National Repository of Grey Literature 91 records found  beginprevious51 - 60nextend  jump to record: Search took 0.00 seconds. 
Role of the segment 400-500 in biological activity of Bordetella pertussis adenylate cyclase toxin
Suková, Anna ; Mašín, Jiří (advisor) ; Krůšek, Jan (referee)
The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in virulence of the whooping cough agent Bordetella pertussis. It translocates an AC enzyme into cytosol of CD11b+ phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. In parallel, CyaA permeabilizes cellular membrane by forming cation-selective pores. The goal of my diploma thesis was an analysis of the mechanism of interaction of the segment linking the invasive adenylate cyclase domain and the RTX hemolysin moiety of CyaA with target membrane. Our data show that the segment linking the AC to the hydrophobic domain of CyaA is directly involved in the interaction of the toxin with the membrane and controls the formation of small cationt-selective pores. Our results generate new knowledge that will be of relevance to the entire field of toxin biology and will enable the design of improved CyaA- based vaccines. Keywords: Bordetella pertussis, adenylate cyclase toxin, membrane translocation, pore- forming activity, black lipid bilayers, liposomes
Identification of the binding sites on transient receptor potential cation channel TRPC6 for Calmodulin and S100A1
Bílý, Jan ; Teisinger, Jan (advisor) ; Krůšek, Jan (referee) ; Pavlíček, Jiří (referee)
Identification of the binding sites on transient receptor potential cation channel TRPC6 for Calmodulin and S100A1 The TRP (transient receptor potential) group of ion channels represents a large subset of membrane receptors. A part of this supergroup are canonical TRPC channels with a sequence homology analogical to TRP receptor first discovered at fruit fly (Drosophila melanogaster). These membrane channels are involved in a variety of physiological functions in different cell types and tissues. TRPC6 is a non-selective cation channel that modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. TRPC6 channel contains binding domain CIBR (Calmodulin inositol binding region), which is also able to adapt to calcium binding protein S100A1. Characterisation of the integrative binding site for calmodulin (CaM) and S100A1 at the C-tail of TRPC6 is presented in this work. Using site-directed mutagenesis, soluble protein fragments TRPC6 CT (801-787) were prepared with intentional changes in amino acid sequence. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by measurement of fluorescence anisotropy influence and their participation in the calcium-dependent binding of CaM and/or S100A1 to...
Localization and characterization of binding sites for Ca2+ binding proteins and phosphatidylinositol phosphates on intracellular termini of TRP channels
Boušová, Kristýna ; Teisinger, Jan (advisor) ; Žáčková, Markéta (referee) ; Krůšek, Jan (referee)
This dissertation concerns with characterization of binding sites for calcium binding protein S100A1 and phosphatidylinositol phosphates on intracellular regions of transient receptor potential channels (TRPs), particular from canonical (TRPC), vaniloid (TRPV) and melastatin (TRPM) families. TRPs represent superfamily of important mediators that play critical roles in sensory physiology: contributions to taste, olfaction, vision, hearing, touch and thermo- and osmo- sensation. They serve as non-selective and nociceptive membrane receptors responsible for the modulation of driving force for cations entry into the cell. TRPs are composed from six transmembrane domains and N- and C- termini intracellular regions. Overall four monomer units form a characteristic assembly of functional channel. It was demonstrated that most of this almost thirty-member family transporters are activated by a variety of different stimuli and function as signal integrators. The most examined intracellular TRPs modulators are cytosolic calcium binding proteins and membrane anchored phosphatidylinositol phosphates (PIPs). These signal integrators bind specific domains in intracellular termini of TRPs, thereby change their structure and activate or inhibit the transportation function of receptor. To identify a novel ligand...
The mechanism of the regulation of phosducin function
Kacířová, Miroslava ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Pavlíček, Jiří (referee)
This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...
Study of interactions of forkhead transcription factor FOXO4 with DNA and the 14-3-3 protein
Vácha, Petr ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee) ; Krůšek, Jan (referee)
CHARLES UNIVERSITY IN PRAGUE THE FACULTY OF NATURAL SCIENCE Department of Physical and Macromolecular Chemistry The summary of the doctoral thesis Study of interactions of forkhead transcription factor FOXO4 with DNA and the 14-3-3 protein RNDr. Petr Vácha Scientific supervisor: Prof. RNDr. Tomáš Obšil, Ph.D. Prague 2015 Abstract This doctoral thesis deals with the interaction of human forkhead transcription factor FOXO4 with DNA and regulating 14-3-3 protein respectively. The main aim of this work was detailed characterization of interaction between DNA binding domain of protein FOXO4 with two canonical DNA sequences and further clarifying the role of the 14-3-3 protein in the regulation of activity of protein FOXO4. FOXO transcription factors are potent activators of the transcription of genes, which affect a variety of cellular processes. FOXO4 protein belongs to the family of forkhead transcription factor, which is a group of several tens of proteins, whose common feature is a highly conserved DNA- binding domain. Summary of the DNA binding specificity of these proteins, namely what precisely determines the small differences in the binding properties of individual forkhead proteins, despite the large amount of available structural data remains still unclear. Therefore, detailed characterization of...
