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Neuroinflammation and mechanisms of neuropathic pain development
Kalynovska, Nataliia ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee) ; Hejnová, Lucie (referee)
Neuropathic pain represents a possible outcome of neural tissue injury; it occurs also as a concomitant symptom of different diseases or as a side effect of several treatments. Up to date, it constitutes a great challenge in clinical practice, as currently available treatments are still unsatisfactory. Mechanism-based treatment approaches are promising strategy in neuropathic pain management. However, there is still a lack of information about the exact mechanisms involved in the development and/or maintenance of neuropathic pain. This Doctoral Thesis is aimed to explore the mechanisms underlying the development of neuropathic pain states in different models. The principal part of this work is focused on the study of anti-inflammatory effect of Angiotensin II receptor type 1 (AT1R) blocker, losartan, in two different models of peripheral neuropathy: paclitaxel-induced peripheral neuropathy (PIPN) and spinal nerve ligation (SNL). The work also aimed to access the involvement of spinal transient receptor potential vanilloid type 1 (TRPV1) channels in the process of neuronal activation induced by paclitaxel (PAC) and chemokine CCL2 treatment. In order to fulfil the abovementioned aims, behavioral, immunohistochemical and molecular methods were used. For every model of peripheral neuropathy, the...
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Study of the differences in the architecture of the binding pockets of two major MDR pumps of yeast Saccharomyces cerevisiae, Pdr5p and Snq2p, using their common substrates
Backová, Lenka ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
Multidrug resistance (MDR) is responsible for the decrease in drug effectiveness on pathogenic microorganisms or tumours. One of the mechanisms of multidrug resistance is drug transport out of the cell (efflux) by membrane transporters - pumps. Main MDR pumps of a yeast species Saccharomyces cerevisiae are Pdr5p and Snq2p, who share high amino acid sequence identity. This thesis focuses on the differences of these pumps, their binding pockets and their arrangement. The binding pocket of Pdr5p is better researched and comparing the results with those of pump Snq2p leads to broader knowledge about the binding pocket of Snq2p. We use disc diffusion assay to determine common substrates of both pumps, ketoconazole and bifonazole. These substrates are used in potentiometric fluorescent probe diS-C3(3) assay. Results of these experiments lead us to the findings that the binding pocket of Snq2p has multiple binding sites. Binding pockets of pump Pdr5p and Snq2p differ in binding sites and their conformation. However, the conformation of both pumps is dynamic, which has been shown after the addition of glucose to supply the pumps with energy. 1
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Structural and pharmacological determinants of NMDA receptor channel gating
Ladislav, Marek ; Krůšek, Jan (advisor) ; Bendová, Zdeňka (referee) ; Zemková, Hana (referee)
N-methyl-D-aspartate receptors (NMDARs) are heterotetramers containing two obligatory glycine-binding (GluN1) and two glutamate/glycine-binding (GluN2/3) subunits. These receptors mediate excitatory synaptic transmission in the central nervous system and play a key role in high order neuronal processes as a learning and formation of memory. It has been shown that dysregulation of NMDARs is involved in the pathophysiology of neurological and psychiatric disorders. Each receptor is composed of four protomers exhibiting a conserved domain organization. The most distal part to the cell membrane is the amino-terminal domain that is linked to the ligand binding domain (LBD), which is connected to the pore-forming transmembrane domain (TMD) communicating with the intracellular carboxy-terminal domain. LBD and TMD are connected via three polypeptide chains - linkers. Channel opening is the key step in the NMDAR gating that allows the flux of ions across the membrane. The energy of agonist binding-evoked conformational changes is transferred via linkers to M3 helices forming an ion channel. The rearrangement of M3 helices in activated receptor makes the central cavity of the channel accessible. The details of energy transfer are not yet fully characterized, although accurate knowledge of the receptor gating...
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Study of transport systems of microorganisms
Jančíková, Iva ; Gášková, Dana (advisor) ; Amler, Evžen (referee) ; Krůšek, Jan (referee)
Title: Study of transport systems of microorganisms Author: Mgr. Iva Jančíková Department: Institute of Physics of Charles University Supervisor: Assoc. Prof. RNDr. Dana Gášková, CSc., Institute of Physics Abstract: Overexpression of drug efflux pumps is responsible for a multidrug resistance (MDR). We used the potentiometric fluorescent probe diS-C3(3), which is a substrate of major MDR pumps in three yeast species, Saccharomyces cerevisiae (ScPdr5p, ScSnq2p), Kluyveromyces lactis (KlPdr5p) and Candida albicans (CaCdr1p, CaCdr2p), to monitor inhibition of selected membrane transporters caused by various chemical stressors (diS-C3(3) assay). The extent of inhibition of probe transport points to a tighter arrangement of the K1Pdr5p binding pocket compared to that of ScPdr5p. Furthermore, we discovered that while deletion of the KlPDR16 gene does not affect K1Pdr5p activity, it only caused cell hyperpolarization, deletion of the KlERG6 gene results in both change in membrane potential and in a suppression of the pump's activity. We developed an effective method to search for inhibitors of MDR proteins of. C. albicans, which is based on pre-screening their potential to block the probe efflux from S. cerevisiae cells. Using this method we identified the substance H, derivative of 1,4-dihydropyridine, which...
