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Neuropharmacology of spatial navigation, cognitive coordination and flexibility tests in animal models
Prokopová, Iva ; Stuchlík, Aleš (advisor) ; Vyklický, Ladislav (referee) ; Blahoš, Jaroslav (referee)
Spatial navigation, cognitive coordination and behavioral flexibility belong amongst cognitive functions, which play a role in many neuropsychiatric disorders. Behavioral tasks have proved to be useful paradigms to test these functions in pharmacological or genetic animal models. First aim was to determine a potential interaction between β-adrenergic and α1-adrenergic or D2-dopaminergic systems. Spatial navigation and coordination were impaired in both studies during co-aplication of subthreshold doses of drugs. Used substances belong to group of widely prescribed drugs, thus our results could be implicated in clinical practice. Another study examined an acute effect of MK-801 (animal model of schizophrenia) on behavioral flexibility in Carousel maze and the Morris water maze (MWM). Carousel maze showed higher sensitivity with impairments from 0.08 mg.kg-1 compared to 0.10 mg.kg- 1 in MWM. The final experiment aimed at testing the effect of reduced expression of Nogo-A protein on spatial navigation and behavioral flexibility of rats. A battery of tests in the Carousel maze revealed impairment in cognitive functions, MWM showed unaffected working memory of rats. Our results support the hypothesis linking Nogo-A knock-down rats with neuropsychiatric symptoms and cognitive disorders. Key words:...
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Characterization of the effect of pregnanolone sulfate and its derivatives on NMDA receptors.
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...
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Muscarinic acetylcholine transmission and Alzheimer's disease
Janíčková, Helena ; Doležal, Vladimír (advisor) ; Blahoš, Jaroslav (referee) ; Rokyta, Richard (referee)
Impairment of the cholinergic neurotransmission system is regularly detected in animal models of Alzheimer's disease as well as in human patients suffering from this serious disease. Moreover, there is increasing amount of evidence suggesting that activation of individual mAChR subtypes specifically influences the cleavage of APP, the precursor for β-amyloid. APP can be processed in an amyloidogenic or non-amyloidogenic pathway and a relative abundance of these patways contributes to establishing the final concentration of neurotoxic β-amyloid in the brain. In this work, I have studied the acute and chronic effects of A β1-42 on binding and functional characteristics of mAChR. I have demonstrated that Aβ1-42 present in cell culture expressing the individual subtypes of mAChR negatively and specifically influences the function of the M1 mAChR subtype. I have also detected a decline in muscarinic receptor-mediated signal transduction in brain tissue of young adult APPswe/PS1dE9 mice, a commonly used animal model of Alzheimer's disease. Demonstration of the impairment of muscarinic transmissin in transgenic mice by soluble β-amyloid that occurs earlier than amyloid pathology and behavioral deficit, and its imitation by soluble Aβ1-42 in vitro lend strong support to the notion of the early involvement...
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Monitoring of bone metabolism affected by selected drugs
Gradošová, Iveta ; Živná, Helena (advisor) ; Broulík, Petr (referee) ; Blahoš, Jaroslav (referee)
Monitoring of bone metabolism affected by selected drugs Osteoporosis is one of the most common metabolic bone diseases, which belong to civilization diseases, and is a major health and socioeconomic problem, particularly in the older age groups. Cardiovascular diseases are one of the great problems of our society and a leading cause of death worldwide. The major risk factors include hypercholesterolemia and arterial hypertension, which can be effectively reduced by several groups of drugs. At the present, not much attention has been paid to whether or how these drugs affect bone metabolism. With increasing age, people are more likely to develop hypertension and hypercholesterolemia with progressive loss of bone leading to osteoporosis. Many studies have suggested that antihypertensive and hypolipidemic drugs in some way influence bone metabolism. The subject of the present thesis was to investigate the effect of selected, frequently prescribed antihypertensive drugs (amlodipine, metoprolol), and hypolipidemic drugs (ezetimibe, atorvastatin) on bone metabolism in healthy male Wistar albino rats and in rats after orchidectomy (Wistar and spontaneously hypertensive rats). During my postgradual study, three experiments in rats with above mentioned drugs were performed. In the first experiment, drugs...
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Role of metabotropic glutamate receptors and their associated proteins in physiology and pathophysiology
Kumpošt, Jiří ; Blahoš, Jaroslav (advisor) ; Dráber, Petr (referee) ; Viklický, Vladimír (referee)
of the thesis Glutamate is a main excitatory neurotransmitter in the brain of mammals, which activates both ionotropic and metabotropic glutamate receptors. Ionotropic receptors are responsible for fast synaptic transmission leading to membrane depolarization and Ca2+ influx into the cell. On the other hand mGlu receptors play an important role in regulation of the transmission via heterotrimeric G-proteins and activation of various signaling pathways. Postsynaptically localized group I mGlu receptors (mGluR1, 5) together with ionotropic NMDA and AMPA receptors share common large receptor signaling complexes, or signalosome facilitating glutamate signal transductions. Individual mGluR1 splice variants are differently associated with signalosome including scaffold proteins like PSD-95 which organize postsynaptic density (PSD). Heterodimerization of different mGluR1 splice variants is a focal point of my thesis together with investigation of recently discovered protein IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) and its role in organization of postsynaptic signalosome. Using biochemical, immunocytochemical and functional assays we showed heterodimers of mGluR1a/1b were expressed on the plasma membrane and that heterodimers are fully functional in the recombinant system. Next we showed...
