improve the possibilities of following autoimmunity and to detect the survival of transplanted pancreatic islet in vivo. Partial aims included vitality testing of isolated islets for transplantation by measurement of respiration activity, observing the process of in vitro labeling of isolated islets with superparamagnetic iron oxide (SPIO) contrast agent for subsequent magnetic resonance imaging (MRI) of islets and observing SPIO particles transport after transplantation. We also studied a new dual paramagnetic contrast agent combined with fluorescein intended for identification of the MRI contrast agent in samples for histology. Further, we assessed autoimmune reaction by evaluation of cytokine response to specific stimulation with auto-antigens. We tried to affect beta-cells destruction by polyclonal anti- thymocyte antibodies in a mouse experimental model. A new method of the islet respiration measurement correlated with other methods of islet quality testing and it was suggested as a diagnostic test before clinical transplantation. Results obtained studying the intercellular...
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