|
|
Modulation of RNA demethylase FTO function in SH-SY5Y cells: the effect on insulin signaling and mitochondrial respiration
Čočková, Zuzana ; Novotný, Jiří (advisor) ; Hlaváčková, Markéta (referee)
Aim of this thesis was to observe changes in oxidative metabolism and expression of important neuroenergetic proteins in human neuroblastoma cell line SH-SY5Y due to inhibition of FTO. FTO is a RNA demethylase that uses N6-methyladenosine as substrate. Differences in enzyme expression are connected to broad area of effects involving energy homeostasis. Mitochondria are cellular powerhouses, a key elements in production of energy and metabolic substrates, yet a source of potentially dangerous reactive oxygen species (ROS) and analogous reactive molecules. In order to better understand FTO purpose in neuronal energetic metabolism, we examined mitochondrial respiratory chain. Using high-resolution respirometry we were capable of observing impairment in mitochondrial respiration after FTO inhibition. There was considerable decline in endogenous respiration, maximal respiration rate and reserve capacity. In order to obtain more detailed view into mitochondrial respiration, expression levels of electron-transport complexes were quantified by Western blot technique. Slight reduction was identified in subunits of complex I and IV. However, the most prominent alteration was seen in complex II subunit. There were no differences in expression of complex III and ATP synthase subunits. Beside disrupted activity...
|
|
|
The role of mitochondria in cardioprotective effect induced by hypoxia in rat
Lomnický, Matouš ; Žurmanová, Jitka (advisor) ; Hlaváčková, Markéta (referee)
Aerobic organisms need sufficient oxygen supply to maintain homeostasis. These organisms are frequently exposed in hypoxic environments naturally, and also occur in hypoxic states in various pathological conditions. Cardioprotective effect of hypoxia had been recognised more than 30 years ago; and later on, cardioprotective effects of ischemic preconditioning were discovered. Long term exposure to hypobaric hypoxia activates cardioprotective mechanisms, which lower the aftermathes of short term ischemia of myocardia and the effects of further health complications. The core of protective mechanisms has not yet been fully clarified. This work deals with the significance of mitochondria on cardioprotection during hypobaric hypoxia adaptation. This work describes physiological adaptive processes on selected animals on natural hypoxic conditions and also molecular mechanisms, examined on experimental models. Molecular mechanisms of the origins of cardioprotective effects discovered so far, mainly indicate PKC signal pathways through thyrosine kinase and mitogenes of activated kinase and also indicate an activation of sarcKATP-channels and mitoKATP-channels. Opening of these channels can protect mitochondria against a Ca2+ overload, or can lead to an increase in mitochondrial capacity which is possibly connected...
|
|
|
Role of protein kinase C isoforms in cardioprotective mechanism of chronic hypoxia
Hlaváčková, Markéta
Cardiovascular diseases, particularly acute myocardial infarction, are one of the leading causes of death in developed countries. It is well known that adaptation to chronic intermittent hypobaric hypoxia (IHH) confers long-lasting cardiac protection against acute ischemia/reperfusion injury. Protein kinase C (PKC) appears to play a role in its cardioprotective mechanism since the administration of general PKC inhibitor completely abolished the improvement of ischemic tolerance in IHH hearts. However, the involvement of individual PKC isoforms remains unclear. Therefore, the primary aim of this study was to investigate the potential involvement of PKCδ and PKCε, the most prevalent PKC isoforms in rat heart, in the mechanism of IHH-induced cardioprotection. We showed that IHH up- regulated PKC protein in left ventricle, enhanced its phosphorylation on Ser643 and increased its co-localization with markers of mitochondrial and sarcolemmal membranes. PKCδ subcellular redistribution induced by IHH as well as the infarct size-limiting effect of IHH was reversed by acute treatment with PKCδ inhibitor rottlerin. These data support the view that PKCδ plays a significant role in IHH-induced cardioprotection. On the other hand, adaptation to IHH decreased the PKC total protein level without affecting its...
