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Functional and structural study of thermally activated TRP ion channels: The role evolutionarily conserved motifs in the TRPA1 modulation
Kádková, Anna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee) ; Obšilová, Veronika (referee)
Ankyrin receptor TRPA1 is an ion channel widely expressed on primary afferent sensory neurons, where it acts as a polymodal sensor of nociceptive stimuli. Apart from pungent chemicals (e. g. isothiocyanates, cinnamaldehyde and its derivatives, acrolein, menthol), it could be activated by cold temperatures, depolarizing voltages or intracellular calcium ions. TRPA1 channel is a homotetramer in which each subunit consists of cytoplasmic N and C termini and a transmembrane region. The transmembrane part is organized into six alpha- helices connected by intra- and extracellular loops. The N terminus comprises a tandem set of 16 to 17 ankyrin repeats (AR), while the C terminus has a substantially shorter, dominantly helical structure. In 2015, a partial cryo-EM structure of TRPA1 was resolved; however, the functional roles of the individual regions of the receptor have not yet been fully understood. This doctoral thesis is concerned to elucidate the role of highly conserved sequence and structural motifs within the cytoplasmic termini and the S4-S5 region of TRPA1 in voltage- and chemical sensitivity of the receptor. The probable binding site for calcium ions that are the most important physiological modulators of TRPA1 was described by using homology modeling, molecular-dynamics simulations,...
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The ion channel TRPV1 and its role in dendritic cells and macrophages
Trnková, Nella ; Krulová, Magdaléna (advisor) ; Vlachová, Viktorie (referee)
This bachelor's thesis deals with the TRPV1 channel and its role in cells of the innate immune system, such as dendritic cells and macrophages. TRPV1 is a membrane ion channel that is activated by physical stimuli such as pH and temperature as well as by substances from the vanilloid group. The most significant of these is capsaicin. Activation of the TRPV1 channel has several effects on different types of cells. Based on the literature review, the bachelor's thesis concludes that the TRPV1 channel tends to lead dendritic cells to have an anti- inflammatory effect. However, there is evidence for both an anti-inflammatory and a pro-inflammatory response in macrophages and the role of TRPV1 is unclear in this cell type. Thus, further research is needed to understand the exact mechanisms of modulation of immune responses and to clarify the role of the TRPV1 channel. Key words: ion channels, TRPV1, capsaicin, dendritic cells, macrophages
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Role of amino acid residues in the first transmembrane domain in deactivation of P2X7 receptor
Knězů, Michal ; Zemková, Hana (advisor) ; Vlachová, Viktorie (referee)
P2X7 receptor is trimeric ligand-gated ion channel activated by extracellular ATP. This receptor is expressed in various tissues and cell types, such as glial cells, immune system cells or Schwann cells, different epithelial tissues and sperm cells, and is involved in many cellular a physiological processes such as is metabolism regulation, phagocytosis or apoptosis. The P2X7 subunit is composed of intracellular N- and C-termini, two transmembrane domains (TM1 and TM2) and a large extracellular ligand-binding domain. P2X7 has low sensitivity for its natural agonist, but prolonged or repeated applications lead to its sensitization and increase in current amplitude. Activation of P2X7 receptor is also known to induce uptake of large organic ions, such as fluorescent dye ethidium bromide. Receptor sensitization is accompanied by prolongation deactivation, mechanism of which is still unkown, that can be measured as current decay evoked by washout of agonist. This work explores the role of individual TM1 residues in deactivation kinetics of the receptor both in naïve and sensitized states. Electrophysiological whole-cell patch clamp method was used to record agonist-stimulated membrane current and it's decay of wildtype P2X7 receptor (P2X7-WT) and TM1 alanine or leucine mutants. Mutations that were...
