The aim of this diploma thesis is to study the effect of morphine withdrawal on catecholaminergic and serotonergic neurotransmitter system in rat brain. Theoretical part of this thesis summarizes basic information known about principles of neurotransmission with focus on the catecholaminergic and serotonergic system, metabolism of its components, their signaling, relevant receptors, their distribution, and especially their effect on morphine dependence and subsequent withdrawal. It also summarizes briefly principles of opioid signaling and outlines the findings yet known about neurochemical analysis of the transmitter systems mentioned above. Experimental part of this thesis is focused on the optimization of the method of high performance liquid chromatography with fluorescence detection and its subsequent use to determine basic components of catecholaminergic and serotonergic neurotransmitter system in samples of different part of brain of rats affected by intraperitoneal administration of morphine sulphate, and its subsequent withdrawal and correspondings unaffected control rats. The expression of selected catecholaminergic receptors in identical samples is also detected. This thesis succesfully implements gradient into the originally isocratic method of high performance liquid chromatography...

Morphine is an opioid agonist, which can exert cardioprotective effects under certain conditions. Lipid rafts are considered important platforms for membrane organization of signaling proteins and, therefore, these structures could play a role in the effects of morphine, which acts through the opioid receptors. The aim of this thesis was to investigate the distribution of the main components of the opioid receptor and Gi/o-mediated signaling pathway in lipid rafts isolated from rat myocardium, which was affected by various doses of morphine. Because we used different isolation techniques with different solubilization agents (Triton X-100, CHAPS, cholate and sodium carbonate) for preparation of lipid rafts, it was of interest to characterize more closely these preparations. Another aim of this study was to investigate how different methods of isolating these structures affect activity of the key target enzyme of the opioid signaling pathway, i.e. adenylyl cyclase. The presence of signaling molecules of the Gi/o/AC pathway of the opioid system in membrane rafts was confirmed and the distribution of selected proteins was dependent on the type of extractant. We also observed the effect of morphine on the localization of proteins in lipid rafts. Different extractants provided different degree of...

G protein-coupled receptors (GPCRs) are the largest group of membrane-bound receptors. Transmission of signals into the cell interior is mediated through the interactions of these receptors with other signaling molecules. Nowadays, a great attention is devoted to biased ligands which are able to alter the conformation of the receptor in a specific way and thus distinctly affect its function. This diploma thesis was focused on a study of µ-opioid receptor (MOR), which is important in nociception. The aim of this study was to find out, how the activation of MOR by specific biased ligands (morphine, endomorphin-2 and DAMGO) affects the function and the interactions of MOR with potential molecular partners (for example G proteins or β-arrestin) A method of siRNA interference was used to knock down the following selected signaling molecules: Gαi1, Gαi2, Gαi3, Gαz and β-arrestin2. The effect of biased ligands on lateral mobility of MOR in the plasma membrane and on activity of adenylyl cyclase (AC) was examined under these conditions. We observed a possible involvement of Gαz subunit in the lateral mobility of MOR after the effect of morphine and endomorphin-2. The lateral mobility of MOR was significantly increased in cells lacking Gαi2 or Gαi3 or β-arrestin2. In this case the MOR was in inactive state....

The diploma thesis is devoted to research state and cure of morphinists and cocainists in lunatics asylums in Bohemia, Moravia, Silesia and Styria in period from 1867 to 1918. The thesis also deal with issue of how often was cure by morphin or cocain in the individual territories, how lunatics asylums cured patient and what was the main reason to illness with morphinismus or cocainismus. For Bohemia was chosen lunetics asylum in Kosmonosy (Zemský ústav pro choromyslné v Kosmonosech), for Moravia in Brno (Zemský ústav pro choromyslné v Brně), for Silesia in Opava (Slezský zemský ústav pro choromyslné v Opavě) and for Styria in Graz (Landes-Irrenanstalt Feldhof bei Graz). Using the issued and unreleased archivel sourses and professional literature.

Morphine is a natural opioid which is used in medicine due to his potent analgesic and sedative effects. In the forefront of scientific interest is a chronic usage of opioids which can lead to a development of drug addiction. Morphine role in oxidative stress was described in last years. It was revealed its protective potencial by many studies. However, some studies described its pro-oxidative effect. The aim of this study was to determinate effect of chronic morphine on cell survival after oxidative stress caused by H202 analog - tBHP in the SH-SY5Y neuroblastoma cell line. The results verified morphine protective effect against oxidative stress. The highest protective effect of morphine was achieved in a concetration of 10 µM. It was desribed that morphine can induce activation of mu-opioid (MOR) and Toll-like 4 (TLR4) receptors signalling pathway on molecular level. The aim of this thesis was to evaluate the role of MOR a TLR4 in protective effect of morphine against oxidative stress by two methods. Firstly, it was used tests of oxidative stress on cell viability. The obtained results demonstrated majority role of TLR4 and minory role of MOR. Afterwards, we assesed changes in the expression of MOR a TLR4 after chronic morphine by SDS-PAGE electrophoresis. Results of these experiments did not...

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...

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...

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.

Infusion pumps administer medications like morphine to pediatric patients in order to manage pain. Drug delivery delays can be the result of flow rate variabilities in the infusion pump system. Due to the risk of over-or underdosing, this could have a high impact on the pediatric population. This study’s aim is to investigate the effect of drug dilution and dosing delays by investigating which factors affect the morphine concentration in the patient. Implementation of a previously developed population pharmacokinetic model was performed in MATLAB. Then combining it with a self-developed model of the infusion pump system, a model which included the infusion pump and the system between the pump and the patient. Simulations were performed to investigate the contributing factors. The results show that dosing delays decrease with an increasing patient’s age. There are larger morphine concentration variations with lower syringe flow rates. A decrease in dosage and a smaller syringe volume result in a shorter time of reaching a steady state concentration. Using the wrong syringe which is not compatible with the machine will lead to an increasing morphine concentration in the patient that does not reach a steady state. A limitation of the study was that no clinical data was used for the simulations. These results are useful for clinicians when making decisions regarding intravenous administration of morphine, potentially leading to fewer medication errors. / Infusionspumpar administrerar läkemedel som morfin till pediatriska patienter för smärtlindring. Fördröjning av läkemedelstillförsel kan vara resultatet av flödeshastighetsvariationer i infusionspumpsystemet. På grund av risken för över- eller underdosering kan detta ha en stor inverkan på den pediatriska populationen. Denna studies syfte är att undersöka effekten av läkemedels- utspädning och -fördröjning genom att undersöka vilka faktorer som påverkar koncentrationen av morfin i patienten. Implementering av en tidigare utvecklad populationsfarmakokinetisk modell gjordes i MATLAB . För att sedan kombinera den med en egenutvecklad modell av infusionspumpsystemet, en modell som inkluderade infusionspumpen och systemet mellan pumpen och patienten. Simuleringar utfördes för att undersöka de bidragande faktorerna. Resultaten visar att doseringsfördröjningar minskar med patientens stigande ålder. Det finns större koncentrationsvariationer med lägre sprutflödeshastig- heter. En minskning av dosen och en mindre sprutvolym resulterar i en kortare tid för att uppnå en steady state-koncentration. Användning av fel spruta som inte är kompatibel med maskinen kommer att leda till en ökad morfinkon- centration hos patienten som inte når ett stabilt tillstånd. En begränsning med studien var att inga klinisk data användes för simuleringarna. Dessa resultat är användbara för läkare när de fattar beslut om intravenös administrering av morfin, vilket potentiellt kan leda till färre medicineringsfel.