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Role of the cannabinoid system in neurodegenerative processes
Akantisová, Lucia ; Novotný, Jiří (advisor) ; Búran, Peter (referee)
The endocannabinoid system (ECD) is involved in a significant number of physiological functions in the central and peripheral nervous system. The ECD consists of cannabinoid receptors (CB1 and CB2), endogenous ligands (ANA, 2-AG) and the enzymatic apparatus required for their synthesis (NAPE and DAGL) and degradation (FAAH, MAG). In the human brain, the cannabinoid receptor CB1 is the most widespread of the group of receptors that bind to G proteins. The signaling mechanisms of these proteins contribute to the overall homeostasis of the organism. With their activity, they affect the concentrations of second messengers, the activity of ion channels, the release of neurotransmitters, and regulate immune responses. In this context, studies in the last 20 years have focused on research in the field of neurodegenerative diseases. Today, i tis known that treatment with cannabinoid compounds improves neurological deficits associated with neuronal damage and alleviates inflammatory processes in animal models of Alzheimer's disease, Parkinson's disease and multiple sclerosis. At the clinical level, treatment with cannabinoids has helped with certain accompanying symptoms occuring in neurodegenerative diseases such as neuropathic pain, insomnia and spasticity. Key words: endocannabinoid system, cannabinoids,...
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Effect of chronic morphine treatment of rats on myocardial signaling systems regulated by trimeric G-proteins
Škrabalová, Jitka ; Novotný, Jiří (advisor) ; Rudajev, Vladimír (referee)
It has recently been discovered that the effect of morphine can significantly reduce the tissue damage that occurs during myocardial ischemia. The molecular mechanisms by which morphine acts on the heart are still little understood. The aim of this thesis was to monitor the effect of chronic 27-day and 10-day administration of low (1 mg/kg/day) and high (10 mg/kg/ day) doses of morphine on the expression of selected G-protein-coupled receptors (GPCR) and on the expression and activity of adenylyl cyclase (AC). Chronic (27 days) morphine treatment reduced the expression of к-opioids receptors, but 10-day morphine exposure did not influence the expression of these receptors. Assessment of β1- and β2-AR by immunoblot technique did not show any significant change in the expression, but the more accurate determination of β-AR expression using the saturation binding studies revealed that 27-day treatment with high doses of morphine appreciable increased the total number of these receptors. Administration of high doses of morphine led to marked up-regulation of adenylyl cyclase (AC) isoforms V/VI, and the amount of AC decreased proportionally with the time of discontinuation of morphine administration. Low doses of morphine up- regulated AC only during 27-day administration. Chronic morphine exposure did...
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka ; Novotný, Jiří (advisor) ; Nováková, Olga (referee) ; Hlaváčková, Markéta (referee)
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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A study of adenosine receptors and their signaling in the rat myocardium
Eichlerová, Lenka ; Novotný, Jiří (advisor) ; Nováková, Olga (referee)
Adenosine plays a critical role in the heart signalling while affecting heart rate, contractility or coronary flow. Nowadays, four adenosine receptor subtypes are distinguished which are present in most of tissues and cells: A1, A2A, A2B and A3. All these receptors belong to the family of G protein-coupled receptors. Upon activation, their main target is an enzyme adenylyl cyclase which produces an important second messenger cAMP. The main goal of this thesis was characterization of adenosine receptors in the rat myocardium, assessment of their distribution, binding properties and signalling. We examined a possible disparity in receptors distribution between the left and right ventricles using SDS-PAGE electrophoresis and Western blotting. The same methods have been used in studies of adenosine receptor distribution in lipid rafts. Samples of lipid rafts and soluble fraction were prepared using a nonionic detergent Triton X-100. We did not find any evidence of different distribution between the left and right ventricles and our results did not confirm compartmentation of the receptors either. For determination of binding properties of the receptors we used radioligand binding assays with the A1 selective radioligand [H3 ]DPCPX. We did not observe any significant difference between the receptor...
