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Simulation of electrophysiological experiments
Kubáček, Svatopluk ; Provazník, Ivo (referee) ; Rychtárik, Milan (advisor)
Simulation of electrophysiological experiments enables to gain valuable data about bioelectrical processes occurring on isolated cells. This thesis deals with basic principals allowing the creation of bioelectrical phenomenons and measuring techniques that need to be used in order to collect information about these phenomenons. The outcome of the thesis is a programme that serves as an instrument for simulation of measuring techniques applied on Luo-Rudy model. The programme contains basic measuring techniques – current clamp and voltage clamp – providing the possibility to change basic parameters of the model. The results of this work are later compared with the accessible literature.
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The fluorescent marking of glass microelectrodes for the patch clamp system
Klepáčová, Ivana ; Ronzhina, Marina (referee) ; Chmelíková, Larisa (advisor)
The paper deals with the issue of the fluorescent marking of glass microelectrodes for the patch clamp system. It analyses in more details the possibilities of creation of glass microelectrodes from the choice of appropriate glass type to the construction of a microelectrode, which can be used in practice in experimental measurements. Phases of creation process of glass microelectrodes document possibilities of the development of the given technique. The description of fluorescence phenomenon includes the classification and description of fluorophores. The paper contains a theoretical analysis of the given issues and presents it in its comprehensive form. The practical part of the thesis suggests the procedure of the external fluorescent marking of glass microelectrodes which can be used in the measurement by means of the patch clamp system. It is concerned with the testing of the established procedure and provides the evaluation of the possible application of the suggested procedure in practice.
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The molecular mechanisms and role of purinergic signaling in hypothalamus
Ivetic, Milorad ; Zemková, Hana (advisor) ; Mysliveček, Jaromír (referee) ; Smejkalová, Terézia (referee)
(EN): Extracellular ATP and purinergic P2 receptors (P2X and P2Y) are involved in a signaling network called "purinergic signaling" which is widely exploited in both somatic and neuronal tissues, and is also operative in endocrine system. The main focus of my thesis is on the role and expression of P2X and P2Y receptors in hypothalamic supraoptic nuclei (SON) producing hormones vasopressin and oxytocin, and the suprachiasmatic nuclei (SCN), the principal circadian pacemaker in mammals. In the first part of my thesis, we tested the hypothesis that P2X and P2Y receptors play a role in the enhanced release of hormones from magnocellular SON neurons stimulated through fasting/refeeding experimental protocol. We studied the effect of 2 h of refeeding after 48 h of fasting on hormone, P2X and P2Y mRNA expression in the SON tissue of 30-day-old rats, and the effect of ATP on electrophysiological properties of SON neurons in brain slices from control and fasted/refed rats. Quantitative real-time PCR revealed that the expression of P2X2 and arginine-vasopressin (AVP) mRNA was increased and P2Y1 mRNA expression was decreased in fasted/refed rats compared to controls, whereas P2X4, P2X7, P2Y2 and oxytocin mRNA levels were not significantly changed. Whole-cell patch clamp recordings showed that the amplitude...
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Identification of new neuroactive steroids that are able to interact with allosteric binding sites on purinergic P2X receptors
Sivčev, Sonja ; Zemková, Hana (advisor) ; Vyklický, Vojtěch (referee) ; Stojilkovic, Stanko S. (referee)
(EN) Purinergic P2X receptors are ATP-gated cation channels with multiple physiological roles and are emerging as important therapeutic targets in a range of diseases. P2X subunit consists of two transmembrane helices (TM1 and TM2), an extracellular ATP-binding domain, and intracellular N- and C- termini. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. P2X are ubiquitously expressed. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain. The activity of P2X depends not only on the presence of ATP but also on allosteric modulators that may inhibit or potentiate the activity of these channels. Our aim was to identify new molecules that could interact with allosteric binding sites on P2X receptors, design and synthesize new analogues of neurosteroids, and define crucial receptor domains and amino acids important for neurosteroid binding. By using a patch-clamp electrophysiology technique we recorded ATP-induced currents in HEK293T cells transfected with rat P2X2, P2X4, and P2X7, as well as in the rat anterior pituitary cells and hypothalamic neurons endogenously expressing these receptors. We found that 17β-ester derivatives of testosterone, namely testosterone butyrate and...
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Characterization of the effect of pregnanolone sulfate and its derivatives on NMDA receptors.
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...
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Biophysical studies of membrane transport proteins from Nramp/MntH family and their function
Ňuňuková, Věra
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...
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Identification of changes in membrane properties of astrocytes in a mouse model of amyotrophic lateral sclerosis
Vaňátko, Ondřej ; Turečková, Jana (advisor) ; Vlachová, Viktorie (referee)
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder of the central nervous system characterized by loss of motor neurons and voluntary muscle degeneration. Astrocytes play a major role in regulation of the disease onset and progression due to their intimate association with neurons. Regulation of ionic homeostasis is one of their key functions and its failure has been linked to several neurological diseases. The aim of this thesis was to explore differences in membrane properties of astrocytes in ALS. To fulfill this aim, a double transgenic mouse strain with ALS-like phenotype and a specific expression of enhanced green fluorescent protein in astrocytes was generated. To phenotype this strain, two sensorimotor tests, wire grid hang test and rotarod test, were conducted. Immunohistochemistry was used to characterize the strain on a cellular level and to explore changes of specific ion channels. Functional properties of astrocytes were explored using the patch clamp technique. The double transgenic strain has the characteristic ALS-like phenotype and is comparable to the original strain with differences in symptom onset and progression between models and sexes. On the cellular level, there are characteristic ALS features, specifically loss of motor neurons and astrogliosis....
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Characterization of the effect of pregnanolone sulfate and its derivatives on NMDA receptors.
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...
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Differentiation potential of polydendrocytes after focal cerebral ischemia
Filipová, Marcela ; Anděrová, Miroslava (advisor) ; Jendelová, Pavla (referee)
Ischemic injury leeds to sequence of pathophysiological events, which are accompanied by a release of growth factors and morphogens that significantly affect cell proliferation, migration and also their differentiation. Following ischemia, besides enhanced neurogenesis and gliogenesis in subventricular zone of the lateral ventricles and gyrus dentatus of the hippocampus, neurogenesis/gliogenesis also occurs in non-neurogenic regions, such as cortex or striatum. Recently, the attention was turned to a new glial cell type, termed polydendrocytes or NG2 glia. Under physiological conditions, these cells are able to divide and differentiate into mature oligodendrocytes due to they have often been equated with oligodendrocyte precursor cells. Based on recent reports, polydendrocytes are also able to generate protoplasmic astrocytes (Zhu et al., 2008) and neurons in vitro (Belachew et al., 2003), however their ability to differentiate into astrocytes or neurons under physiological or pathological conditions is still highly debated. Therefore, we have investigated the effect of different growth factors and morphogens, specifically brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and a morphogen sonic hedgehog (Shh), on...
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Biophysical studies of membrane transport proteins from Nramp/MntH family and their function
Ňuňuková, Věra
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...
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