N-methyl-D-aspartate (NMDA) receptors are glutamatergic ionotropic receptors involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They are heteromeric complexes of GluN1 combined with GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Many allosteric modulators can influence the activity of these receptors including neurosteroids. Pregnanolone sulfáte (3α5βS) is an endogenous neurosteroid that inhibits NMDA receptors in a use-dependent manner and has neuroprotective effect. Binding site for 3α5βS on the NMDA receptor molecule is still not indentified. The aim of my work was to contribute to the identification of the biding site by kinetic analysis of rate of response return from 3α5βS inhibition. Using the point mutation we also attempted to identify the amino acids residues that could be involved in the neurosteroid binding. In order to study the effect of 3α5βS on NMDA receptors the electropfysiological recordings on human embryonic kidney 293T cells expressing recombinant GluN1/GluN2B receptors was performed. We confirm that the effect of 3α5βS on GluN1/GluN2B receptors is voltage-independent. The results of my work indicate that steroids can reach the binding site on the NMDA receptors through the membrane rather than directly from the aqueous...

Toll like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to pathogens. Primarily they are responsible for induction and regulation of the innate and adaptive immune responses whereby the effector function is executed chiefly by differentiated myeloid cells. Somewhat unexpectedly, TLRs have been also shown to be involved in direct pathogen sensing by bone marrow-derived hematopoietic stem cells (HSCs) and hematopoietic progenitors when, under inflammatory conditions, the rapid generation of innate immune effector cells that effectively combat the infection is of utmost priority. While it has been recognized that the release of inflammatory cytokines from inflamed tissues along with the changes in proportions of differentiating cells in the bone marrow (BM) as well as the BM niche can nudge the differentiation of adult BM-derived cells towards myeloid cells and granulocytes, a direct role of TLRs expressed by HSCs in this process has been demonstrated only recently. However, whether a similar mechanism operates also during embryonic hematopoiesis is unknown. Here we show that TLRs and their adaptor proteins are functionally expressed during early stages of embryogenesis by short-lived maternally-transferred...

Binding of sperm to the oocyte is mediated by complementary molecules on the surface of both gametes and involves the interaction of sperm protein receptors with the zona pellucida (ZP) saccharide structures. It has been shown that many proteins receptors are involved in the sperm-ZP interaction, and potential primary sperm receptors for ZP glycoproteins have been investigated in various mammals. The majority of proteins with identified sperm-ZP binding activity belong to the plasma membrane proteins. However, the exact methods for isolation of sperm membrane proteins are still to be standardized. This study is focused on investigating how employment of various isolation protocols leads to acquisition of various protein mixtures. Further in the work, two possible approaches towards identification of potential ZP-binding partners are implemented, in order to disclose novel primary ZP-binding receptor candidates. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated by: Triton X-100, Triton X-114, acetic acid, sodium dodecyl sulphate (SDS), N-octyl-β-D- glucopyranoside (OBG), rehydration buffer (RHB), and finally by freezing-thawing extraction and they were characterized by 1-D, 2-D protein profiles, glycoprotein staining and substrate zymographic methods. The results have...

It has been reported that opioid receptor activation mimics ischemic preconditioning, which may protect the heart from the development of infarction. Toll-like receptor 4 (TLR-4) during infarction stimulates cytokine production leading to inflammation and injury of the heart tissue. Our aim was to study the effect of morphine in vitro on the viability and oxidative state of H9c2 cells (rat cardiomyoblasts) and the role of TLR-4 during oxidative stress. Our experiments showed that pretreatment with morphine before tert-butylhydroperoxide (t-BHP)-, 2,2'-bipyridyl (BP)- and lipopolysaccharide (LPS)-induced oxidative stess had protective effect on the viability of H9c2 cells and markedly reduced the production of reactive oxygen species (ROS). The protective effect of morphine was diminished after naloxone treatment, which confirms the role of opioid receptors in preconditioning. TLR-4 inhibition by TAK-242 pretreatment and silencing TLR-4 by RNA interference resulted in a partial increase in cell viability but significant attenuation of ROS production after t-BHP and BP treatment. The action of LPS was reduced in response to TLR-4 silencing. Interestingly, naloxone pretreatment and suppression of TLR-4 markedly alleviated oxidative stress and resulted in a significant improvement of cell viability. We...

(EN) Research in interactions between the nervous and immune systems is focused mainly on mammals, while in other vertebrates, including birds, it remains neglected. Two types of cannabinoid receptors interconnect the nervous and immune systems: CB1, which is in mammals involved in regulation of neural processes, and CB2, which is in mammals involved in regulation of immune processes. However, little is presently known about the roles of these receptors in nervous and especially immune processes in birds. Therefore, in this work I focus on the expression of cannabinoid receptors in cognitively advanced bird species (parrots, passerines) during induced sterile peritoneal inflammation. Unlike passerines, parrots appear to lack the gene for CB2, which may affect the inflammation regulation. I have revealed no changes in the expression of these receptors during peritoneal inflammation neither in parrots, nor in songbirds. Nevertheless, the increase in expression of the proinflammatory cytokine IL- 1β in the brain in parrots confirms the importance of neuroimmune interaction and mutual influences along the gut-brain axis. This work suggests that even in birds, the central nervous system is affected by inflammation through the gut-brain axis. The expression of cannabinoid receptors does not change much...

Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12) Abstract Natural killer (NK) cells constitute the subpopulation of large granular lymphocytes which mediate spontaneous immune response against infected, transformed or allogeneic cells and thus represent an important component of the innate immunity. NK cells express a wide repertoir of surface receptors which can be either activating or inhibitory and which mediate NK cell recognition and regulation of cytolytic activity. NKR-P1 and Ly49 receptor families belong to the most important murine NK receptors. Both NKR-P1 and Ly49 families are members of C-type lectin-like superfamily of receptors encoded by natural killer gene complex (NKC) on chromosome 6 and include both activating and inhibitory members. The aim of this diploma thesis was to elucidate the impact of Nkr-p1c gene divergence on Ly49 receptors expression and to find out whether the Ly49 and Nkr-p1 gene clusters (which are localized on opposite ends of NKC) are inherited independently or whether the NKC domain is inherited as a complex. The second research interest was to illustrate the influence of the above mentioned divergence on cytotoxic activity of NK cells and tumor growth. In this study, inbred mouse strains C57BL/6 and Balb/c...

Endogenous cardiac protection against acute ischemia/reperfusion injury can by increased by cardiac adaptation to various forms of chronic hypoxia. Chronic hypoxia induces a large variety of adaptive changes in the myocardium that could be considered as protective, but the exact mechanism of increased ischemic tolerance is unknown. Different studies suggest that catecholamine release and their effect on -adrenergic signaling after adaptation to chronic hypoxia contributes to cardioprotection. In this study we focused on characterization of -adrenergic receptors ( -ARs) in the myocardium of rats after adaptation to three different hypoxic conditions: 1. intermittent normobaric hypoxia - INH/R (23 h hypoxia, 1 h reoxygenation), 2. intermittent normobaric hypoxia - INH (8 h hypoxia, 16 h normoxia), 3. continuous normobaric hypoxia - CNH (24 h hypoxia). We compared how each hypoxic model affects the total number of -adrenergic receptors and proportion of individual subtypes ( 1-and 2-ARs) in the left and right ventricles compared control normoxic rats. The INH model had apparently no effect on -ARs in either ventricles. On the other hand, adaptation to INH/R and CNH was accompanied by a significant decrease (by about 25%) in the total number of -adrenergic receptors in the right ventricles. Our present...

(english) Natural Killers - NK cells play an important role in immune surveilance and regulation either by direct cytotoxicity towards infected, transformed or otherwise damaged cells, or by production of cytokines and chemokines. The resulting response of NK cells is given by the sum of stimulating and inhibiting signals, tranduced by a wide array of receptors. Killer Ig-like receptors KIR2DL4 and LILRB1, which recognize self HLA-G molecules in pregnancy, as well as NKR-P1 receptors, which differ in the number of isotypes, are species-dependent and reduced during phylogenesis. NKG2D, reacting to stress-inducible proteins, and adenosine receptors (AR), which supress the inflamatory reaction, remain evolutionary conserved. The aim of this work was to study the involvement of NK cells and their receptors in several immune disorders and in various species, to provide new insights into their function and posisible immune modulation. We have shown here, that the choice of species in the study of NK cell effector functions may be crucial in some cases. The reaction to glycans, using synthetic GlcNAc-terminated glycomimetics GN8P, exerted opposing effects on NK cell function in humans and C57Bl/6 mice. In humans, the glycomimetic decreased cytotoxic activity of high NKR-P1A expressing NK cells, while in...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Jan Dušek Supervisor Prof. PharmDr. Petr Pávek, Ph.D. Title of Doctoral Thesis Development of novel Constitutive androstane receptor (CAR) ligands Constitutive androstane receptor is nowadays known as the established nuclear receptor that regulates the expression of several key cytochrome P450 enzymes, predominantly CYP3A4 and CYP2B6. Recently it has been shown that CAR has also essential role in the regulation of endogenous metabolism of glucose, lipids, cholesterole or bile acids. Simultaneously, this receptor is considered to have proliferative effect on human hepatocytes and protective effects against toxic and dietary damage of liver parenchyme. Given the possible therapeutic utilization of CAR, its therapeutic options are being intensively studied. Unfortunately, currently known ligands of human or mouse CAR are either poorly selective or indirect. The aim of my doctoral thesis was to find new ligands of mouse and human CAR, that would enable more detailed study of the receptor.

Transcriptional regulation of gene expression in eukaryotes has evolved over millions of years. The regulatory pathways of nuclear receptors represent an evolutionarily ancient, but conserved mechanism with associated accessory proteins, many of them forming a functional nexus known as the Mediator complex involved in transcription. Despite the versatility of the pathway, e.g. through the adoption of new regulatory functions in phylogenetically more recent Metazoa, we hypothesise that the intrinsic potential of the NR-Mediator axis to directly translate a stimulus to a biological response is conserved across species, and additional regulation could also be achieved through secondary functions of its essential members. To support the hypothesis, we assessed the ligand-binding capability of retinoic X receptor in Trichoplax adhaerens and provided evidence to support the concept that this capability was already present at the base of metazoan evolution. With regards to the potential secondary functions, we took inspiration from previous research and identified the Mediator subunit 28 (MED28) as the only known member having documented nuclear and cytoplasmic dual roles, and thus possessing the potential to transmit signals from the cellular structural states to the nucleus. Due to the lack of...