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The metabolic role of pulmonary arterial fibroblasts in the activation of immune system during development of pulmonary arterial hypertension
Křivonosková, Monika ; Plecitá, Lydie (advisor) ; Zadražil, Zdeněk (referee)
The development of inflammation in the small distal pulmonary vessels plays an important role in the development of pulmonary arterial hypertension (PAH). One of the cell types found in the pulmonary vessels are fibroblasts, which, according to the "outside-in" theory, may be the first to respond to stimuli in the vessel, modulating remodeling of the pulmonary vessels toward the inner layers of the vessel and attracting other immune cells to the site. In addition to inflammation, the so- called Warburg effect also occurs in vessels affected by PAH, in which metabolism shifts toward glycolysis and lactate production. Among other changes, a pro-oxidative state is induced within the cell by mitochondrial metabolism and NADPH oxidase, leading to an imbalance in reactive oxygen species production. We therefore wanted to test whether calf lung fibroblasts with hypoxia-induced PAH have an active inflammasome, with which they would produce mature interleukin 1β (IL-1β) and to clarify the effect of a pro-oxidant environment on this expression. In our in vitro model we have confirmed IL-1β mRNA expression, but we were unable to detect its expression at the protein level. This was consistent with the inability to detect inflammasome activity. We believe that the inability to detect the protein form of IL-1β...
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The effect of endothelial cell proliferation on susceptibility to mitochondrially controlled apoptosis and oxidative stress.
Blecha, Jan ; Novák, Petr (advisor) ; Plecitá, Lydie (referee)
Mitochondria are multifunctional organelles playing a key role in energy metabolism and cell death induction. Mitochondria, and specifically their respiratory chain, are also the main producers of reactive oxygen species (ROS) in cells. Metabolism can be affected by the state of cellular proliferation and certain ROS-inducing agents have an antiangiogenic effect based on the preferential elimination of proliferating endothelial cells (EC). Therefore, in this work we investigated, whether mitochondria could be responsible for different sensitivity of proliferation and confluent EC to cell death. We mainly focused on systems that regulate ROS level and apoptosis: respiratory chain (ROS production), antioxidant defense (ROS detoxification) and Bcl-2 family of proteins (apoptosis regulation). First, we treated EC with functional and nonfunctional respiratory chain with various oxidative stress- and apoptosis-inducing agents and determined ROS production and susceptibility to apoptosis in proliferating and confluent cells. Our results show that functional respiratory chain greatly increases the susceptibility of proliferating cells to ROS induction and apoptosis, whereas in qiescent cells it protects against cell death. Given these findings, we assessed the activity of respiratory chain in proliferating...
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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 metabolism and immune system in pulmonary hypertension
Křivonosková, Monika ; Plecitá, Lydie (advisor) ; Vašek, Daniel (referee)
Pulmonary arterial hypertension is a low-incidence, fatal, disease that affects the pulmonary vessels, which remodel, thicken and gradually reduce lumen. During this signaling molecules are excreted from endothelial cells, which act vasoconstrictively and proliferatively on smooth muscle cells. There is also a reduction in the production of vaodilators, which again has a vasoconstrictive effect. Accumulation of immune cells in adventitia promotes smooth muscle cell proliferation, migration and differentiation of fibroblasts into myofibroblasts, which causes changes in the composition of the extracellular matrix. Changes in metabolism within all artery layers related to the transition to glycolysis and increased glucose uptake promote cell proliferation and modulate signaling leading to infiltration and activation of immune cells resulting in artery remodeling.
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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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The effect of endothelial cell proliferation on susceptibility to mitochondrially controlled apoptosis and oxidative stress.
Blecha, Jan ; Novák, Petr (advisor) ; Plecitá, Lydie (referee)
Mitochondria are multifunctional organelles playing a key role in energy metabolism and cell death induction. Mitochondria, and specifically their respiratory chain, are also the main producers of reactive oxygen species (ROS) in cells. Metabolism can be affected by the state of cellular proliferation and certain ROS-inducing agents have an antiangiogenic effect based on the preferential elimination of proliferating endothelial cells (EC). Therefore, in this work we investigated, whether mitochondria could be responsible for different sensitivity of proliferation and confluent EC to cell death. We mainly focused on systems that regulate ROS level and apoptosis: respiratory chain (ROS production), antioxidant defense (ROS detoxification) and Bcl-2 family of proteins (apoptosis regulation). First, we treated EC with functional and nonfunctional respiratory chain with various oxidative stress- and apoptosis-inducing agents and determined ROS production and susceptibility to apoptosis in proliferating and confluent cells. Our results show that functional respiratory chain greatly increases the susceptibility of proliferating cells to ROS induction and apoptosis, whereas in qiescent cells it protects against cell death. Given these findings, we assessed the activity of respiratory chain in proliferating...
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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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