(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...

Schizophrenia is a neurological disorder with a complex psychopathology, which is far from fully elucidated. In the patients with this disorder, changes on anatomical, cellular, and neurotransmitter level have been found. The aim of this work is to elucidate the function of specific ionotropic glutamate receptors in NG2 glia in the hippocampus of a mouse model of schizophrenia. For this purpose, a mouse model of schizophrenia was generated and validated using immunohistochemistry and behavioural testing. Mice with NG2 glia labelled by a fluorescent protein with a calcium indicator also in NG2 glia were used to observe the activity of glutamate channels and the properties of the extracellular space in these mice. Changes were found in the schizophrenic animals when compared to control animals in the numbers of hippocampal oligodendrocyte lineage cells, in prepulse inhibition and in both volume fraction and tortuosity of the extracellular space in hippocampus. Moreover, the percentage of cells responding to glutamate receptor agonists in NG2 glia in hippocampus also differed significantly between the schizophrenic and the control animals. In conclusion, it can be said that we were able to observe significant changes in the mouse model of schizophrenia that we generated in comparison to control...

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.

Charles university in Prague, Pharmaceutical faculty in Hradci Králové, Department of biological and medical sciences Rheinische Friedrich-Wilhelms-University Bonn, Institute of Pharmacology and Toxicology Candidate: Michaela Čajková Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: Dr. Linda Sarah Hoffmann Title of diploma thesis: Role of Gq-coupled receptors in brown adipocytes In my diploma thesis, we focused on four Gq-coupled receptors (F2R, LPHN1, α1DAR, TSHR) in brown adipocytes (BAs), which were identified in the screen as the highest expressed in immature and mature BAs. Our goal was to validate suggestion, that Thyroid stimulating hormone receptor (TSHR) plays a key role in differentiation of BAs and that F2R, LPHN1, α1D-AR might be important for BAs. In our study, we investigated gene expression of these four receptors in BAs, using analytical methodsquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Results from analysis revealed, that expression of TSHR was increased in mature BAs, it means, that TSHR induce differentiation of BAs. The BAs transduced with short hairpin RNA (sh-RNA) against TSHR were less differentiated, this we proved also with Oil Red-O staining. Expression of adipocyte Protein 2 (aP2), peroxisome proliferator-activated...

In this work, the detailed analysis of GABAB-R/G protein coupling in the course of pre- and postnatal development of rat brain cortex indicated the significant intrinsic efficacy of GABAB-receptors already shortly after the birth: at postnatal day 1 and 2. Subsequently, both baclofen and SKF97541-stimulated G protein activity, measured as the high-affinity [35 S]GTPγS binding, was increased. The highest level of agonist-stimulated [35 S]GTPγS binding was detected at postnatal days 14 and 15. In older rats, the efficacy, i.e. the maximum response of baclofen- and SKF97541-stimulated [35 S]GTPγS binding was continuously decreased so, that the level in adult, 90-days old rats was not different from that in newborn animals. The potency of G protein response to baclofen stimulation, characterized by EC50 values, was also high at birth but unchanged by further development. The individual variance among the agonists was observed in this respect, as the potency of SKF97541 response was decreased when compared in 2-days old and adult rats. The highest plasma membrane density of GABAB-R, determined by saturation binding assay with specific antagonist [3 H]CGP54626AA, was observed in 1-day old animals. The further development was reflected in decrease of receptor number. The adult level was ≈3- fold lower than...

Reproduction is an essential feature of all animals and a fundamental step to produce new generations. Study of sperm proteins is crucial for understanding of the sperm-egg recognition. We searched out sperm surface proteins involving in the zona pellucida (ZP) binding and studied whether these proteins are preserved throughout mammalian species. Indirect immunofluorescent technique was used to test a panel of monoclonal antibodies prepared against boar sperm surface proteins on spermatozoa of bull and mice. We found a cross-reactivity of some antibodies against boar sperm with bull ejaculated and mouse epididymal spermatozoa. Further, we isolated sperm proteins from different mammalian species, such as pig, bull, dog, cat, mouse and human. Proteins were separated by SDS- electrophoresis and protein/glycoprotein profiles from epididymal, ejaculated and in vitro capacitated sperm were compared. The interaction of sperm with ZP was studied on electrophoretically-separated sperm surface proteins from pig and bull with biotin-labeled ZP glycoproteins. Antibodies, which reacted with boar sperm surface proteins with ZP- binding activity, therefore could be potential egg-binding receptors, were used for monitoring of the sperm protein origin in reproductive fluids and tissues. (In Czech) Keywords: sperm...

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...

To preserve homeostasis and proper function in every living organism, it is important for cells to communicate with each other and their environment. Cells are constantly processing a huge amount of extracellular stimuli through proteins called receptors. Receptors can transduce the signal from extracellular to intracellular compartments. G- protein coupled receptors are the biggest group, in which also belongs Cannabinoid receptor type 1 (CB1R). Endocannabinoid system regulates many biological processes such as learning, food intake, and movement. Obesity is a serious issue nowadays and in cases of claryfing its molecular-genetic basis, there was found Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 has a role in the regulation of energetic balance and its overexpression is leading to a development of obesity. SGIP1 was detected as an interaction partner of CB1R and it had been found that it is involved in internalization via clathrin-mediated endocytosis (CME). Key proteins for initiation and early phase of CME are FCHO1/2, with which SGIP1 shares high sequential homology. However, effect of SGIP1 on internalization of activated CB1R is inhibitory unlike FCHO1/2,wheras detailed mechanism of its function remains unclear. The aim of this...

To preserve homeostasis and proper function in every living organism, it is important for cells to communicate with each other and their environment. Cells are constantly processing a huge amount of extracellular stimuli through proteins called receptors. Receptors can transduce the signal from extracellular to intracellular compartments. G-protein coupled receptors are the biggest group, in which also belongs Cannabinoid receptor type 1 (CB1R). Endocannabinoid system regulates many biological processes such as learning, food intake, and movement. Obesity is a serious issue nowadays and in cases of searching for candidate molecules, there was found Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 has a role in the regulation of energetic balance and its overexpression is leading to a development of obesity. SGIP1 was detected as an interaction partner of CB1R and it had been found that it is involved in internalization via clathrin-mediated endocytosis. SGIP1 is very homological with FCHO1/2 - important proteins which participate on early stages of endocytosis. Mechanism of inhibitory effect of SGIP1 on internalization remains unclear. The aim of this study is to clarify the role of distinct domains of SGIP1 in context of endocytosis. Key...

IN ENGLISH LANGUAGE Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmacology and Toxicology Candidate: Mgr. Alžbeta Štefela Supervisor: Prof. PharmDr. Petr Pávek, PhD. Title of the doctoral thesis: Novel bile acid derivatives as promising therapeutic approach Bile acids (BAs) are amphipathic steroidal molecules that are traditionally known to facilitate intestinal digestion and absorption of lipids and fat-soluble substances. On top, the recent findings have revealed that they represent important signaling agents involved in the orchestration of lipid, glucose and energy metabolism and immune response. BAs exhibit these roles by activating intracellular nuclear receptors such as farnesoid X (FXR), pregnane X (PXR) vitamin D receptors. Furthermore, BAs act as endocrine signaling molecules and activate numerous biological cascades via a membrane G-protein-coupled receptor, termed TGR5. Therefore, the extensive modulation of BA scaffold underwent to identify compounds with specific targeting of above-mentioned receptors as a promising therapeutic approach for the treatment of various liver and metabolic disorders including cholestasis, biliary cirrhosis, nonalcoholic steatohepatitis or diabetes. The principal aim of this doctoral thesis was to investigate the structure...