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The molecular mechanisms and role of purinergic signaling in hypothalamus
Ivetic, Milorad ; Zemková, Hana (advisor) ; Mysliveček, Jaromír (referee) ; Smejkalová, Terézia (referee)
(EN): Extracellular ATP and purinergic P2 receptors (P2X and P2Y) are involved in a signaling network called "purinergic signaling" which is widely exploited in both somatic and neuronal tissues, and is also operative in endocrine system. The main focus of my thesis is on the role and expression of P2X and P2Y receptors in hypothalamic supraoptic nuclei (SON) producing hormones vasopressin and oxytocin, and the suprachiasmatic nuclei (SCN), the principal circadian pacemaker in mammals. In the first part of my thesis, we tested the hypothesis that P2X and P2Y receptors play a role in the enhanced release of hormones from magnocellular SON neurons stimulated through fasting/refeeding experimental protocol. We studied the effect of 2 h of refeeding after 48 h of fasting on hormone, P2X and P2Y mRNA expression in the SON tissue of 30-day-old rats, and the effect of ATP on electrophysiological properties of SON neurons in brain slices from control and fasted/refed rats. Quantitative real-time PCR revealed that the expression of P2X2 and arginine-vasopressin (AVP) mRNA was increased and P2Y1 mRNA expression was decreased in fasted/refed rats compared to controls, whereas P2X4, P2X7, P2Y2 and oxytocin mRNA levels were not significantly changed. Whole-cell patch clamp recordings showed that the amplitude...
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Struktura a funkce rekombinantního P2X4 receptoru
Rokič, Miloš ; Zemková, Hana (advisor) ; Vlachová, Viktorie (referee) ; Bendová, Zdeňka (referee)
4 Abstract Purinergic P2X receptors are membrane ion channels activated by extracellular ATP. There are seven isoforms of mammalian P2X receptors designated as P2X1-7, which according to their structure represent a specific family of ligand gated ionic channels, with extraordinary structural/functional properties. The P2X receptor consists of three subunits and each subunit has two transmembrane domains. Crystalographic data demonstrate that ionic channel pore is situated between the second transmembrane domains. Crystal structure of P2X4 receptor from the zebrafish (Danio rerio) is available in both open and closed state of the channel and the exact structure of ATP binding site is solved. The aim of this thesis was to study the structure-function relationships in a model of recombinant P2X4 receptor of the rat. By employing the point mutagenesis and electrophysiological recording, the functional importance of conserved cysteine residues in the ectodomain and amino acid residues which form the extracellular vestibule was investigated. All ten cysteins were substituted one by one with alanine or threonine and ATP-induced currents were measured from HEK293T cells expressing wild type (WT) and mutated P2X4 receptors. The results indicate that C116A, C126A, C149A and C165A mutations disrupt two disulfide bonds...
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