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Modulation of nociceptive synaptic transmission
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee) ; Hejnová, Lucie (referee)
Modulation of synaptic transmission in the spinal cord dorsal horn plays an important role in development and maintenance of pathological pain states. The indisputable part of this modulation is conducted via activity of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and the cannabinoid receptor 1 (CB1), expressed on presynaptic endings of primary afferents in the superficial spinal cord dorsal horn. Under physiological conditions, activation of TRPV1 receptors is pronociceptive while CB1 receptor activation leads to attenuation of nociceptive signalling. However, both receptors share also one endogenous agonist anandamide (AEA) that may be produced from N-arachidonoyl phosphatidylethanolamine (20:4-NAPE). Main objective of this thesis focuses on the effect of 20:4-NAPE on nociceptive synaptic transmission in spinal cord slices under naïve and inflammatory conditions and consequent on the possible interaction of TRPV1 and CB1 receptors. First, 20:4-NAPE application induced significant release of anandamide from spinal cord slices under in vitro conditions. Next, patch- clamp recordings of excitatory postsynaptic currents (mEPSC and sEPSC) from superficial dorsal horn (DH) neurons in acute spinal cord slices were used. 20:4-NAPE application under the physiological...
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Molecular mechanisms and functions of 14-3-3 proteins
Šilhán, Jan ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Schneider, Bohdan (referee)
Závěr Hlavním cílem této doktorské práce bylo objasnění molekulárních mechanismů funkce 14-3-3 proteinů a vlivu na proteiny FOXO4 a tyrosinhydroxylasu. V první časti této práce (publikace I) byla potvrzena předložená hypotéza polohy Cterminálního konce molekuly 14-3-3. Bylo ukázáno, že v nepřítomnosti ligandu se Cterminální konec nachází ve vazebném místě a brání tak vstupu ligandů. Po vazbě fosforylovaných ligandů, dochází k velmi silné vazbě a vytěsnění C-terminálního konce 14-3- 3 proteinu z vazebného místa. Tyto výsledky jsou v souladu s původními pracemi, které navrhly důležitost tohoto segmentu jako inhibitoru nepatřičných ligandů. Druhá část této doktorské práce poskytuje rozsáhlý popis vlivu 14-3-3 proteinů na transkripční faktory FOXO4. S použitím stacionární a časově-rozlišené fluorescence byla studována interakce 14-3-3 proteinu s fosforylovaným transkripčním faktorem FOXO4. Navázání 14-3-3 proteinu způsobuje rozpad komplexu FOXO4:DNA. Tato část práce charakterizuje interakci 14-3-3 proteinu s DNA-vazebnou doménou FOXO4. Výsledky neprokázaly výrazné konformační změny v rámci DNA-vazebné domény. Spíše dochází ke sterickému bránění vazby DNA (publikace II). Ve třetí části se práce zabývá studiem interakcí 14-3-3 s fosforylovaným ligandem odvozeným od C-konce enzymu serotonin N-acetyltransferasa...
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Characterization of native and heterologously expressed membrane transporters in yeast using fluorescent probes
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Cebecauer, Marek (referee) ; Krůšek, Jan (referee)
Yeast plasma membrane transporters play crucial roles in many cellular processes, including detoxification and build-up and maintenance of the plasma membrane potential (ΔΨ). The former development of the diS-C3(3) fluorescence assay by the Biophysics Group of the Institute of Physics, Charles University, enabled us to conveniently study both, including their changes, using a simple fluorescent probe diS-C3(3). Many studies carried out on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using the diS- C3(3) assay for multidrug-resistance pump inhibitors in a set of isogenic yeast pdr5 and snq2 deletion mutants we found that n-alcohols (from ethanol to hexanol) exhibit an inhibitory effect on both pumps, increasing with the length of the alcohol carbon chain. The inhibition is not connected with loss of plasma membrane structural or functional integrity and is fully reversible. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins. Tok1p is a highly specific...
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Study of membrane transport processes in yeast using potentiometric fluorescent porbe diS-C3(3)
Bartl, Tomáš ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
1 Title: Study of membrane transport processes in yeast using potentiometric fluorescent probe diS-C3(3) Author: Tomáš Bartl Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Dana Gášková, CSc., Institute of Physics of Charles University Abstract: Yeast membranes contain a number of transporters. Some are responsible for flow of nutrients to the inside of the cell, others for disposing of waste and foreign substances and some for transport of small ions or protons across the membrane. The focus of this work is on the activity of specific transport membrane proteins, so-called MDR pumps, which are responsible for transport of foreign substances or drugs, out of the cell. Using the series of mutant strains of the yeast Saccharomyces cerevisiae (AD1-3, AD1-8 and AD12) differentiated in the presence of specific MDR pumps in their membrane, an influence of various chemical substances on the intracellular concentration of the potentiometric fluorescent probe diS-C3(3), which is actively being transported out of the cell by some of the MDR pumps, was observed. By the examination of the effect of 2-deoxyglucose we proved the active contribution of not only the main MDR pump, Pdr5p, but also of some other pumps, in lowering the intracellular probe concentration. It was observed that...
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