|
|
|
HPLC method devolopment for betacarotene determination in nutraceuticals
Hlaváčková, Markéta ; Šatínský, Dalibor (advisor) ; Chocholoušová Havlíková, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Markéta Hlaváčková Consultant: Doc. RNDr. Dalibor Šatínský, Ph.D. Diploma Thesis Title: Development of HPLC Method for the Determination of Betacarotene in Nutritional Supplements A new HPLC method was optimized and developed for the determination of betacarotene contents in nutritional supplements Betakaroten Farmax, GS Betakaroten FORTE (GreenSwan pharmaceuticals), Walmark Betakaroten, Bioaktivní karoten (Pharma Nord Denmark), Beta karoten Max, BETAVID + LUTEIN (Naturvita), Karovit (Vitabalans Oy), SELZINK PLUS (PRO.MED.CS), Pupalkový olej (Aromatica). The method is based on using Ascentis Express C18 column (30 x 4.6 mm; 2.7 µm) and UV detection at 450 nm. There was used isocratic elution of the mobile phase 100% methanol at a flow-rate of 1.5 ml/min. Column oven temperature was set at 60 řC during the measurement. The retention time of betacarotene under the optimized and validated conditions was 2.185 min. Keywords: betacarotene, HPLC, Betakaroten Farmax, GS Betakaroten FORTE (GreenSwan pharmaceuticals), Walmark Betakaroten, Bioaktivní karoten (Pharma Nord Denmark), Beta karoten Max, BETAVID + LUTEIN (Naturvita), Karovit (Vitabalans Oy), SELZINK PLUS (PRO.MED.CS), Pupalkový olej...
|
|
| |
|
|
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...
|
|
| |
|
|
Role of protein kinase C isoforms in cardioprotective mechanism of chronic hypoxia
Hlaváčková, Markéta
Cardiovascular diseases, particularly acute myocardial infarction, are one of the leading causes of death in developed countries. It is well known that adaptation to chronic intermittent hypobaric hypoxia (IHH) confers long-lasting cardiac protection against acute ischemia/reperfusion injury. Protein kinase C (PKC) appears to play a role in its cardioprotective mechanism since the administration of general PKC inhibitor completely abolished the improvement of ischemic tolerance in IHH hearts. However, the involvement of individual PKC isoforms remains unclear. Therefore, the primary aim of this study was to investigate the potential involvement of PKCδ and PKCε, the most prevalent PKC isoforms in rat heart, in the mechanism of IHH-induced cardioprotection. We showed that IHH up- regulated PKC protein in left ventricle, enhanced its phosphorylation on Ser643 and increased its co-localization with markers of mitochondrial and sarcolemmal membranes. PKCδ subcellular redistribution induced by IHH as well as the infarct size-limiting effect of IHH was reversed by acute treatment with PKCδ inhibitor rottlerin. These data support the view that PKCδ plays a significant role in IHH-induced cardioprotection. On the other hand, adaptation to IHH decreased the PKC total protein level without affecting its...
|
|
|
Modulation of RNA demethylase FTO function in SH-SY5Y cells: the effect on insulin signaling and mitochondrial respiration
Čočková, Zuzana ; Novotný, Jiří (advisor) ; Hlaváčková, Markéta (referee)
Aim of this thesis was to observe changes in oxidative metabolism and expression of important neuroenergetic proteins in human neuroblastoma cell line SH-SY5Y due to inhibition of FTO. FTO is a RNA demethylase that uses N6-methyladenosine as substrate. Differences in enzyme expression are connected to broad area of effects involving energy homeostasis. Mitochondria are cellular powerhouses, a key elements in production of energy and metabolic substrates, yet a source of potentially dangerous reactive oxygen species (ROS) and analogous reactive molecules. In order to better understand FTO purpose in neuronal energetic metabolism, we examined mitochondrial respiratory chain. Using high-resolution respirometry we were capable of observing impairment in mitochondrial respiration after FTO inhibition. There was considerable decline in endogenous respiration, maximal respiration rate and reserve capacity. In order to obtain more detailed view into mitochondrial respiration, expression levels of electron-transport complexes were quantified by Western blot technique. Slight reduction was identified in subunits of complex I and IV. However, the most prominent alteration was seen in complex II subunit. There were no differences in expression of complex III and ATP synthase subunits. Beside disrupted activity...
|
|
| |
guest ::
RSS feed.