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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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Characterization of molecular components in cannabinoid signaling pathways.
Hájková, Alena ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Maletínská, Lenka (referee)
The cannabinoid receptor 1 (CB1R), a member of the G-protein coupled receptors superfamily, is a key player in endocannabinoid signalling. The CB1R is found presynaptically in neurones where it modulates synaptic plasticity. Precise description of the molecular mechanisms of synaptic neurotransmission is crucial for understanding of brain diseases and development of new therapeutic aproaches. Possible pharmacological targets of CB1R signalling include the treatment of various ailments such as energy imbalance disorders (anorexia, obesity), drug addiction, pain, insomnia, and some psychiatric conditions. This study reveals the "Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1" (SGIP1) as a novel interacting partner of the CB1R. The SGIP1 is an intracellular neuronal protein localized predominantly in axon terminals and is involved in clathrin mediated endocytosis. The overexpression of SGIP1 imbalance energy homeostasis and leads to obesity. We show that SGIP1 affects CB1R signalling via ERK1/2 whereas G-protein signallization remains unaltered. The SGIP1 also hinders CB1R internalization from the cell surface and supports its interaction with β-arrestin2. Also, we demonstrated heterodimerization of the main splice variants of metabotropic glutamate...
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Glutamatergic synaptic transmission, targeting and distribution of metabotropic glutamate receptor in neuronal cells
Techlovská, Šárka ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Fusek, Martin (referee)
Stránka 7│146 ABSTRACT An evolutionary developed eukaryotic proteins for transformation of extracellular signal into intracellular targets are receptors. There are known receptors integrated to intracellular organelle membranes e.g. inositol-3-phosphate receptor (Inositol-3-phoshpate receptor; IP3R) specific for endoplasmic reticulum or nuclear receptors e.g. receptors for gonad hormones transforming the intracellular signals, however the vast majority of receptors are present on plasmatic membrane and recognize an extracellular signals. High percentage of these receptors are included in family of proteins called G-protein coupled receptors, (G-protein coupled receptors, GPCRs) due to their signal mediators heterotrimeric G-proteins. Each receptor of this family has the preference to specific type of G-protein influencing the intracellular concentrations of Ca2+ , cyclic adenosinmonophosphate or inositol-3- phosphate. Some of them are able to signalise by dual manner or through G-protein independent pathways. Independently on cellular target membrane, GPCRs integration into membranes is enables by the content of GPRCs-specific 7-transmembrane domain (7-transmembrane domain, 7TM) which is consist of hydrophobic amino acids. The relation between the structure and functionality is very tightly related. Many...
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Function of Zinc finger protein 644 (Zfp644) in mouse organism.
Szczerkowska, Katarzyna Izabela ; Sedláček, Radislav (advisor) ; Komrsková, Kateřina (referee) ; Blahoš, Jaroslav (referee)
ZNF644 (Zinc Finger Protein 644) is a C2H2 zinc finger gene encoding a putative transcription regulator, of which a point mutation (S672G) is associated with inherited high myopia in humans. It is also described to be a partner of the G9a/GLP (G9a- euchromatic histone- lysine N-methyltransferase 2, EHMT2; GLP - euchromatic histone-lysine N-methyltransferase 1, EHMT1) complex, known for its essential role in histone methylation, specifically H3K9me1and H3K9me2. It was reported that another transcription factor, WIZ (Widely-Interspaced Zinc Finger-Containing Protein), can bind to this complex and cooperate in gene silencing simultaneously. In order to study Zfp644 impact on myopia, we generated a mouse model, Zfp644S673G that mimics human mutation. In addition, a mouse with a persuasive truncated form of the protein, Zfp644Δ8 was created. Both mouse models went through an examination of retinal function and morphology. Moreover, with use of ultrasonography, different ocular parameters were examined. We conclude, that Zfp644 gene is causative for myopia in mice. Further examinations of Zfp644Δ8 animals show severe symptoms in metabolism and female fertility. To describe the impact of Zfp644 in mouse fertility we performed various experiments including analysis of expression of Zfp644 in reproductive...
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Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1
Dvořáková, Michaela ; Blahoš, Jaroslav (advisor) ; Konvalinka, Jan (referee) ; Stuchlík, Aleš (referee)
Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1 Abstract Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1) has been identified as an interacting partner of cannabinoid receptor 1 (CB1R). Their protein-protein interaction was confirmed by co-immunoprecipitation. SGIP1 hinders the internalization of activated CB1R and modulates its signaling in HEK293 cells. Employing whole-cell patch-clamp electrophysiology, we have shown that SGIP1 affects CB1R signaling in autaptic hippocampal neurons. Using a battery of behavioral tests in SGIP1 constitutive knock-out (SGIP1-/- ) and WT mice, we investigated the consequences of SGIP1 deletion on behavior regulated by the endocannabinoid system. In SGIP1-/- mice, exploratory levels, working memory and sensorimotor gating were unaltered. SGIP1-/- mice showed decreased anxiety-like and depressive-like behaviors. Fear extinction to tone was enhanced in SGIP1-/- females. Several cannabinoid tetrad behaviors were altered in the absence of SGIP1. SGIP1-/- males exhibited abnormal THC withdrawal behaviors. SGIP1 deletion also reduced acute nociception, and SGIP1-/- mice were more sensitive to antinociceptive effects of CB1R agonists and morphine. CB1R-SGIP1 interaction results in profound modification of CB1R...
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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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Bone remodeling in rheumatic diseases: Bone loss in juvenile idiopathic arthritis
Brábníková Marešová, Kristýna ; Štěpán, Jan (advisor) ; Blahoš, Jaroslav (referee) ; Hrnčíř, Zbyněk (referee)
Introduction: The inflammation plays the essential role in the bone loss in juvenile idiopathic arthritis (JIA). Proinflammatory cytokines and also glucocorticoids (GCs) may activate bone resorption by osteoclasts. Simultaneously, bone formation can be attenuated, especially by inhibitors of proteins, which control the osteoblast differentiation. The aim was to verify the hypothesis that in patients with highly active JIA, reduction of bone formation via Wingless (Wnt) proteins inhibitors - Dickkopf 1 (Dkk-1) and sclerostin could be found. Except the densitometry measurements of bone and lean mass, we assessed markers of disease activity, bone metabolism and remodeling in young adult patients with JIA before and during 2 years of anti TNFα (tumour necrosis factor α) treatment, which decreases disease activity. Results: In patients with JIA before antiTNFα treatment, bone mineral density (BMD, g/cmš) was significantly reduced compared to controls. Values of BMD and body composition in JIA significantly depended on disease duration and GCs treatment. Serum concentration of sclerostin was significantly elevated in JIA compared to values in healthy controls. Values of the other monitored markers did not differ between JIA and controls. In patients with JIA, Dkk-1 correlated positively with C-reactive...
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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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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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