IRS-2 is the inhibitor of serine proteases from the Ixodes ricinus tick. My task in this thesis was to find out the effect of the IRS-2 on dendritic cells activated by TLR4 ligand or by Borrelia afzelii. This effect was studied on several levels. I focused on the cytokine production, the expression of costimulatory molecules and cell signaling pathways. The results show that the IRS-2 may inhibit the expression of costimulatory molecules CD-80 a CD-86 on the cell surface, but this finding needs to be confirmed again. The production of cytokines was not affected by the IRS-2. The effect of the IRS-2 on the activity of p38, Erk1/2 nor NF-?B in LPS stimulated cells vas not observed. The fosforylation of STAT 3 in cells activated by the B. afzelii was lowered by the IRS-2.

Tick saliva contains a lot of molecules with antihemostatic and immunosupressive effects.The goal of this thesis is to test the effects of tick salivary cystatins from I.ricinus and I.scapularis on TLR - induced maturation of bone-marrow derived dendritic cells and production of chosen cytokines. Over all, the supressive effect of tick cystatins was observed in relation to TLR-induced maturation of DC. In addition, cystatins enhanced production of IL-10 and attenuated induction of IL-12 cytokines.

To find out if the haematopoietic system is common feature of vertebrates, we decided to examine haematopoiesis in a sea lamprey (Petromyzon marinus). All blood cells arises from the haematopoietic stem cells in higher vertebrates. We assume that this is common for the higher vertebrates and a jawless vertebrates, but nobody was interested in the jawless haematopoiesis since 1970. Using a reverse genetic, we identify homologues of important hematopoietic of higher vertebrates in transcriptome of the sea lamprey with emphasis on important receptors or transcription factors, because they can be used as the specific markers of different blood cells and their progenitors. Then we use those sequences for cloning, expression measurements and other work. We picked up sea lamprey as model organism because its unique phylogenetic position, important foe evo-devo studies, but also because lack of elementary knowledge about sea lamprey haematopoiesis. Key words: Petromyzon marinus, haematopoiesis, HSC, evo-devo

Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...

Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.

Contrary to the classical mouse inbred strains with unnatural genetic variability, wild-derived strains offer a more suitable model for evolutionary immunology. Toll-like receptors (TLRs) belong to initial detectors of invading pathogens. Although TLRs recognise conserved structures they were shown to be polymorphic. This polymorphism is associated with various diseases. In my thesis, I describe variability of Tlr1, 2 and 6 in 24 inbred strains derived from two subspecies of house mouse (Mus m. musculus and M. m. domesticus). These Tlrs exhibit different levels in variability among the strains. In Tlr1 the polymorphic sites are spread along the whole exodomain. Tlr6 is quite conserved (a lower amount of substitutions located far from the binding region and with minor modifications in the amino acid residue properties). Tlr2, on the contrary, contains some substitutions with substantial alternations of residue properties that are located within or nearby the binding region and the subspecies differ at these sites. All alleles of M. m. domesticus and M. m. musculus, except for Tlr1 PWD, Tlr2 STAIL, are phylogenetically separated. The strains and the subspecies vary in the production of IL-1β, IL-12 a NO after stimulation by TLR1, 2 and 6 ligands. This trend is, however, presumably influenced by the effect of...

Toll-like receptors (TLRs) are important receptor family of innate immunity. They enable fast recognition of infection through so called pathogen associated molecular patterns (PAMPs). In this thesis, we studied interaction of mouse polyomavirus (MPyV) with TLRs of mouse embryonic fibroblasts (MEF cells). We observed that inhibition of TLR4 signaling abolished response of MEF cells to MPyV. This suggested that TLR4 plays a role in MEF cells recognition of MPyV. To detect response of MEF cell to MPyV, we measured IL-6 production by ELISA. Next, we investigated effect of TLR4 signalization on MPyV infection. Inhibition of TLR4 signaling with CLI-095 inhibitor did not affect number of infected cells. Presence of TLR4 antagonist, LPS-RS, led to significant decrease in quantity of infected cells 20 hours post infection. Decrease in number of infected cells was also observed in presence of LPS. Viral infection was also inhibited by TLR9 antagonist ODN 2088. We also investigated role of MAP kinases in MPyV infection. We tested, whether inhibition of selected MAP kinases would affect number of infected cells. Inhibition of kinase p38 did not affect infection. On the other hand, inhibition of MEK kinase or JNK resulted in decrease of number of cells infected by MPyV.

Glutamate is the main excitatory neurotransmitter in the mammalian brain and its transmission is responsible for higher brain functions, such as learning, memory and cognition. Glutamate action is mediated by a variety of glutamate receptors, though their properties were until now studied predominantly in neurons. Glutamate receptors are expressed also in NG2-glia, however their role under physiological conditions as well as in pathological states of the central nervous system is not fully understood. The aim of this work is to elucidate the presence, composition and function of these receptors in NG2-glia under physiological conditions and following focal cerebral ischemia. For this purpose we used transgenic mice, in which NG2-glia are labeled by a fluorescent protein for their precise identification. To analyze the expression pattern of glutamate receptors in NG2-glia we employed single-cell RT-qPCR. Furthermore, we used calcium imaging to characterize their functional properties.

The process of self-nonself discrimination by the immune system is a fundamental attribute of healthy organisms. Since T-cell receptors (TCRs) are generated by the random process of somatic recombination without regard to its targets, the newly developed T-cell clones could recognize either self or nonself antigens. The mechanisms of central tolerance robustly limit the self-reactive repertoire within the T-cell population via deletion of clones that express self-reactive TCRs or their deviation into the regulatory T-cells (Tregs). These processes occur mainly in the thymic medulla where the TCR reactivity to self-antigens is tested by various types of antigen-presenting cells, mainly medullary thymic epithelial cells (mTECs), dendritic cells (DCs), and B-cells. The cooperation between these cell-types has been shown to be essential for the establishment of thymic tolerance. A key molecule regulating the production of self-antigens is the autoimmune regulator (AIRE), which is thought to be expressed primarily by mTECs and its mutations are associated with the development of severe autoimmune disorders. In this context, the presented thesis describes the novel regulatory pathways important for the development of a functional and "harmless" repertoire of T-cells and for enforcement of tolerance....

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.