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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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Circadian changes of the cholinergic system in various areas of the central nervous system
Křížová, Monika ; Mysliveček, Jaromír (advisor) ; Jakubík, Jan (referee)
Muscarinic acetylcholine receptors type 4 are one of the five subtypes of metabotropic muscarinic receptors whose knockout led to disruption of the biorhythm represented by locomotion in female mice. In this thesis, we attempted to determine the biorhythmic activity of selected components of the cholinergic system in structures of the central nervous system which are suspected to regulate circadian locomotion. These selected structures were the striatum, motor cortex, thalamus, intergeniculate leaflet, subparaventricular zone and posterior hypothalamic area. Changes of muscarinic receptor density in wild type and M4 receptor knockout female mice were measured by using autoradiography throughout the day. In the striatum, motor cortex, and thalamus, the density of muscarinic receptors type 1 and the activity of acetyl- and butyrylcholinesterases were also measured by autoradiography and the Ellman's method. Rhythmic oscillations of muscarinic receptor density were observed in all structures selected in both types of mice. The main brain structure responsible for the differences in the biological rhythm of wild type and M4 receptor knockout mice was identified as the striatum and motor cortex region, where the percentage of M4 receptors was highest. Diurnal oscillations were also exhibited by...
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Role of M4 muscarinic receptors in the central nervous system
Křížová, Monika ; Mysliveček, Jaromír (advisor) ; Slepička, Jakub (referee)
Muscarinic receptors type 4 are one of the five subtypes of muscarinic receptors, that are members of cholinergic receptors family, together with nicotinic receptors. An acetylcholine binding on the receptor triggers the receptor activation and the signal transmission into the cell by G-proteins located on plasmatic membrane, in the case of muscarinic receptors type 4 by Gi/o proteins. The M4 receptors are, just like other subtypes on muscarinic receptors, located in central and peripheral nervous system. In the central nervous system, they are mostly located in striatum and hippocampus. The M4 receptors have a whole range of regulative functions, the most significant one is most likely the regulation of a locomotion in striatum by the dopaminergic system. The muscarinic receptors type 4 are involved in many behavioural and cognitive processes and are therefore studied as potential drug target for the treatment of mental disorders. Key words: muscarinic receptors, M4 muscarinic receptors, GPCRs, cholinergic system, central nervous system
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Adaptace centrálního nervového systému na chybění acetylcholinesterázy
Farár, Vladimír ; Mysliveček, Jaromír (advisor) ; Jakubík, Jan (referee) ; Cordero-Erausquin, Matilde (referee)
Acetylcholinesterase (AChE) effectively hydrolyzes acetylcholine (ACh). The inhibition of AChE is generally lethal and mice without AChE in all tissues (AChE KO) have severe impairments. In the brain, AChE is anchored in the plasma membrane by proline-rich membrane anchor (PRiMA), while in the muscles, AChE is anchored by collagen Q (ColQ) in the basal lamina. We report here that the PRiMA KO mice, in which AChE is essentially eliminated in the brain, show very little changes in behavior despite an excess of ACh in the brain and adaptation of ACh receptors comparable to those seen in AChE KO mice. Moreover, when AChE cannot interact with ColQ and PRiMA, the phenotype resembles that of AChE KO mice, but the biochemical changes in the brain are similar to those in PRiMA KO mice. PRiMA KO mice also differ from other AChE-deficit mice strains in their responses to AChE inhibitor. Our results suggest that AChE in the peripheral tissues is the major target of AChE inhibitors and AChE absence in the peripheral tissues is the leading cause of the phenotype of AChE KO mice.
