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Inhibition of nicotinic acetylcholine receptors by tacrine and its derivatives
Skřenková, Kristýna ; Krůšek, Jan (advisor) ; Doležal, Vladimír (referee)
Nicotinic acetylcholine receptors are ligand-gated ion channels which are located on neuromuscular junction and in central and perifric nervous system. Activity of nicotinic receptor might be modulated by variety of pharmacological agents. In this work, we have focused on the study of the inhibition effect of tacrine and its derivatives on the nicotinic acetycholine receptors of muscle and neuronal type. These derivatives function as acetylcholinesterase inhibitors and also interact with nicotinic acetylcholine receptors. The majority of current forms of treatment of Alzheimer's disease is based on cholinesterase inhibitors. We have studied the mechanism of tacrine and its derivatives by using patch clamp method in the configuration of whole-cell recording. Powered by TCPDF (www.tcpdf.org)
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Muscarinic acetylcholine transmission and Alzheimer's disease
Janíčková, Helena ; Doležal, Vladimír (advisor) ; Blahoš, Jaroslav (referee) ; Rokyta, Richard (referee)
Impairment of the cholinergic neurotransmission system is regularly detected in animal models of Alzheimer's disease as well as in human patients suffering from this serious disease. Moreover, there is increasing amount of evidence suggesting that activation of individual mAChR subtypes specifically influences the cleavage of APP, the precursor for β-amyloid. APP can be processed in an amyloidogenic or non-amyloidogenic pathway and a relative abundance of these patways contributes to establishing the final concentration of neurotoxic β-amyloid in the brain. In this work, I have studied the acute and chronic effects of A β1-42 on binding and functional characteristics of mAChR. I have demonstrated that Aβ1-42 present in cell culture expressing the individual subtypes of mAChR negatively and specifically influences the function of the M1 mAChR subtype. I have also detected a decline in muscarinic receptor-mediated signal transduction in brain tissue of young adult APPswe/PS1dE9 mice, a commonly used animal model of Alzheimer's disease. Demonstration of the impairment of muscarinic transmissin in transgenic mice by soluble β-amyloid that occurs earlier than amyloid pathology and behavioral deficit, and its imitation by soluble Aβ1-42 in vitro lend strong support to the notion of the early involvement...
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Assesment of stress and psychotropic induced changes of trimetric G protein subunit levels and cellular functions in vitro and in vivo in the context of depressive disorder
Páv, Marek ; Kovářů, Hana (advisor) ; Höschl, Cyril (referee) ; Doležal, Vladimír (referee)
Depression is a complex systemic disorder with exhibiting biological, cognitive and psychopathological symptoms. There are changes in hormonal regulation, immune changes and disturbance in function of especially monoamine and indoleamine neurotransmitter circuits in the CNS. Pharmacologic intervention with antidepressants is treating depressive syndrome as well as homeostatic imbalance. These neurotransmitter systems use in the signal transduction from membrane into the cell receptors coupled with heterotrimeric G proteins (GTP binding proteins).This thesis studies changes in G protein subunit levels induced by stress and psychotropic drugs in the CNS and immune system of experimental mice and antidepressant induced changes in vitro in the C6 glioma cell line an in vivo in the rat tissue. Immobilization stress induces prominent changes in the G protein subunit levels in the spleen and CNS of experimental mice with analogical profile of response. Results show importance of stress dopaminergic component in the leukocyte function regulation and also demonstrate importance of dopaminergic regulation without stress exposition. Findings obtained in the glioma C6 cell line show antidepressant induced drug specific changes of the G subunit profiles and demonstrate thus mode of action independent on the...
