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Influence of cytokines on peripheral metabolism of glucocorticoids
Vitáčková, Kateřina ; Pácha, Jiří (advisor) ; Liberda, Jiří (referee)
11β-hydroxysteroid dehydrogenase (11βHSD) participates in prereceptor metabolism of glucocorticoids by conversion of their active forms (cortisol or corticosterone) to inactive forms (cortisone or 11-dehydrocorticosterone), thereby regulating concentration and activity of glucocorticoids in organism. 11βHSD type 1 (11βHSD1) activates glucocorticoids from their biologically inactive keto-forms, whereas the role of 11βHSD type 2 (11βHSD2) is their biological reinactivation. Previous in vivo studies showed that mRNA expression and enzyme activity of 11βHSD1 or 2 can be modified by influence of these cytokines, for example IL-1β and TNF-α. The aim of this study was to determine whether these cytokines play a role in regulation of 11βHSD expression in distal colon organ cultures and whether the changes of 11βHSD2 expression are induced by a direct effect of cytokines on epithelial cells. Total RNA was isolated from all samples and RNA was transformed to cDNA by reverse transcription. Subsequently mRNA of 11βHSD1 or 2 and the given housekeeping gene was measured by Q-PCR. Incubation of distal colon organ cultures with TNF-α (10 ng/ml; 48 hours) caused significant increase of 11βHSD1 expression, but no influence on 11βHSD2 expression was observed. In contrast, the expression of 11βHSD2 but not 11βHSD1 was...
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The role of transport systems for potassium cations in the physiology of Saccharomyces cerevisiae yeast
Marešová, Lydie ; Sychrová, Hana (advisor) ; Pácha, Jiří (referee) ; Janderová, Blanka (referee)
[n this work, we present the characteristics of kha 1 6,. strains in the background of various multiple mutations in genes encoding alkali-metal-cation transporters. Two main phenotype manifestations of the khal deletion were growth defect on high externa\ pH and hygromycin sensitivity. The correlation between these phenotypes and the kha 1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of GFP-tagged Kha 1 p showed that this antiporter is localized preferentially intracellu\arly (in contrast to the plasma-membrane Na+/H+ antiporter Nha 1 p). Based on these findings, Kha 1 p is probably not localized in plasma membrane and do es not mediate efflux of alkali metal cations from cells (as published before in RammÍrez el aJ., 1998), but is important for the regulation of intracelular cation homeostasis and optima\ pH contro l, similarly as the Nhx l p. The khal deletion phenotypes were complemented by heterologous expression of a plant antiporter AtChx 17, showing that the proteins AfChx 17 and SeKhal could have similar ťunction and that S cerevisiae khal deletion mutants could serve for heterologous expression and characterization of some plant transporters in yeast, especially those localized intracellularly. We also showed that the presence of the Tokl channel strongly infl uences...
