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Interactions between gut microorganisms and brain
Fajstová, Alena ; Pácha, Jiří (advisor) ; Hock, Miroslav (referee)
Intestinal microbiota communicates with brain via various cooperating pathways including neuronal, endocrine and immune. Pathogenic and commensal bacteria produce great amounts of neurotransmitters and various other metabolites which can interact with brain. Presence of bacteria can also induce immune system response which can influence brain through cytokines and other mediators. Last but not least the communication can be mediated through nerves, especially the vagus nerve. The brain can influence the intestines through sympathic and parasympathic efferent nerves and through hormones. Gut colonization by nonpathogenic commensal bacteria is crucial for proper brain development. If this doesn't happen in certain period psychiatric disorders such as depression or autism can occur later in life. Various pathological conditions might be ameliorated or fully reversed by administration of probiotic bacteria. Aim of this thesis is to briefly review factors influencing gut microbiota, its influence on the brain development and the role of probiotics in the therapy of intestinal and psychiatric diseases. Keywords: gut microbiome, brain, neuroendocrine regulation, probiotic, neurotransmitter
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CIrcadian regulation of miRNA and clock-controlled genes in tumorigenesis
Balounová, Kateřina ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee)
The circadian clock generates circadian rhythms, which participate on regulation of a number of signalling pathways. Disruption of the circadian regulatory mechanism is linked to a development and a progression of certain types of cancer including colorectal tumorigenesis. Progression of tumorigenesis depends on the cell cycle machinery related to cell proliferation and apoptosis. MiRNAs play a role in initiation and progression of tumorigenesis because they interfere in regulatory pathways associated with tumorigenesis. The aim of the thesis was to determinate existence of circadian rhytms in clock controlled genes (Tef, Dbp), miRNAs (miR-1-3p, miR-16-5p, miR-34a-5p, miR-155-5p, miR-192-3p) and genes of the cell cycle machinery (Ccnd1, Ccne1, Ccna1, Ccnb1) and apoptosis (Casp3, Bcl2, Bad). Further, to compare detected circadian rhythms during aging and neoplastic transformation of colon by quantitative RT-PCR. We have observed circadian expression of Tef, Dbp, Ccne1, Ccna1, Ccnb1, Casp3 and Bcl2 in young mice colon, Tef, Dbp, miR-1-3p, Ccne1, Ccna1 in old mice colon and Tef and Dbp in colorectal tumors. In summary, circadian expression of clock controlled genes varied but was maintained in mice colorectal tumors. In aging we demonstrated weakening of circadian rhythms of the genes of the cell...
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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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Role of microRNA in regulations of circadian rhythms and tumorigenesis
Balounová, Kateřina ; Pácha, Jiří (advisor) ; Červená, Kateřina (referee)
MicroRNAs (miRNAs) are ~22nt long single-strand RNA found in all groups of organisms, where they affect biochemical, physiological and behavioral pathways by regulation of protein expression. Regulation of protein expression is mediated by silencing mRNA of target genes in one of two processes, translation repression or degradation of mRNA. Changed expression of miRNA can lead to aberrant regulatory pathways resulting in various pathophysiological conditions like cardiovascular diseases, cancer or neurological disorders. MiRNA can play a role in cancer both as an oncogen or as a tumor suppressor, and it is tissue and cancer-type specific. In colorectal cancer miRNAs downregulate or upregulate signaling pathways including key processes involved in cancer development, like proliferation, cell cycle, apoptosis and metastasis formation. Circadian clock in mammals synchronizes cellular and physiological processes by transcriptional-translational feedback loops. Not only miRNAs regulate the levels of key clock genes and clock controlled genes, but also a number of miRNAs exhibit circadian expression. Therefore aberrant circadian rhythms increase risk of colorectal cancer also by altered expression of miRNAs. The main aim of the thesis was to identify miRNAs, which regulate both tumorigenesis and circadian...
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Phenotypic plasticity of smooth muscle cells
Misárková, Eliška ; Zicha, Josef (advisor) ; Pácha, Jiří (referee)
Vascular smooth muscle cells display a certain level of phenotype plasticity. Under specific conditions fully differentiated cells are able to undergo dedifferentiation and to restart growth and proliferation. An organ culture method is a useful technique for the analysis of dedifferentiation of vascular smooth muscle cells, because it provides an opportunity for studying the changes in cell phenotype. The aim of this study was to investigate the basic contractile characteristics in rat femoral arteries cultured for different time periods (from one to three days). In addition, the effects of fetal bovine serum (FBS), that contains various growth factors and other biological active molecules, on contractile function were studied. We also tried to attenuate cell dedifferentiation by lowering the calcium influx, because calcium is an important second messenger participating in cell growth and proliferation. To achieve this goal we used cultivation with nifedipine, a voltage-dependent calcium channel inhibitor. The cultivation without FBS slightly decreased arterial contractility, whereas the cultivation with FBS decreased arterial contractility considerably. The major change in contractility of arteries cultivated with FBS occurred approximately within 24 hours of cultivation. The cultivation with...