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Monitoring of bone metabolism affected by selected drugs
Gradošová, Iveta ; Živná, Helena (advisor) ; Broulík, Petr (referee) ; Blahoš, Jaroslav (referee)
Monitoring of bone metabolism affected by selected drugs Osteoporosis is one of the most common metabolic bone diseases, which belong to civilization diseases, and is a major health and socioeconomic problem, particularly in the older age groups. Cardiovascular diseases are one of the great problems of our society and a leading cause of death worldwide. The major risk factors include hypercholesterolemia and arterial hypertension, which can be effectively reduced by several groups of drugs. At the present, not much attention has been paid to whether or how these drugs affect bone metabolism. With increasing age, people are more likely to develop hypertension and hypercholesterolemia with progressive loss of bone leading to osteoporosis. Many studies have suggested that antihypertensive and hypolipidemic drugs in some way influence bone metabolism. The subject of the present thesis was to investigate the effect of selected, frequently prescribed antihypertensive drugs (amlodipine, metoprolol), and hypolipidemic drugs (ezetimibe, atorvastatin) on bone metabolism in healthy male Wistar albino rats and in rats after orchidectomy (Wistar and spontaneously hypertensive rats). During my postgradual study, three experiments in rats with above mentioned drugs were performed. In the first experiment, drugs...
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Role of NMDA NR1 subunit in pathophysiology of schizophrenia
Vrajová, Monika ; Horáček, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Vyklický, Ladislav (referee)
Our work is focused on the role of NR1 subunit of N-methyl-D-aspartate receptor in pathophysiology of schizophrenia. In animal model using separately or in combination, antisense oligodeoxynucleotide (aODN) for NR1, NR2A and NR2B subunit of NMDAR, we affected expression of these proteins in rat hippocampus. We assessed prepulse inhibition of acoustic startle reaction (PPI) in rats and protein expression of NMDAR subunits and expression of PSD proteins. There were significant differences in expression of PSD-95 and NR1 between groups. Application of aODN (NR2A, NR2B) was associated with a significant decrease of PSD-95. PPI and expression of NR2A, NR2B and PSD-93 were not changed after aODN application.The next part of the work concentrates on a human post mortem study. To assess actual changes in the expression of the NR1 subunit and its isoforms, we measured absolute differences in the levels of mRNA/protein for panNR1, as well as the individual mRNA/protein isoforms in the post mortem left/right hippocampus of patients with schizophrenia in comparison with non-psychiatric subjects. There were no significant differences in the panNR1 subunit mRNA expression, but the absolute left/right differences were much more pronounced in the patients with schizophrenia. The expression of splice variants in the...
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A study of the modulation of recombinant and native NMDA receptors by physical and chemical factors
Cais, Ondřej ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee) ; Chvátal, Alexandr (referee) ; Šťastný, František (referee)
Excitatory synaptic transmission in mammalian CNS is mostly mediated by ionotropic glutamate receptors. NMDA receptors, one of three subclasses of this ligand-activated ion channels family, are involved in memory formation and learning and also play a role in the pathogenesis of various neurodegenerative diseases. Current knowledge about NMDA receptors function is predominantly based upon results of in vitro experiments conducted at room temperature, far from physiological. The aim of this PhD thesis was to describe the temperature dependence of NMDA receptors. We determined the rate constants that characterise each step in the mechanism of recombinant NR1/NR2B receptors activation in the temperature range 25-45řC. The receptor desensitization, resensitization and glutamate unbinding turned out to be the most temperature sensitive of these processes. In addition to that, we described the temperature dependence of deactivation kinetics in various experimental models of NMDA receptors (both recombinant and native). The second part of the thesis focused on the modulation of NMDA receptors function by steroid compounds derived from pregnanolone sulfate, an endogenously occuring neurosteroid. We tested 21 steroids that showed various degree of ability to inhibit (or, in one case, potentiate) the current...
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Study of Structure-Function Relationship of Temperature-Gated TRP Channels
Benedikt, Jan ; Vlachová, Viktorie (advisor) ; Kršiak, Miloslav (referee) ; Blahoš, Jaroslav (referee)
Sensory physiology research was heavily influenced by molecular identification of transient receptor potential (TRP) ion channel family. Discovery of these unique family of membrane receptors allowed detailed study of their structure-function relationship. TRP channel expression in sensory neurons, but also apparently in keratinocytes provides living organisms with the ability to fast and accurately detect noxious thermal and chemical stimuli and to transmit this noxious signaling to higher nervous system structures. Despite recent efforts to elucidate molecular mechanisms of temperature or chemical activation of these non-selective cation channels, there is still no unifying hypothesis that is able to explain complex behaviour of these receptors. This dissertation aims to investigate three aspects of the TRP channel function: 1. Molecular characterization of acute desensitization of vanilloid receptor TRPV1 and investigation of the role of phosphorylation sites for calmodulin kinase II. 2. To characterize mechanisms of etanol-induced inhibition of menthol receptor TRPM8 and to find out possible physiological consequences of this inhibition. 3. To explore the role of inner pore region in activation gating of ankyrin receptor TRPA1 and identify amino acids involved in this process. Our findings contribute to...
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