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Local metabolism of glucocorticoids in female Prague hereditary hypertriglyceridemic rats
Klusoňová, Petra ; Pácha, Jiří (advisor) ; Kopecký, Jan (referee) ; Haluzík, Martin (referee)
11-hydroxysteroid dehydrogenase (11HSD1) is an oxidoreductase which catalyzes conversion of inactive 11-oxo steroid derivatives into active 11-hydroxy forms. 11HSD1 elevates intracellular level of active glucocorticoid (GC) hormones: cortisol in human tissues and corticosterone in rodents, therefore local level of active GCs can be set independently from systemic secretion driven by hypothalamo-pituitary-adrenal axis (HPA axis). Chronic systemic excess of GCs results in development of Cushing's syndrome which is characterised by central obesity and other metabolic disturbances. Despite normal serum levels of GCs, the patients with idiopathic obesity also develop metabolic syndrome. It was suggested that GCs could be elevated locally in target tissues due to enhanced 11HSD1 activity. This hypothesis was confirmed in transgenic rodent models. Prague hereditary hypertriglyceridemic (HHTg) rats represent a non-obese model of metabolic syndrome without genetic manipulations or specific mutations. The strain was bred by cross-mating of Wistar rat individuals with elevated serum levels of triglycerides (TGs). The strain exhibit hypertriglyceridemia and hypertension. When kept on high carbohydrate diet HHTg rats exhibit alterations in glucose homeostasis. Since there are no data that would describe...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee) ; Herichová, Iveta (referee)
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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The effect of stress on regulation and regeneration of glucocorticoids in animal models differing in response of hypothalamo-pituitary-adrenal axis
Vodička, Martin ; Pácha, Jiří (advisor) ; Svoboda, Jan (referee) ; Bendová, Zdeňka (referee)
Stress reaction is usually activated by the brain, when homeostasis is or perceived to be threatened. The stress signals are transmitted from the brain by two main branches; the sympathoadrenomedullary and the hypothalamo-pituitary-adrenal (HPA) axes and employ neural, humoral and immune pathways to cope with the stressor. Because of its potency, the stress reaction has to be precisely regulated. The HPA axis is regulated by feedback loops where its end product, corticosterone in laboratory rat and mouse, inhibits its activity. The effect of corticosterone does not depend only on the concentration of corticosterone but also on local metabolism of glucocorticoids via oxo-reduction catalyzed by the enzyme 11β -hydroxysteroid dehydrogenase 1 (encoded by the Hsd11b1 gene), which intracellularly regenerates active corticosterone from inactive 11-dehydrocorticosterone, or by extra-adrenal de novo steroidogenesis of glucocorticoids. We focused on analysis of stress response in experimental animals differing in HPA axis responsivity (Fischer 344 rats (F344) vs. Lewis rats (LEW) and germ-free (GF) vs. specific pathogen free mice (SPF)) with special emphasis on regulation of stress response, glucocorticoid regeneration and influence of gut microbiota. We found that stress modulated local regeneration of...
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Sulfation and desulfation of neuroactive steroids in normal and neoplastic primate brain tissues
Kříž, Lubomír ; Hampl, Richard (advisor) ; Lapčík, Oldřich (referee) ; Pácha, Jiří (referee)
A novel methodology for the determination of steroid sulfatase (STS) and sulfotransferase (SULT) activities in primate brain tissue have been developed and evaluated. STS activity determination was based on enzyme assay followed by liquid-liquid extraction ofthe reaction product, its subsequent derivatization and gas chromatography-mass spectrometry analysis. Indirect determination including product hydrolysis and determination by radioimmunoassay was used for assessment of SULT activity. For both, STS and SULT assays, optimal reaction conditions were found while strong structure-activity relationships for both enzyme activities were observed. Additionally, so far unidentified high-substrate inhibition for STS was revealed wíth a hypothesis suggesting its physiotogícal relevance' Determinations of STS and SULT activities in monkey brain regions revealed differences between genders and brain regions. Due to low number of samples, significance was not described. For STS kinetic characteristics were determined. In human brain tumors, STS and SULT activities showed highly significant differences according to diagnoses. Furthermore, tendency to cluster formation with respect to both enzyme activities emerged. It indicated specific metabolic characteristics especially for glioblastomas considered as the...
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Effect of microbiota on the activity of HPA axis
Fajstová, Alena ; Pácha, Jiří (advisor) ; Telenský, Petr (referee)
Recent research shows, that gut microbiome can influence various functions of the organism and is able to communicate with the brain. The data also show that changes in the composition of gut microbiome can influence behavior and stress reactions and vice versa, psychological state of the organism can cause changes in gut microbiome. The aim of this master's thesis was to examine changes of HPA activation and local metabolism of glucocorticoids caused by stress in the presence or absence of gut microbiome. We therefore used germ-free mice and studied the effect of stress in pituitary, adrenal gland, colon and spleen. We found that, stress has different impact on gene expression in adrenal gland, colon and spleen in the presence or absence of gut microbiome. In contrast, there wasn't any significant effect of stress on pituitary in germ free mice and their conventionaly colonized counterparts.
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