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Cholinergic neurons and Alzheimer's disease
Machová, Eva ; Doležal, Vladimír (advisor) ; Krištofíková, Zdena (referee) ; Zvolský, Petr (referee)
Alzheimer's disease is a degenerative brain disorder. The incidence of the disease increases with age and every year the total number of affected persons is higher. The malfunction of various mental functions is a typical feature of Alzheimer's disease, including loss of short-term memory and development of personality changes. It is now generally accepted that the main cause of the disease is increased production of r3-amyloid fragments that mediate toxic effects and lead to r3-amyloid plaques formation. Alzheimer's disease and also normal aging are accompanied by a loss of cholinergic neurons and weakened cholinergic neurotransmission. In my thesis I dealt with changes in the brain chol inergic system during aging in control mice and in a mouse model of Alzheimer's disease (APPswe/PS1 dE9). In this context I also investigated in vitro influence of docosahexaenoic acid (w-3 essential fatty acid) changes in membrane cholesterol content on the expression of cholinergic phenotype in the NG108-1 5 cholinergic cell line. I also performed ex vivo experiments on rat brain cortex to investigate the characteristics of action of muscarin ic agonist xanomeline that was developed as a selective muscarinic agonist to strengthen signalization through the muscarinic M1 receptor in Alzheimer's disease. The experiments on...
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The role of conserved residues in the S4/S4-S5 region of vanilloid TRP channels
Boukalová, Štěpána ; Vlachová, Viktorie (advisor) ; Doležal, Vladimír (referee)
The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat or highly depolarizing voltages. In TRPV1 channel, the charged residues in the S4 region and intracellular S4-S5 linker have been proposed to constitute a part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. Molecular basis of this gating event are poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and related vanilloid receptors to identify potential voltage-sensing residues. The functionality of most of the TRPV1 mutants was altered with respect to voltage, capsaicin, heat and/or their interactions. We identified two amino acid residues (R557 and D576) that could potentially constitute part of TRPV1 voltage sensor. The non-functional charge-reversing mutations R557E and R579E were partially rescued by charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2aminoethoxydiphenyl...
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Expression of cholinergic gene locus in a mouse model of Alzheimer's disease
Zimčík, Pavel ; Doležal, Vladimír (advisor) ; Teisinger, Jan (referee)
(anglický jazyk) The most common senile dementia, Alzheimer disease (AD), is characterized by a decline of memory and high cognitive functions. Typical post-mortem brain lesions are extracellular amyloid deposits, intracellular neurofibrilary tangles and ruined cholinergic and other neurotransmitters systems. Connection between damaged central cholinergic system and beta-amyloid accumulation remains obscure. We examined parietal cortex of young adult (7- month-old) female APPswe/PS1dE9 double transgenic mice which develope beta-amyloid fragments at high rate. Cholinergic synapses of these mice demonstrate functional presynaptic (stimulated acetylcholine release) as well as postsynaptic (muscarinic receptor-induced G- protein activation) deficits and reduction of cholinergic markers. The mRNA levels of choline acetyltransferase, vesicular acetylcholine transporter and M1 to M4 subtypes of muscarinic receptors were determined in transgenic and littermate controls using qPCR. Obtained experimental data does not show any changes in measured mRNA levels. These observations indicate that reduction of cholinergic synaptic markers and function is due to posttranscriptional events.
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Gen expresion of high affinity choline transporter in mouse model of Alzheimer's disease
Kurfürst, Helena ; Krištofíková, Zdena (referee) ; Doležal, Vladimír (advisor)
: Choline is being used in all mammalian cells as a precursor for synthesis of a major phospholipide phosphatidylcholine and as a donor of acetyl residues. Cholinergic neurons in addition require choline to synthesize neuromediator acetylcholine. The ability of cells to create choline via de novo synthesis is limited and therefore they need to transport choline from extracellular space. Limited availability of choline in brain leads specifically to diminished function of cholinergic neurons and in general to impaired reparation of biological membranes. Dysfunctions of cholinergic signaling in brain is characteristic for Alzheimer's disease. Aim of this work was to investigate whether gene and protein expression of high- affinity cholinergic transporters is altered in 5-6 months old APPswe/PS1dE9 mouse model of Alzheimer's disease. Expression of specific high-affinity cholinergic transporter CHT1 (responsible for transport of choline to be used for acetylcholine synthesis) and putative high-afinity choline transporter CTL1 (generally present in all cells and related to high affinity choline transport for phospholipide synthesis) in cerebral cortex was measured. Compared to non-trangenic littermates, no changes in the expression of both genes were detected at either mRNA (quantitative PCR) or protein...
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