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Cellular and molecular mechanisms of TRPC5 regulation
Ptáková, Alexandra ; Vlachová, Viktorie (advisor) ; Tureček, Rostislav (referee)
The TRPC5 ion channel is a molecular cold detector involved in the development of neuro- pathic and inflammatory pain in the peripheral nervous system. Its possible involvement in the mechanisms of the development of cold allodynia emerging as a side effect of chemotherapeutic treatment has not yet been investigated. The activation properties of the human TRPC5 receptor expressed in HEK293T cells in the context of exposure to low temperatures and oxaliplatin - widely used cytostatic drug, which side effects often manifest as a peripheral neuropathy trig- gered or enhanced by cold temperatures, were investigated using patch clamp electrophysiology and calcium imaging. Molecular modelling was used to explore possible mechanisms of action of oxaliplatin and lysophosphatidylcholine 18:1 whose levels are increased after oxaliplatin treatment. Low temperature (5 řC) decreased the amplitude of agonist-evoked membrane cur- rent responses and slowed down their deactivation. Analysis of single-channel recordings re- vealed increased open probability of the channel as well as prolonged mean open dwell time and reduced mean closed dwell time upon cooling. The highest temperature sensitivity of the gating of the channel was approximately between 16-11 řC with the corresponding value of the temperature...
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Cellular mechanisms of TRPA1 channel regulation
Barvíková, Kristýna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee)
TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...
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Cellular mechanisms of TRPA1 channel regulation
Barvíková, Kristýna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee)
TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...
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The role of protein kinase C in the pathogenesis of insulin resistance and its complications
Marková, Irena ; Novák, František (advisor) ; Zeman, Miroslav (referee) ; Zídková, Jarmila (referee)
18 effects of TZDs are probably due to the remodeling of adipose tissue and increased adiponectin secretion. SUMMARY Studying the pathogenesis of insulin resistance and the role of PKC in insulin resistance In HHTg rats, elevated serum triglycerides and FFA were associated with the ectopic accumulation of triglycerides in tissues and reduced insulin sensitivity of peripheral tissues. Impaired glucose utilization in the peripheral tissues was associated with the reduced activity of GS in skeletal muscle. Decreased GS activity and glucose utilization in peripheral tissues indicate a possible defect in insulin signal transduction. In line with this, our results show that skeletal muscle IR was associated with the increased activation and translocation of PKC θ. Nutritionally induced obesity of HHTg rats resulted, in many cases, in the further deterioration of metabolic abnormalities associated with IR. We found that PKC θ, in particular, could contribute to the metabolic abnormalities associated with IR and obesity. The age-related increase in IR and deterioration of some parametres of carbohydrate and lipid metabolism, were not associated, in HHTg rats, with obesity but with increased serum levels of triglycerides and FFA. The age-related worsening of IR in HHTg rats was accompanied by increased...
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