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Molecular mechanisms of activation and modulation of TRPV3 receptor
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Novotný, Jiří (referee)
Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...
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Behavioral expression of pain in rats
Zámečník, Jakub ; Vaculín, Šimon (advisor) ; Boukalová, Štěpána (referee)
The aim of this work is to summarize the current knowledge regarding the behavioral expressions of pain in rat. The work is focused on rats, because they are one of the most used laboratory animals, and may experience pain in some experiments. Behavioral expressions of pain were selected, because they are quite easily recognizable and measurable. The reader will be first acquainted with the necessary basics associated with the phenomenon of pain, with its conception in animals, with classification, physiology and for example with the ways in which are in rats experimentally induced pain. Then in the second part, there are shown individual behavioral expressions with examples in which cases were these manifestations observed. So everyone who is interested can learn something about pain in connection with the avoidance reaction, pose, appearance, specific movements of rat, vocalization, changes in eating and drinking, changes in activity, anxiety, cognitive functions and many other expressions. The work can be valuable contribution for all workers with animals and with its character can contribute to animal welfare. Powered by TCPDF (www.tcpdf.org)
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Methods for measuring pain in animals
Štěrbová, Petra ; Vaculín, Šimon (advisor) ; Nerandžič, Vladimír (referee)
Pain in animals was almost completely overlooked in the last years. It was assumed that animals do not suffer because they are not able to tell it anyway. In a recent time we know that it's not truth and the animals can physically feel pain as we do. This thesis is summarization of the basic knowledge about the pain measurement of animals. However, there are two different approaches to the measurement of pain. The first one is based on the threshold of pain, the another one observes changes in behavior. There have been number of animal models developed in order to pain detection. Exploration of the pain in animals can help us to clarify mechanisms and treatments for serious and chronic neuropathic pain. From an ethical point of view, it's almost impossible to expose a person to serious neuropathic injury for experimental purposes. Therefore, in these cases, the animals are commonly used. And besides this fact, nowadays animals represent a full member of the family for many people, so it is something like moral obligation for us to relieve a pain in animals.
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The role of synaptic modulation in pain states.
Adámek, Pavel ; Paleček, Jiří (advisor) ; Moravec, Jan (referee)
Everybody has experienced pain. Pain by definition is an unpleasant sensory and emotional experience associated with actual or potential tissue damage. In the peripheral tissues acute painful stimuli activate specialized endings of afferent neurons called nociceptors. The information about tissue damage is then transmitted to the cell bodies of these dorsal root ganglion neurons by unmyelinated or thinly myelinated axons (C and A fibers, respectively). The central branches of these neurons form synapses with superficial dorsal horn neurons in the spinal cord. The information is conveyed at the synaptic connections by neurotransmitters such as glutamate and many others neuromodulators. Important is the subsequent activation of projection neurons that transmit the information to supraspinal brain areas. Activity of excitatory and inhibitory interneurons, glial cells and descending pathways from the CNS are also important for the modulation of nociceptive information at the spinal cord level. After peripheral tissue damage and in other pathological states, increased sensitivity to peripheral stimuli may develop. As results of this change innocuous stimuli are perceived as painful (alodynia) and increased pain is perceived after noxious stimuli (hyperalgesia). The underlying mechanisms of these changes may be...
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Physiological significance of pain
Zámostná, Kateřina ; Vaculín, Šimon (advisor) ; Telenský, Petr (referee)
Pain is defined as a physiological process associated with negative emotional experience which warns our body about impending damage. Apart from neuropathic pain, which by definition has no purpose, pain in effect provides a defensive function. However, this role is already being fulfilled by a different mechanism - the defensive reflex, which is evolutionarily older. Therefore we can ask, whether pain is only an emotional experience attached to this reflex, or if it has its own functions. To determine the physiological purpose of pain a rare syndrome of congenital analgesia, characterized by complete absence of pain, will be used. Based on a comparison of clinical cases of the syndrome with a population without pain disorders individual functions of pain will be described and connected to the individual components of pain - sensory- discriminatiory and affective-motivational. The sensory-discriminatory component provides a defensive, diagnostic and an informative function. The defensive function is analogous to the defensive reflex. The informative function transmits information about a physiological process, where pain plays a key role - labour. Diagnostic function applies in medicine and is based on verbal description of the defensive function. Functions of the SD component are involved at the time of...
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Joint configuration changes pressure pain threshold-centered and decentered position
Jevič, Filip ; Čech, Zdeněk (advisor) ; Vaculín, Šimon (referee)
The term "joint centration" is recently perceived primarily in its biomechanical context. This paper suggests a possible neurophysiological point of view and examines the effect of centered or decentered position on pressure pain treshold (PPT) of three muscles (m. temporalis, m. tibialis anterior, m. interosseus dorsalis I) in sitting and lying position, researched on healthy volunteers. PPT was measured in four randomly ordered positions with 49 people (21 women, 28 men). Significantly higher PPT was detected in all three muscles (p=0,001; 0,0016; 0,00009). In percent the PPT change resembles some of studies working with therapeutical techniques (mobilization, joint manipulation, exercise). Influence of higher postural position (sitting compared to lying down) on PPT was not proved. Cenetered joint position increases the PPT of healthy young adults compared to decentered position. This newly proved fact opens further possibilities of conceiving the key physiotherapeutical notion of "joint centration".
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Modulation of synaptic transmission, studies on spinal cord slices in vitro
Mrózková, Petra ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of a synaptic transmission in the spinal cord dorsal horn plays a key role in nociceptive signalling, especially in states of pathological pain. The goal of this study was to develop a method for calcium imaging in spinal cord slices in vitro. This method allowed us to record changes of intracellular free calcium ions concentration (iCa2+ ), that are a major mediator of neuronal plasticity. In this work, we have focused on application of this method in a conventional fluorescence microscope and on the role of different neuromodulators of synaptic activity. Changes of iCa2+ induced by dorsal root electrical stimulation were recorded altogether in 744 dorsal horn (lamina I and II) neurons. In the first series of experiments, stimulation protocols activating preferentially A and A + C dorsal root fibers were used and long-term stability of the calcium responses was verified. The dorsal root stimulation induced in the neurons fast and delayed type of calcium response. Application of AMPA and NMDA receptors antagonists, CNQX (50μM) and MK801 (45μM), reduced the calcium response amplitude and confirmed the importance of glutamate receptors in synaptic activation. In several experiments the effect of capsaicin a TRPV1 receptors agonist, application was tested. Application of even low...
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Pathological pain states, the role of synaptic modulation at spinal cord level
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...
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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
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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