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Peripheral metabolism of glucocoricoids in immune cells
Ergang, Peter ; Pácha, Jiří (advisor) ; Kalous, Martin (referee) ; Teisinger, Jan (referee)
4 Abstract Glucocorticoids are hormones that regulate a variety of homeostatic processes including metabolism, cell proliferation, differentiation and immune functions, including inflammation. Acute inflammatory response is associated with an increase in glucocorticoid levels via the stimulation of pro-inflammatory cytokines and activation of the hypothalamo- pituitary-adrenal axis. Within target cells or tissues the glucocorticoid action depends not only on the plasma level of the hormone, its receptors and receptor-effector coupling, but also on the local metabolism of glucocorticoids. Two distinct types of this enzyme have been cloned and characterized. Type 1 (11HSD1) is a NADP+ (H)-dependent enzyme whose reductase activity predominates in intact cells. This enzyme activates cortisol and corticosterone from their 11-keto derivatives and thus increases the local concentration of active glucocorticoid. In contrast, type 2 (11HSD2) requires NAD+ as a co-substrate and possesses only dehydrogenase activity, thereby inactivating endogenous glucocorticoid hormones. We have demonstrated that inflammation (arthritis or experimental colitis) is accompanied by elevated 11-reductase activity and the expression of 11HSD1 mRNA, moreover in the case of colitis also with a decrease in the expression of 11HSD2....
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Inhibition of 11β-hydroxysteroid dehydrogenase 1 in hippocampus
Kvapilová, Pavlína ; Pácha, Jiří (advisor) ; Horníková, Daniela (referee)
This diploma thesis deals with the inhibition of 11-hydroxysteroid dehydrogenase 1 in the hippocampus. The aim was to determine whether the selected inhibitors with intraperitoneal administration pass through the blood-brain barrier, to determine the time course of inhibition of hippocampal 11-HSD1, to compare the efficacy of different inhibitors and to find out plasma concentrations of the most effective inhibitors and compare them with plasma concentrations of glucocorticoids. Selected inhibitors were emodin, quercetin, glycyrrhetinic acid and substituted adamantyl C544. The most significant effect on the inhibition of 11-HSD1 demonstrated nonselective inhibitor glycyrrhetinic acid and selective inhibitor substituted adamantyl C544. By comparing the plasma levels of these inhibitors, and the plasma levels of glucocorticoids was found that the selective inhibition by the C544 does not affect the overall level of corticosterone, and thus the activity of the HPA axis, which makes it important potent inhibitor of 11-HSD1 suitable for further studies. Key words 11-hydroxysteroiddehydrogenase 1, HPA axis, glucocorticoids, hippocampus, inhibition, emodin, quercetin, glycyrrhetinic acid, substituted adamantyl C544
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Relationship between corticosteroid metabolism, ontogenesis and stress response
Makal, Jakub ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee)
Stress is a widespread phenomenon in the western society of these days. It is a risky factor for health and well-being of the majority of people. Based on these facts, it is the main subject for the field of "stress physiology" research, which aims to study processes occurring during stress response and tries to elucidate mechanisms leading to stress-induced health impairment. The first aim of this thesis was to describe effects of psycho-social stress on organism. The second aim was to find out if can stress applied in juvenile age affect the stress response in adulthood. If so, how is the role of glucocorticoid-metabolism enzyme 11β-HSD1 in this influence? To answer these questions, two different animal models inducing stress response in the laboratory rat were used. The first one is the model of mild social stress based on the resident-intruder paradigm. Our results show efficancy of this model. Fisher 344 male rats treated under this model for seven consecutive days show highly elevated plasma corticosterone concentrations and elevated expression of the glucocorticoid receptor gene in the pituitary. Behavioral analysis demonstrates a decreased social behavioral profile of the intruders, suggesting submisive social position of these animals in the resident-intruder paradigm. The second model used is...
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Effect of stress on expression of 11β-hydroxysteroid dehydrogenase in rat brain
Kuželová, Andrea ; Pácha, Jiří (advisor) ; Vybíral, Stanislav (referee)
This thesis examines the influence of stress on the activity of hippocampal CA1 area. The main task was to determine whether the stress load affects the changes of the local metabolism of glucocorticoids, and whether the levels of corticosteroid receptors in the CA1 hippocampus are modulated in response to stress. In order to answer these questions, the experiments were carried out using three different rat strains - Fisher, Lewis and Wistar which differ in their activities of hypothalamic-pituitary-adrenal axis. Our results demonstrate that stress has no effect on expression of MR mRNA. Conversely, stress reduces the levels of GR mRNA in CA1 area of the dorsal hippocampus. Moreover, we confirmed that the Lewis and Wistar rats didn't change metabolism of glucocorticoids after stress response. By the Fisher rats increased levels of 11β-HSD1 mRNA expression and therefore increased the metabolism of corticosterone.
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