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Study of structure and interaction of human lymphocyte receptors
Bláha, Jan ; Vaněk, Ondřej (advisor) ; Šulc, Miroslav (referee) ; Obšil, Tomáš (referee)
Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...
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Effect of pH on initiation of viral infection
Vomáčka, Petr ; Španielová, Hana (advisor) ; Šmahelová, Jana (referee)
In organisms, acidic pH occurs either in the intracellular or extracellular environment. Intracellularly an acidic pH is found in particular cellular compartments, such as early and late endosomes or lysosomes. In the extracellular space, the acidic pH is found in hypoxic tissues, or in tissues where inflammation or tumour growth occurs. The value of the pH is usually in the range of approximately from 5.5 up to 6.5. This work focuses on the description of the conformation changes of viral proteins, which occur due to a decreased pH and lead to escape of viral components from membrane compartments. Changes in pH in organelles of endocytic pathways as well as changes in extracellular pH affect significantly the infection of many viruses. Research on the role of pH during the viral can bring important information for the development of antiviral and anticancer treatment. Key words: virus, conformation, pH, receptor, endocytosis
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Preparation of strategy for studying interaction of human constitutive androstane receptor with steroid receptor coactivator-1
Šachová, Ivana ; Martin, Jan (advisor) ; Mladěnka, Přemysl (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Ivana Šachová Supervisors: PharmDr. Jan Martin, PhD., Dipl. Ing. Mgr. Ferdinand Molnár, PhD. Title of diploma thesis: Preparation of strategy for studying interaction of human constitutive androstane receptor with steroid receptor coactivator-1 The aim of this study was to express wild type and mutated synthetic human SRC1 central domains in E. coli BL21 DE3 cells. Expressed recombinant proteins were purified afterwards and used for investigation of SRC1-CAR protein interaction using pull down experiments. The constitutive androstane receptor (CAR), a nuclear receptor (NR), is a transcriptional regulator, which influences the expression of various proteins, such as enzymes of biotransformation and transporters important for metabolism of both endogenous and exogenous compounds. CAR can be fully active only in presence of coactivators, such as steroid receptor coactivator-1 (SRC1). The most important fragment of SRC1 for binding to NRs is the central domain, made of three α-helical Leu-X-X-Leu-Leu (LXXLL) L1, L2, L3 motifs, in where L is a leucine and X represents any amino acid. hSRC1, as well as hCAR, can be successfully produced by expression of cDNA of interest in E. coli strains. In case of SRC1,...
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Increasing affinity of Interferon gamma receptor 1 to Interferon gamma by combining molecular modeling and experimental methods
Mikulecký, Pavel ; Schneider, Bohdan (advisor) ; Šulc, Miroslav (referee) ; Vaněk, Ondřej (referee)
Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...
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Molecular mechanisms of the glycinergic signalization in the central nervous system
Melichar, Adolf ; Králíková, Michaela (advisor) ; Ladislav, Marek (referee)
The amino acid neurotransmitter glycine, which mediates the inhibitory transmission and regulates the excitatory signalization, is found in many regions of the central nervous system. It is produced especially the spinal cord and the brainstem, where glycine activates the presynaptic and postsynaptic Gly receptors (GlyR). In other regions of the brain, glycine is released in the area of occurrence of NMDA receptors (NMDAR), which it activates together with the excitatory neurotransmitter glutamate. The binding of the glycine leads to the opening of the channel of these ionotropic receptors and thus also to a change in the electrochemical properties of the membrane, such as permeability for Ca2+ ions, membrane potential and shunting. The expression of different subunits of NMDAR and GlyR is specific in terms of location and time, which enables various responses to the presence of glycine in the synaptic cleft and its surroundings. The concentration of glycine in synaptic cleft and glycinergic terminals is controlled especially by glycine transporters (GLYT), that are found in two functionally different types. The destabilisation of the equilibrium and signal mechanisms of glycine may lead to serious diseases such as hyperekplexia that can be caused by mutations in the glycine receptors and...
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Increasing affinity of Interferon gamma receptor 1 to Interferon gamma by combining molecular modeling and experimental methods
Mikulecký, Pavel
Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...
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Interaction of retroviral env glycoproteins with receptors and virus entry into the cell
Přikryl, David ; Pečenka, Vladimír (advisor) ; Horníková, Lenka (referee)
Humankind has adjusted many animals, plants and bacteria to serve their needs. Now the time has come to exploit also viruses. The most significant applications for them seem to lie in the sphere of human health. Particularly the family of retroviruses represent a perfekt vectors for gene delivery to cure genetic diseases or to destroy undesirable, e.g. cancer cells. In order to efficiently use these vehicles, a series of problems must be overcome and some segments of retroviral life cycle must be more deeply explored. This work describes some of them along with possible ways to solve them. The first described area is nonspecific adsorption of virions on the cell surface, the second is securing effective interaction between envelope glycoproteins and receptors highly specific for the targeted cell type and finally driven fusion of membranes. Key words: retrovirus, adsorption, env, receptor, phusion
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Preparation of expression vectors for receptor NKp30 and its ligands B7-H6 and BAG-6
Pažický, Samuel ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
NK cells, the cells of non-adaptive imune system, are able to recognise viraly infected or oncogenous cells by many inhibiting and activating receptors that are expressed on their surface and eliminate them consequently. For NKp30, activating receptor of NK cells included in NCR (Natural Killer Cell Receptors) family, lately there were identified several ligands, including membrane protein B7-H6 expressed on oncogenous cells surface and BAG- 6, cell core protein with wide spectrum of functions. Aim of this thesis was preparation of expression vectors coding for receptor NKp30 and his ligands B7-H6 and BAG-6, enabling expression of these proteins in HEK293 cell line. Keywords: NK cell, plasmid, receptor, NKp30, B7-H6, BAG-6
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Translation in the Dialogue: The Process of Encoding and Decoding of Film Style in a Remake of the Original Work
Gladiš, Michal ; Svatoňová, Kateřina (advisor) ; Kokeš, Radomír D. (referee)
The following bachelor's thesis concerns the remake, in the context of its use in the history of cinema and in film theory. I discuss the various definitions and possible forms of the remake, which has existed since the inception of film. Each theoretical consideration of the remake brings about a different choice of perspective and method of examination. Thus, the main part of the thesis is comprised of an effort to acquire a new apparatus through which one can think of the remake as a variant interpretation of the original work. In this regard, I transpose approaches from literary theory to film discourse, mainly referencing the theoretical methods of Gérard Genette, Julia Kristeva, Seymour Chatman, Umberto Eco and the concepts of intertextuality and interpretation. The last chapter analyzes two specific films, positioning Takashi Miike's Visitor Q as a remake of Pier Paolo Pasolini's Teorema. The relationship between remake and original develops new readings of both works. A remake is, in fact, based on the filmmaker's unique reading of an original work. This reading marks the transition between the filmmaker's experience as a viewer of the original and their position as creator of the remake.
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Structural biology of complex of rat NK cell receptors NKR-P1B and Clrb
Dvorská, Anna ; Vaněk, Ondřej (advisor) ; Ingr, Marek (referee)
The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...
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