Activation mechanisms of muscarinic M1 receptor by atypical agonists
Randáková, Alena ; Doležal, Vladimír (advisor) ; Mysliveček, Jaromír (referee) ; Krůšek, Jan (referee)
Atypical agonists of muscarinic receptors bind to individual receptor subtypes with comparable affinity but activate them selectively to a certain extent. Molecular mechanism underlying this "functional selectivity" is not known and its elucidation may contribute to development of new atypical functionally selective agonists suitable for therapeutic use. Functional selectivity of atypical muscarinic agonists may be caused by a distinct molecular mechanism(s) of how these compounds activate the receptor. Agonist-specific conformations induced by structurally complex atypical agonists may lead to utilization of a parallel activation mechanism that is different than the activation mechanism induced by non-selective classical agonists. In order to examine this possibility we investigated whether the M1 receptor preferring atypical agonists xanomeline and N-desmethylclozapine, and the classical orthosteric agonists carbachol and oxotremorine, activate the M1 receptor through a common cascade of transmission switches. To this end we mutated key amino acids of the M1 receptor that are essential for ligand binding to the orthosteric binding site (D1053.32 , D993.26 ), receptor activation (transmission switch, D712.50 ), or interaction with G-protein (ionic lock switch, R1233.50 D1223.49 ). We compared...
Pore-forming properties of Bordetella pertussis CyaA toxin and composition of the lipid bilayer.
Rädisch, Robert ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Bordetella pertussis produces many virulent factors including adenylate cyclase toxin (CyaA) This toxin preferentially invades cells of immune system with integrin receptor CD11b/CD18 and weakens the immune system of the host. CyaA affects invaded cells in two ways. First, CyaA creates a cation-selective pores in the membrane of invaded cell and causes colloidal osmotic lysis. Second, CyaA converts cytosolic ATP into signal molecule cAMP, which causes a loss of physiological function of invaded cell and also leads to cellular death. The aim of my thesis was to test a suitability of a new model system composed from synthetic lipids - diphytanoyls, for a characterization of pore-forming properties of adenylate cyclase toxin. In the past, asolectin model system comprising many different lipid was used for characterization but it was found to be too complex for defining the role of individual lipids in CyaA activity. Further the effect of cholesterol for activity of CyaA was studied in a new model system because it was found recently that translocation of adenylate cyclase domain takes place at lipids rafts with high concentration of cholesterol. The last aim of my thesis was to characterize a newly discovered type of channel with the two conductance levels. Key words: Bordetella pertussis, adenylate...
The influence of zinc ions on ionotropic glutamate receptors
Fraňková, Denisa ; Krůšek, Jan (advisor) ; Adámek, Pavel (referee)
Zinc is one of the most abundant divalent metal ion in the central nervous system, where it serves as regulator of many proteins (channels, receptors, pump...), product of neurosecretion or cofactor. The highest concentration of zinc in synaptic vesicles of the specific neurons, which are called zinc-containing neurons and it is subset of glutamatergic neurons. The cumulation of zinc in the synaptic neurons is arranged mostly by transporter ZnT3. A concentration of zinc in the synaptic's vesicles is about 1mmol/l and maybe higher. The zinc-containing neurons are mostly in forebrain, where create complex and neuronal network in mammalian's brain, which conect most of cerebral cortex and limbic system (Frederickson et al. 2000). Zinc is released from synapses after a stimulation (Vogt et al. 2000) and then it affects a lot of receptors, for example postsynaptic NMDA receptors (Koh & Choi 1994) and Ca2+ permeable AMPA and cainate receptors (Hong Z. Yin 1995), voltage-gated Ca2+ channels (Atar et al. 1995) and GABAA receptors (Ruiz et al. 2004). This bachelor thesis summarizes the efekt Zn2+ on the ionotropic glutamate receptors. It is mostly adressed on the NMDA receptors, which are the most affected by zinc. The first chapter is about structure of ionotropic glutamate receptors, which is followed up other...
Inhibition of nicotinic acetylcholine receptors by tacrine and its derivatives
Skřenková, Kristýna ; Krůšek, Jan (advisor) ; Doležal, Vladimír (referee)
Nicotinic acetylcholine receptors are ligand-gated ion channels which are located on neuromuscular junction and in central and perifric nervous system. Activity of nicotinic receptor might be modulated by variety of pharmacological agents. In this work, we have focused on the study of the inhibition effect of tacrine and its derivatives on the nicotinic acetycholine receptors of muscle and neuronal type. These derivatives function as acetylcholinesterase inhibitors and also interact with nicotinic acetylcholine receptors. The majority of current forms of treatment of Alzheimer's disease is based on cholinesterase inhibitors. We have studied the mechanism of tacrine and its derivatives by using patch clamp method in the configuration of whole-cell recording. Powered by TCPDF (www.tcpdf.org)
The role of TRPV1 receptors in chemokine CCL2 induced modulation of nociceptive synaptic transmission at spinal cord level
Adámek, Pavel ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn is a significant mechanism in the development and maintenance of different pathological pain states. Accumulating evidence indicates that the TRPV1 (transient receptor potential vanilloid 1) receptor and chemokine CCL2 (C-C motif ligand 2) may play a critical role in this process. The aim of this diploma thesis was to investigate the CCL2 induced modulation of nociceptive synaptic transmission in the dorsal horn of spinal cord and the role of the TRPV1 receptors. To investigate this aim patch-clamp recordings of spontaneous and miniature excitatory postsynaptic currents (sEPSC, mEPSC) from superficial dorsal horn neurons in acute rat lumbar spinal cord slices were used. After acute application of CCL2 on the slice preparation from naïve animals, a frequency increase of both sEPSC and mEPSC was present. This CCL2 induced increase in both sEPSC and mEPSC frequency was prevented by the TRPV1 receptor antagonist SB366791 application. No changes were observed in the amplitudes of sEPSC or mEPSC after application of the CCL2, SB366791, or co-application of CCL2 and SB366791. This suggests that the observed changes were mediated predominantly by presynaptic mechanisms. The preliminary results indicate that after chronic constriction...

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