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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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Influence of dietary components and redox enzymes on intestinal microbiota proliferation in the tick \kur{Ixodes ricinus}
KUČERA, Matěj
In this work, we have analysed the temporal dynamics of gut-dwelling bacteria and Borrelia in the gut of the deer tick Ixodes ricinus. Using quantitative PCR, we have shown that levels of the tick intestinal microflora are profoundly decreased at later stages of feeding on whole blood but not on serum. Even though we noted that host complement system manages to interfere with Borrelia viability in vitro, we did not see any effect of host complement on Borrelia acquisition in adult ticks in vivo. However, we revealed that host hemoglobin is essential for Borrelia proliferation in the tick gut. All together, these data imply that, during feeding, levels of gut-dwelling bacteria and Borrelia are determined by the host. While hemoglobin seems to be detrimental for gut-dwelling bacteria, Borrelia require it in order to proliferate. During off-host stage, we showed that levels of gut-dwelling microflora are regulated by an intestinal transmembrane enzyme Dual oxidase. In conclusion, we aimed, and mostly succeeded, to perform pilot experiments describing the biology of a complex process of regulating gut microflora in the vector Ixodes ricinus and extend it by its impact on Borrelia acquisition
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Molecular characterization of NADPH oxidase in the gut of the tick \kur{Ixodes ricinus}
KUČERA, Matěj
This thesis focuses on characterization of one member of NADPH oxidases - dual oxidase (DUOX) which has been described as the main factor of epithelial immunity in the gut of model organism Drosophila melanogaster. We have identified an orthologous gene coding for DUOX in the tick I. ricinus and described its tissue expression profile. The DUOX is mainly expressed in the gut of unfed ticks and seems to be downregulated upon artificial microbial infection. A fragment of tick DUOX was prepared as recombinant protein and used for preparation of specific antibodies to be used for further characterization of the enzyme. Our main aim is to highlight the importance of tick DUOX producing the reactive oxygen species and their role in the defence against pathogenic organisms within the tick gut.
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