7 Abstract With the first appearance of life on Earth, organisms had to adapt to an ever-changing surrounding environment in order to survive. Since the emergence of metazoan multi- cellularity, subsets of cells could adapt to perform specific biological tasks beneficial to the whole organism, necessitating not only spatiotemporal regulation of gene expression during development, but also integration of tissue specific needs with overall organis- mal status. Within the set of evolutionary conserved regulatory systems, the family of nuclear receptor (NR) transcription factors stands out due to its high degree of evolu- tionary conservation, plasticity and uniqueness to the metazoan kingdom, regulating gene expression in response to, or in the absence of a ligand by genomic and non- genomic actions. With an increasing number of different compounds being recognized as ligands to NRs, it is now thought that ancient NRs were probably characterized by low ligand binding specificity, eventually serving as environmental sensors, integrating nutrient availability and gene expression at the base of metazoan evolution. Characteri- zation of the NR network in one of the simplest metazoan organisms, Trichoplax ad- haerens, revealed not only a functional network and sub-specialization of NR dependent gene regulation, but...

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1 ABSTRACT In this Thesis, we studied properties of μ-, δ-, and κ-opioid receptors in lymphocytes isolated from rat spleen. This splenocytes were exposed to mitogen concanavalin A or opiate morphine and cultivated for 48 hours. Under physiological conditions, level of opioid receptors in immune cells is very low. Due to various factors such as presence of opioids, mitogens, long-term exposition to stress, expression of these receptors can be amplified. In this study we demonstrated, that concanavalin A causes up-regulation of μ-, δ- and κ-opioid receptors in lymphocytes isolated from rat spleen. In control cells no significant signal of μ- or δ-receptors was observed. In contrast, κ-opioid receptors were detected already in control cells. Concanavalin A stimulation caused a 2.4 - fold increase of these receptors. In lymphocytes treated with morphine only μ-opioid receptors were up-regulated, whereas in control cells, there was no signal for these receptor type. δ-opioid receptors were not detected in control or morphine treated cells. κ-opioid receptors were determined in control and also in morphine affected lymphocytes but the amount of these receptors wasn't changed by morphine. Detection of μ-, δ- and κ-opioid receptors using Western blot technique in lymphocytes isolated from rat spleen, that were...

N-methyl-D-aspartate receptors (NMDARs) are heterotetramers containing two obligatory glycine-binding (GluN1) and two glutamate/glycine-binding (GluN2/3) subunits. These receptors mediate excitatory synaptic transmission in the central nervous system and play a key role in high order neuronal processes as a learning and formation of memory. It has been shown that dysregulation of NMDARs is involved in the pathophysiology of neurological and psychiatric disorders. Each receptor is composed of four protomers exhibiting a conserved domain organization. The most distal part to the cell membrane is the amino-terminal domain that is linked to the ligand binding domain (LBD), which is connected to the pore-forming transmembrane domain (TMD) communicating with the intracellular carboxy-terminal domain. LBD and TMD are connected via three polypeptide chains - linkers. Channel opening is the key step in the NMDAR gating that allows the flux of ions across the membrane. The energy of agonist binding-evoked conformational changes is transferred via linkers to M3 helices forming an ion channel. The rearrangement of M3 helices in activated receptor makes the central cavity of the channel accessible. The details of energy transfer are not yet fully characterized, although accurate knowledge of the receptor gating...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Draský Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Pharmacological influence of nuclear receptors in diabetes mellitus therapy Nuclear receptors belong to the superfamily of transcription factors, their main functions include regulating the expression of target genes. In my work I focused mainly on the group of orphan receptors, namely the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). A common feature of these receptors is their activation by a specific ligand. Both CAR and PXR have an essential function as biological sensors of hydrophobic xenobiotics when they induce enzymes I and II. phase of metabolism. They are also essential in the regulation of gluconeogenesis, insulin response, adipogenesis, cholesterol homeostasis, fatty acids, triglycerides and glycogen. The aim of this experimental work was to introduce a luciferase reporter assay method for two DNA constructs containing the promoter region of the PEPCK and CYP7A1 genes. We used the known agonist rifampicin and the antagonist SPA70 to activate/deactivate PXR. We used CITCO as a CAR receptor agonist. We first verified the functionality of the luciferase reporter gene assay...

The IEM 1460 is a potential age-specific anticonvulsant and an indicator of the distribution of AMPA receptor subtypes among rat brain cells. It is a derivative of adamantane, that was tested in previous studies on models of human myoclonic and generalized tonic-clonic seizures with promising results. In this thesis we evaluated its effect on the motor activity of rat offspring in the age of 12, 18 and 25 days, we used 90 animals in total . The effect was evaluated 30 minutes after intraperitoneal administration of IEM 1460 in two doses, 10 mg/kg or 20 mg/kg, and was compared to the control animals with physiological solution applied intraperitoneally in amount of 20 mg/kg. To test the animals we used Open field test, righting reflex, negative geotaxis, horizontal bar test, rope climbing test, regular and irregular horizontal ladder test. The tests were applied to animals in mentioned order. There were found significant changes influencing motor behaviour, primarily in the 12 days old animals with the dose of 20 mg/kg IEM 1460 and in the 25 days old animals with both doses of IEM 1460, 10 and 20 mg/kg. In the 18 days old animals the results were less significant. Powered by TCPDF (www.tcpdf.org)

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...

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...

Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...

NK cells, which are part of the innate immune system, are increasingly gaining attention, especially due to their cytotoxic ability to kill tumor cells of certain lines and certain viral, bacterial or parasitic infestation of the body. They lay a role in organ transplantation, the fight against HIV and other autoimmune diseases. NK cells have been studied since the 70th of the 20th century, but the structures and physiological ligands of their receptors remain only partially understood, as does the exact role of these cells in the organism. They communicate with others through their receptors, that recognize the lack of expression of MHC class I glycoproteins on the surface of target cells, thereby preventing cell recognition by cytotoxic lymphocytes. This diploma thesis deals with the research of receptors from cattle (Bos taurus), which is not a traditional laboratory animal and my task was to contribute to research of the structure of this group of proteins. I dealt with the recombinant production of some of the most important representatives of NK receptors CD69, NKRP1 and NKG2D in bacterial cells. The findings published in this thesis are a continuation of my bachelor thesis and together can be beneficial for further research into structural proteins and thus may help as in veterinary medicine...