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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The effect of morphine withdrawal on the adenylyl cylclase signaling system in rat brain
Nováková, Daniela ; Novotný, Jiří (advisor) ; Rudajev, Vladimír (referee)
The aim of this bachelor thesis is to study the effect of morphine withdrawal on the adenylyl cyclase signal system in the rat brain. Theoretical part of this thesis summarizes basic information known about opioids and its leading representative morphine, molecular mechanisms of its effect on the organism and yet determined knowledge about effects of morphine addiction and subsequent withdrawal on the organism and above all on the central nervous system. There is still much unknown about exact mechanism of development of morphine addiction and withdrawal. This thesis also summarizes briefly the Western blot method used in analysing the effect of morphine withdrawal on selected proteins of the adenylyl cyclase signal system in the rat brain. Experimental part of this thesis is based on the detection of expression of key components of the adenylyl cyclase pathway in selected regions of the rat brain after long-term administration and subsequent withdrawal of increasing doses of morphine. Results of this study did not reveal statistically significant differences in the expression of adenylyl cyclase signaling system components between the withdrawal and the corresponding control groups of animals, confirming high ability of the organism to withstand morphine administration and to return to...
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka ; Novotný, Jiří (advisor) ; Nováková, Olga (referee) ; Hlaváčková, Markéta (referee)
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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Beta-adrenergic receptors and their desensitization
Biriczová, Lilla ; Novotný, Jiří (advisor) ; Kolář, David (referee)
β-Adrenergic receptors (β-ARs) are G-protein-coupled receptors (GPCR), widely present in the animal organism and mediate catecholamine pathways leading to diverse physiological responses. The family of β-ARs consists of β1-AR, β2-AR and β3-AR, which are distinguished by their affinity to adrenaline and noradrenaline. A typical model of β-AR signalling includes binding of the ligand, G-protein coupling, activation of adenylyl cyclase (AC) resulting in production of the second messenger cAMP and activation of protein kinase A (PKA) that phosphorylates downstream proteins leading to physiological responses. Beacause excessive catecholamine signalling can cause undesirable consequences, a mechanism has evolved, which attenuates the function of β-ARs in spite of further stimulation, so called desensitization. The classic course of desensitization consists of characteristic steps including phosphorylation of the receptor, β-arrestin attachement and uncoupling of the G-protein from β-AR. Restoration of the signalling ability is allowed through resensitization of β-AR when the receptor is sequestrated and dephosphorylated. Given that β-ARs are structurally and genetically different, it is reasonable to consider that each step of the desenstization process may happen differently among the different subtypes...
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Compartmentalization of the beta-adrenergic signaling system in cardiac cells: the effect of hypoxia
Karlovská, Ivana ; Novotný, Jiří (advisor) ; Nováková, Olga (referee)
The aim of this thesis was to study the changes that occur in cell line H9c2 after exposure to an oxygen level reduced to 2 % for 24 hours. We monitored changes in compartmentation of chosen members of β-adrenergic signaling system. We found an increase in expression of β1AR and β2AR. Only β2AR showed change in compartmentation after hypoxia, as they relocate from membrane rafts to non-rafts fractions of membrane. AC also showed an increase of expression and was located in membrane rafts. The next aim of this work was to monitore apoptotic markers to determine whether there are activated pro-apoptotic or anti-apoptotic signals under chosen conditions of hypoxia. There was an increase in expression of both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. We compare ratios of Bcl-2 to Bax and we found that there is a bigger increase in protein Bax expression. Another apoptotic marker, caspase 3, was tested and we also found that there was an increase in expression of caspase 3 in cells after hypoxia. Furthermore, we studied possible activation of kinase signaling pathways that may contribute to protective effects of hypoxia. Expression of Akt and ERK kinases was increased after hypoxia, but we did not confirm activation by phosphorylation of these kinases. Levels of phosphorylated Akt...
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