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Modulation of central cholinergic neurotransmission
Valušková, Paulína ; Mysliveček, Jaromír (advisor) ; Cendelín, Jan (referee) ; Jakubík, Jan (referee)
Introduction: Central cholinergic system plays a key role in control of different brain functions such as learning, memory, attention, locomotion and rewards. Disrupted integrity, regulation or capacity of cholinergic signalling is closely connected with cognitive symptoms of several neurodegenerative and neuropsychiatric diseases, as Alzheimer disease, Parkinson disease, attention deficit hyperactivity disorder (ADHD), depression, schizophrenia and increased distractibility. The major neurotransmitter of cholinergic neurons is acetylcholine (ACh) and regulation of ACh levels is main pharmacotherapeutic approach to the treatment of diseases associated with central cholinergic system. The aim of the thesis was to study the changes of central cholinergic neurotransmission with respect to various aspects of modulation of ACh levels in the brain by controlling its release through M4 muscarinic receptors (MR), its hydrolysis by acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) and after hydrolysis in the synapse, regulation of the uptake of metabolite choline by high affinity choline transporter (CHT). Methods: Here we used telemetry to measure locomotor activity and body temperature in mice with selective deletion of M4 MR (M4KO) and their wild type (M4WT) controls under the basal conditions...
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The effects of stress on the periphery and in the central nervous system with respect to the role of muscarinic receptors
Vařejková, Eva ; Mysliveček, Jaromír (advisor) ; Kuncová, Jitka (referee) ; Bakoš, Ján (referee)
The aim of our work was to analyse the role of muscarinic M2 receptors (M2MRs) in the organism at rest and under stress. In the experiments, changes in animals with expressed M2MRs are compared with M2KO animals who lack these receptors. The heart rate (HR) is determined by the level of the sympathetic and parasympathetic tones, the resulting HR is being the interaction of adrenergic and cholinergic receptors, which are among the receptors attached to G proteins. Their activity is influenced by structures of the autonomic nervous system with the highest centers with integrative function in the hypothalamus. The organism responds to the absence of M2MRs by decreasing the number of adrenergic receptors; heart rate in resting conditions does not change significantly. However, in restraint stress, there are differences both during the stress response and in the post-stress period (more pronounced and prolonged tachycardia). The treatment with carbachol (MR agonist) in M2KO animals induced tachycardia, strongly suggesting the presence of cardioexcitatory MRs. Ultradian heart rate rhythm is the superstructure of the circadian rhythm controlled by the suprachiasmatic nucleus. Autonomous regulation can affect other hypothalamic functions - circadian rhythms, endocrine regulation, thermoregulation,...
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Photodynamic Therapy of Xenotransplanted Human Tumours
Sutoris, Karol ; Gürlich, Robert (advisor) ; Mysliveček, Jaromír (referee) ; Šimša, Jaromír (referee)
SUTORIS, Karol. Fotodynamická terapie xenotransplantovaných lidských tumorů. [Photodynamic therapy of xenotransplanted human tumours]. Prague, 2015. 168 pp. Doctoral Thesis. Charles University in Prague, Third Faculty of Medicine, Department of Surgery Faculty Hospital Královské Vinohrady. Lector GÜRLICH, Robert. Language Czech. In todays clinical practice oncological indications of photodynamic therapy (PDT) are limited primarily to palliative treatment and are used as an adjunct to conventional oncosurgical routines with the aim of improving the quality of life and prolonging patient survival. The efficacy of experimental PDT on xenotransplanted human tumours has been proven in our in vivo study on nu/nu mice. One particular cell line of mammary carcinoma (MDA-MB-231) and two biologically different cell lines of prostate carcinoma (LNCaP, PC- 3) were tested. The key aspect of our experiment was the application of newly developed photosensitizer - hydroxy-aluminum phthalocyanine (AlOH-Pc) in the form of liposomal gel designed for locotopical application. Therapy achieved complete remission in 90% of mice with mammary carcinoma xenografts and in 100% of those with prostate carcinoma xenografts. The new photosensitizer, unlike the older ones, has minimal drug-light interval and does not cause...
