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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan ; Honys, David (advisor) ; Paleček, Jan (referee) ; Smýkal, Petr (referee)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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The role of nociceptive synaptic transmission modulation
Heleš, Mário ; Paleček, Jiří (advisor) ; Rokyta, Richard (referee) ; Krůšek, Jan (referee)
Pain represents a major symptom in a multitude of medical conditions and can often become the main negative factor in a patient's low quality of life. The complex issue of pain management is further underscored by the reduced efficacy of conventional analgesics in conditions such as neuropathic pain. Neuropathic pain, unlike acute nociceptive pain, originates from damage to the peripheral or central nervous system and often develops into chronic pain syndrome. Most analgesics available today provide only limited and unsatisfactory analgesia in chronic neuropathic pain and are often associated with severe adverse effects. Modulation of nociceptive transmission in spinal cord dorsal horn (SCDH) stands out in recent research as a pivotal mechanism, especially in chronic pain development and maintenance. The major aim of this doctoral thesis was to investigate how pain-associated processes interfere with opioid-induced analgesia, with the main focus on the interaction between chemokine (C-C motif) ligand 2 (CCL2), transient receptor potential vanilloid type 1 (TRPV1), and μ-opioid receptor (MOR). To achieve a better insight into opioid signaling in SCDH we studied the following issues: (I.) How does CCL2 modulate MOR-mediated effects on nociceptive synaptic transmission in SCDH neurons and in vivo...
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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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název v anglickém jazyce není uveden
Pospíšilová, Eva ; Paleček, Jiří (advisor) ; Kršiak, Miloslav (referee) ; Rokyta, Richard (referee)
Tissue injury leads to increased sensitivity to noxious and innocuous stimuli due to mechanisms of peripheral sensitisation of primary nociceptors and central sensitization of neurons in the spinal dorsal horns. The subpopulation of capsaicin - sensitive sensory neurons plays an important role in this process. The capsaicin - sensitive neurons express capsaicin TRPV1 receptors (transient receptor potential 1) on their peripheral and central terminals, local high concentration capsaicin treatment can induce regional destruction of these endings. It is well estabilished that TRPV1 receptors play the key role in neural transmission of nociceptive information and its modulation. The aims of this study were to investigate the role of capsaicin - sensitive primary afferent fibers and the involvement of peripheral and central TRPV1 receptors in the development of hypersensitivity after surgical tissue trauma, to test the effect of high concentration capsaicin and the specific TRPV1 antagonist treatment on postoperative pain and to enlighten the function of central TRPV1 receptors in the neural mechanisms of nociception. Using behavioral testing methods, the responses to mechanical (Von Frey filaments) and thermal stimuli (radiant heat source) were tested on the rat plantar incision model of surgical pain...
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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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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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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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Modulation of synaptic transmission in the development of painful states
Slepička, Jakub ; Paleček, Jiří (advisor) ; Hejnová, Lucie (referee)
My thesis introduces the topic of nociceptive signalisation and processes involved in the formation and spreading of neuropathic pain. This study focuses on the mechanisms of nociceptive synaptic transmission mechanisms in the level of spinal dorsal horn and its modulation by paclitaxel, a chemotherapeutic drug inducing neuropathic changes. The attention is put especially on the possibility of glial activity participation in paclitaxel side effects. This idea stems from the existing hypothesis of the functional connection between TLR4 and TRPV1 receptor activity. TRPV1 is well known for its participation in chemical, thermal and nociceptive sensory transmission. Minocycline antibiotic is considered as an inhibitor of microglial activation therefore it was used for blocking neuroinflammation. The experimental part is comparing an impact of substances applied to the model of tachyphylaxis used for monitoring of nociceptive transmission changes according to decreasing activity of TRPV1 receptors. Electrophysiological recording of miniature excitatory postsynaptic currents from neurons in the Rexed laminae I. and II. of spinal dorsal horn was used. The results of my measurements show that minocycline is able to suppress acute effects of paclitaxel application in vitro if the spinal slice is incubated...
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The role of TRPV1 receptors in chemokine CCL2 induced modulation of nociceptive synaptic transmission at spinal cord level
Adámek, Pavel ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn is a significant mechanism in the development and maintenance of different pathological pain states. Accumulating evidence indicates that the TRPV1 (transient receptor potential vanilloid 1) receptor and chemokine CCL2 (C-C motif ligand 2) may play a critical role in this process. The aim of this diploma thesis was to investigate the CCL2 induced modulation of nociceptive synaptic transmission in the dorsal horn of spinal cord and the role of the TRPV1 receptors. To investigate this aim patch-clamp recordings of spontaneous and miniature excitatory postsynaptic currents (sEPSC, mEPSC) from superficial dorsal horn neurons in acute rat lumbar spinal cord slices were used. After acute application of CCL2 on the slice preparation from naïve animals, a frequency increase of both sEPSC and mEPSC was present. This CCL2 induced increase in both sEPSC and mEPSC frequency was prevented by the TRPV1 receptor antagonist SB366791 application. No changes were observed in the amplitudes of sEPSC or mEPSC after application of the CCL2, SB366791, or co-application of CCL2 and SB366791. This suggests that the observed changes were mediated predominantly by presynaptic mechanisms. The preliminary results indicate that after chronic constriction...
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