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Cellular and molecular mechanisms of TRPC5 regulation
Ptáková, Alexandra ; Vlachová, Viktorie (advisor) ; Tureček, Rostislav (referee)
The TRPC5 ion channel is a molecular cold detector involved in the development of neuro- pathic and inflammatory pain in the peripheral nervous system. Its possible involvement in the mechanisms of the development of cold allodynia emerging as a side effect of chemotherapeutic treatment has not yet been investigated. The activation properties of the human TRPC5 receptor expressed in HEK293T cells in the context of exposure to low temperatures and oxaliplatin - widely used cytostatic drug, which side effects often manifest as a peripheral neuropathy trig- gered or enhanced by cold temperatures, were investigated using patch clamp electrophysiology and calcium imaging. Molecular modelling was used to explore possible mechanisms of action of oxaliplatin and lysophosphatidylcholine 18:1 whose levels are increased after oxaliplatin treatment. Low temperature (5 řC) decreased the amplitude of agonist-evoked membrane cur- rent responses and slowed down their deactivation. Analysis of single-channel recordings re- vealed increased open probability of the channel as well as prolonged mean open dwell time and reduced mean closed dwell time upon cooling. The highest temperature sensitivity of the gating of the channel was approximately between 16-11 řC with the corresponding value of the temperature...
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The role of aquaporins in the Alzheimer's disease
Kubísková, Monika ; Turečková, Jana (advisor) ; Vlachová, Viktorie (referee)
Alzheimer's disease (AD) is a progressive neurodegenerative disorder with complex pathophysiology affecting the central nervous system (CNS). In progress of the disease, various pathological changes occur in the brain, leading to neurodegeneration and subsequent impairment of physiological and cognitive functions. Although it is the most common cause of dementia in elderly, currently, there is no effective treatment for AD that that targets its underlying mechanisms. There are different theories as to which process is the key trigger for the development of AD. The generally accepted theory considers increased production of amyloid β (Aβ), its accumulation in the ECS and the formation of amyloid plaques as the main cause of the disease. However, recent studies show that the primary cause of amyloid plaque formation is not increased Aβ production, but rather its impaired clearance through the glymphatic system, the main component of which are aquaporin water channels, specifically aquaporin-4 (AQP4). The goal of this thesis is to provide an overview of the available knowledge on the involvement of aquaporins in AD pathophysiology, with a particular focus on AQP4 and its role in the glymphatic system. Key words: Alzheimer's disease, neurodegeneration, central nervous system, astrocytes, aquaporins,...
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Thermally gated TRP channels in nociceptive neurones
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Mrózková, Petra (referee)
Transduction ion channels are gated in response to a variety of external stimuli and this process is critical for the proper functioning of sensory neurons. These specialized proteins enable the survival of any organism, which depends on having adequate information about the external environment. The thermosensitive TRP (transient receptor potential) ion channels, whose molecular structure has been identified during last decades, enable the transduction of thermal stimuli in primary nociceptive neurons. During the last decade, molecular biological techniques have provided new tools for studying the structure of these specialized transduction ion channels in relation to their function and to understand more deeply their physiological roles. The aim of this bachelor thesis is to give an overview of recent evidence regarding the functional and physiological properties of sensory-neuron specific mammalian TRP ion channels that are activated by thermal stimuli: heat and cold.
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The role of nociceptive synaptic transmission modulation at the spinal cord level in different pain states
Adámek, Pavel ; Paleček, Jiří (advisor) ; Vaculín, Šimon (referee) ; Vlachová, Viktorie (referee)
Pain is a common symptom of many clinical syndromes and diseases. In particular, the treatment of neuropathic pain represents a serious public health issue because currently available analgesia is ineffective in many cases or it has adverse effects. Treatment of pain-related suffering requires knowledge of how pain signals are initially generated and subsequently transmitted by the nervous system. A nociceptive system plays a key role in this process of encoding and transmission of pain signals. Modulation of the nociceptive synaptic transmission in the spinal cord dorsal horn represents an important mechanism in the development and maintenance of different pathological pain states. This doctoral thesis has aimed to investigate and clarify some of the mechanisms involved in the modulation of the spinal nociceptive processing in different pain states. The main attention was paid to study the following issues: (I.) Which is the role of Transient Receptor Potential Vanilloid type 1 channels (TRPV1), Toll-Like Receptors 4 (TLR4), and phosphatidylinositol 3-kinase (PI3K) in the development of neuropathic pain induced by paclitaxel (PAC) chemotherapy in acute in vitro, and subchronic in vivo murine model of PAC-induced peripheral neuropathy (PIPN)? (II.) How is affected spinal inhibitory synaptic control...
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