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The role of whiskers in compensation of visual deficit and the influence of a neurodegenerative disorder on cross-modal compensation in a mousse model of retinal and olivocerebellar degeneration
Voller, Jaroslav ; Vožeh, František (advisor) ; Jagla, Fedor (referee) ; Mysliveček, Jaromír (referee)
Sensory deprivation in one modality can enhance the development of the remaining modalities via mechanisms of synaptic plasticity. Mice of C3H strain suffers from RD1 retinal degeneration that leads to visual impairment at weaning age. Independently on the retinal degeneration there is also present olivocerebellar degeneration caused by Lurcher mutation. This neurodegenerative disorder causes motor deficits, increased CNS excitability as well as changes in synaptic plasticity. The aim of this study was to evaluate a role of whiskers in compensation of the visual deficit and to assess the influence of the olivocerebellar degeneration on this process. To differentiate contribution of the whiskers from other mechanisms that can take part in the compensation, we investigated the effect of both chronic and acute tactile deprivation. We focused on motor skills (rotarod, beam walking test), gait control (CatWalk system), spontaneous motor activity (open field) and the CNS excitability (audiogenic epilepsy). In the seeing mice without olivocerebellar degeneration, the removal of the whiskers had no effect. In the blind animals without olivocerebellar degeneration, chronic tactile deprivation caused changes in gait and impaired the performance in motor tests. Some other compensatory mechanisms were involved but the...
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Characterisation and regulation of muscarinic and adrenergic receptors Subtitle: The effect of stress on muscarinic and adrenergic receptors in the lung and in the heart
Nováková, Martina ; Mysliveček, Jaromír (advisor) ; Hynie, Sixtus (referee) ; Mravec, Boris (referee)
The aim of this thesis was to clarify the influence of the stress on the adrenergic and muscarinic receptors in the heart and in the lungs. Research was perform on rat hearts and lungs and on the hearts and lungs of the CRH KO mice. First, we assessed mRNA levels of all α- and β-adrenergic receptor and muscarinic receptor subtypes. Subsequently, we performed the radioligand-binding studies to determine densities of these receptors. We identified all three α1-adrenergic receptor subtypes in the rat lungs. In the lungs of WT mice, we found that the amount of α1-adrenergic and muscarinic receptors was sex-dependent. Densities of the former were higher in females and those of the latter were higher in males. There was no difference between males and females in β-adrenergic receptor density. As for CRH KO mice, the basal densities of studied receptors were lower than in CRH WT mice (except β1-adrenergic receptors in females). The main purpose of the thesis was to detect the immobilization-induced changes in the studied receptors in the kontrol (WT) and CRH KO mice. Short-term and long-term immobilization caused decrease in all α1-adrenergic receptor subtypes in females, whereas only α1A-adrenergic receptors decreased in males. The amount of β1-adrenergic receptors decreased in males and remained without...
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Regulace receptorů spřažených s G proteiny Studie muskarinových a β-adrenergních receptorů u M2KO myší
Beneš, Jan ; Mysliveček, Jaromír (advisor) ; Kuncová, Jitka (referee) ; Nováková, Marie (referee)
(in English): The aim of the work was to perform in-depth analysis of M2KO mice both at baseline and upon a challenge with a cold stress and to explore the role of opposing receptors (i.e. adrenoceptors) in adaptation to lacking M2-receptors in the heart. We have performed receptor binding studies, study of receptor gene expression, echocardiography, telemetric monitoring of heart rate, body temperature and activity, heart rate variability and biorhythm analysis, analysis of heart rate response to the application of drugs (carbachol, atropine, isoprenaline, propranolol), assessment of adenylyl cyclase and NO synthase activity, measurement of catecholamine blood concentration and gene expression of catecholamine-synthesizing enzymes. We have found that the disruption of M2-receptor gene caused a compensatory decrease of cardiostimulatory β1-adrenoceptors and β2-adrenoceptors with corresponding down-regulation of the gene expression, M3-receptors were down-regulated as well. Missing M2-receptors were functionally replaced by the main cardioinhibitory β3-adrenoceptors that were up-regulated, not by cardioinhibitory M4-receptors. β3-adrenoceptors were found to signal through adenylyl cyclase instead of NO synthase. All these changes were found in the left ventricle only, so heterologous regulation is...
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