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Study of transport proteins of the Nramp family
Surá, Lucie ; Chaloupka, Roman (advisor) ; Holoubek, Aleš (referee)
The Natural Resistance-Associated Macrophage Proteins (Nramp) form functionally conserved family of proton-dependent divalent metal ion transporters. In the present study, we investigated transport properties of a prokaryotic Nramp homolog - MntH transporter from Escherichia coli. H+ transport mediated by MntH was monitored in a bacterial model system using pH-sensitive green fluorescent protein (pHluorin). Our experimental conditions enabled us to observe an uncoupled H+ transport mediated by MntH. Uncoupled H+ flux had been previously described in eukaryotic Nramp proteins, nevertheless this is the first observation of this phenomenon in a prokaryotic homolog. We demonstrated that the uncoupled H+ transport is pH- and temperature- dependent. The uncoupled transport H+ is also affected by specific single-point mutations at functionally important residues Asp34, His211 and Asn401. The second part of the work focused on effect of different ions, which are not MntH substrates, on transport properties of MntH. It was shown that addition of excess calcium or magnesium resulted in increase of H+ transport induced by divalent metal ions, but on the other hand our data suggest that calcium inhibits uncoupled H+ transport.
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Documentation of Barová cave - central part
Chaloupka, Roman ; Machotka, Radovan (referee) ; Kratochvíl, Radim (advisor)
The aim of this bachelor thesis is the graphic documentation of Barová cave – central part in the form of a plan and transection in a scale of 1:250. The thesis contains, including brief description of the area, all steps from reconnaisasance and completion of the existing measuring network to the measuring and processing part leading to the creation of the resulting map.
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Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors
Kaniaková, Martina ; Krůšek, Jan (advisor) ; Chaloupka, Roman (referee) ; Zemková, Hana (referee)
Title: Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors Author: Mgr. Martina Kaniaková Department: Institute of Physiology AS CR, v.v.i. Supervisor: RNDr. Jan Krůšek, CSc., Institute of Physiology AS CR, v.v.i. Abstract: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. We use the whole-cell patch-clamp technique to study functional and pharmacological properties of muscle and neuronal nicotinic receptors. Rat neuronal receptors were heterologously expressed in COS cells and human embryonic muscle receptors were studied in TE671 cells. Lobeline, a plant alkaloid with a long history of therapeutic use, interacts with the classical agonist-binding site of nAChRs. The final result of this interaction depends on the receptor subtype, lobeline and other agonists concentrations and the time schedule of application. Generally, lobeline is a very weak partial agonist eliciting deep desensitization at several subtypes of nAChRs. In combination with other agonists, lobeline acts as a competitive antagonist or coagonist. Using point mutation procedure we studied the functional role of negatively charged amino acids in the F-loop of β2 and β4 subunits of neuronal receptors. Neutralising mutations in β4 subunit led to up to eighteen-fold increase in the...
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Stoichiometry of transport mediated by bacterial membrane transport protein MntH of E. coli
Doležalová, Iva ; Chaloupka, Roman (advisor) ; Marešová, Lydie (referee)
The Nramp family (Natural Resistance-Associated Macrophage Protein) includes secondary active transport proteins transporting divalent metal ions together with protons into a cell. Divalent metal ions play a major role in the cell metabolism and participate in many intracellular processes. We have studied bacterial Nramp ortholog - MntH (Proton-dependent Manganese Transporter), its transport stoichiometry and other functionally important characteristics. We have found that the external pH affected the transport stoichiometry. The functional differences between the wild type and the protein with mutation N401G appears only in lower external pH. Moreover, uncoupled proton flux mediated by MntH under specific experimental conditions is inhibited by calcium, whereas coupled proton flux increases after the addition of calcium ions. This can be connected to the fact that calcium is also transported by MntH. Surprisingly, the transport stoichiometry of neither the wild type nor the protein with mutation N401G is affected by the addition of calcium or the change of the external medium.
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Biophysical studies of membrane transport proteins from Nramp/MntH family and their function
Ňuňuková, Věra ; Chaloupka, Roman (advisor) ; Krůšek, Jan (referee) ; Kubala, Martin (referee)
Three synthetic peptides corresponding to transmembrane segments TMS1, TMS3 and TMS6 of secondary-active transporter MntH from Escherichia coli were used as a suitable alternative model enabling to study TMS structure, TMS interaction with membranes, TMS mutual interaction and also function of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformation in lipid membranes. Electrophysiological experiments demonstrated that individual TMS were able under certain conditions to form ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS1 and TMS6 channels, influenced the transition between closed and open states and affected the conformation of all studied peptides. For TMS3 Mn2+ was crucial for formation of ion channels. It was shown that a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings can contribute to understanding of structure-function relationship at the molecular level. However, it remains unclear to what extent...
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Regulace vnitřního pH kvasinek - vliv vybraných transportních proteinů
Zalom, Peter ; Chaloupka, Roman (advisor) ; Holoubek, Aleš (referee)
Intracellular pH affects nearly all biochemical processes in yeast, the processes regulating the cytosolic pH includes function of many transport proteins. In this work, the impact of selected sodium transporters on cytosolic pH has been studied in two yeast species: Saccharomyces cerevisiae and Zygosaccharomyces rouxii including wild-type and mutants with affected sodium transport. Measurements of cytosolic pH and buffering capacity have been performed using fluorescent protein probe pHluorin - a pH sensitive derivate of green fluorescence protein. Several procedures for calibration of pHluorin fluorescence response have been compared and the importance of a proper correction of the calibration curve has been demonstrated. It has been shown that cytosolic pH is influenced by the function of Nha1 transport protein in S. cerevisiae as well as in Z. rouxii but not by Sod2-22 transporter in Z. rouxii. It has been demonstrated that the buffering capacity of cytosol decrease in the presence of glucose in all strains studied.
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