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Transport mechanisms of secondary metabolites across membranes of plant cells
Kubeš, Jan ; Tůmová, Lenka (advisor) ; Babula, Petr (referee) ; Obložinský, Marek (referee)
Faculty of Pharmacy in Hradec Králové, Charles University Department of pharmacognosy Candidate: Mgr. Jan Kubeš Supervisor: PharmDr. Lenka Tůmová, CSc. Title of Doctoral thesis: Transport mechanisms of secondary metabolites across membranes of plant cells An attention was focused on the metabolism of isoflavones in recent years, including their transport within plants and cultures in vitro. A research is focused on the transport mechanism identification across cell or vacuolar membrane. There are localized different transporters as or the transport of some metabolites can take place through membrane vesicules. The release of five isoflavones (genistin, genistein, biochanin A, daidzein and formononetin) into nutrient medium was observed in suspension culture of Genista tinctoria L and Trifolium pratense L. These cultures were treated with NH4VO3 solutions (1 a 10 M concentration) when isoflavon content in medium was higher after 24 hours. This increased volume of isoflavones was supressed by different transport mechanism inhibitors. The transport of isoflavones in T. pratense was influenced by ABC inhibitors from MRP subfamily, but genistein concentration in medium was lower after treatment with MDR subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased concentration of...
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The role of adrenergic signaling in cardioprotection induced by adaptation to chronic hypoxia
Hahnová, Klára ; Novotný, Jiří (advisor) ; Polák, Jan (referee) ; Babula, Petr (referee)
Cardiovascular diseases are currently one of the most common causes of morbidity and mortality in the Western world. Adaptation to chronic hypoxia can contribute to the improvement of ischemic tolerance of myocardium but exact molecular mechanisms leading to the development of a cardioprotective phenotype are still unclear. There are some indications that point to the possible role of β-adrenergic signaling in these processes. In the first part of the thesis, we examined the effect of protective continuous (CNH; 24 h/day) and nonprotective intermittent (INH; 23 h/day hypoxia, 1 h/day reoxygenation) normobaric hypoxia on β-adrenergic signaling in the right (RV) and left ventricles (LV) of Wistar rats. Both hypoxic models led to decrease in the number of β1-adrenergic receptors (β1-ARs) in the RV. There were no significant changes in β-ARs in LV preparations. Although adenylyl cyclase (AC) activity stimulated through Gs proteins was decreased in the RV and increased in the LV after adaptation to CNH and INH, there were no significant changes in the expression of dominant AC 5/6 isoforms. Expression of Gs proteins was decreased in RV in both hypoxic models. These results suggest that chronic normobaric hypoxia may have a strong effect on myocardial β-adrenergic signaling without differences between...
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Antioxidant system in hypoxic heart
Sotáková, Dita ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee) ; Babula, Petr (referee)
The cardiovascular disease, particularly acute myocardial infarction, is the most common cause of death worldwide. It is well documented that adaptation to chronic hypoxia increases resistance to ischemia-reperfusion (I/R) injury in heart tissue. Reactive oxygen species (ROS) play an important signalling role by the activation of the protective pathways during I/R, although, the excess of ROS during reperfusion leads to cardiac tissue injury. As the cellular antioxidant system is responsible for the maintenance of redox homeostasis, the main aim of this thesis was to investigate the relationship between myocardial tolerance to I/R injury and regulation of main components of antioxidant systems, related transcription factors and their target genes in protective and non- protective regimens of chronic hypoxia. We found differences in cardioprotective phenotype in rats exposed to three regimens of chronic normobaric hypoxia (FiO2 0.1, 3 weeks). The adaptation to continual (CNH) and intermittent (CNH-8; 8 h/day) regimen of hypoxia increased myocardial resistance to I/R damage, whereas 1-hour daily interruption of hypoxic adaptation (INH-23) abolished cardioprotective effect and decreased the ratio of reduced and oxidized glutathione (GSH/GSSG). Both cardioprotective regimens significantly increased...
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The role of cold acclimation in activation of adrenegic and thyrode signalling pathway in rat myocardium
Tibenská, Veronika ; Žurmanová, Jitka (advisor) ; Babula, Petr (referee) ; Tribulová, Narcisa (referee)
Despite advances in research and therapy, cardiovascular diseases are still the leading cause of death worldwide. A closer understanding of the endogenous protective mechanism may improve pharmacological interventions for the treatment of heart diseases. Cold acclimation or hardening has strong potential for reducing cardiovascular risk and the literature shows that it stimulates the β-adrenergic and thyroid systems in tissues. At the same time, the adrenergic system in the heart is one of the main regulators of cardiac activity. However, these signaling pathways have surprisingly not been studied at the protein level in the heart yet, and no studies can be found on the subject matter in current literature. Our results show a reduced infarct size induced by ischemic injury in cold-acclimated rats (CA) at 8 ř C for 5 weeks and then returning to normothermic conditions for 2 weeks (CAR). The aim of this dissertation is to determine, the degree of involvement of the adrenergic system in the myocardium during acclimation after 3 days, 10 days, 5 weeks of CA and subsequent CAR at the level of all three β-adrenergic receptor isoforms (β-ARs) and their signaling pathways. The results show unchanged signaling of β1-AR-Gs-adenylyl cyclase-protein kinase A in the cardioprotective regimes CA and CAR, whose...
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Antioxidant system in hypoxic heart
Sotáková, Dita ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee) ; Babula, Petr (referee)
The cardiovascular disease, particularly acute myocardial infarction, is the most common cause of death worldwide. It is well documented that adaptation to chronic hypoxia increases resistance to ischemia-reperfusion (I/R) injury in heart tissue. Reactive oxygen species (ROS) play an important signalling role by the activation of the protective pathways during I/R, although, the excess of ROS during reperfusion leads to cardiac tissue injury. As the cellular antioxidant system is responsible for the maintenance of redox homeostasis, the main aim of this thesis was to investigate the relationship between myocardial tolerance to I/R injury and regulation of main components of antioxidant systems, related transcription factors and their target genes in protective and non- protective regimens of chronic hypoxia. We found differences in cardioprotective phenotype in rats exposed to three regimens of chronic normobaric hypoxia (FiO2 0.1, 3 weeks). The adaptation to continual (CNH) and intermittent (CNH-8; 8 h/day) regimen of hypoxia increased myocardial resistance to I/R damage, whereas 1-hour daily interruption of hypoxic adaptation (INH-23) abolished cardioprotective effect and decreased the ratio of reduced and oxidized glutathione (GSH/GSSG). Both cardioprotective regimens significantly increased...
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The role of adrenergic signaling in cardioprotection induced by adaptation to chronic hypoxia
Hahnová, Klára ; Novotný, Jiří (advisor) ; Polák, Jan (referee) ; Babula, Petr (referee)
Cardiovascular diseases are currently one of the most common causes of morbidity and mortality in the Western world. Adaptation to chronic hypoxia can contribute to the improvement of ischemic tolerance of myocardium but exact molecular mechanisms leading to the development of a cardioprotective phenotype are still unclear. There are some indications that point to the possible role of β-adrenergic signaling in these processes. In the first part of the thesis, we examined the effect of protective continuous (CNH; 24 h/day) and nonprotective intermittent (INH; 23 h/day hypoxia, 1 h/day reoxygenation) normobaric hypoxia on β-adrenergic signaling in the right (RV) and left ventricles (LV) of Wistar rats. Both hypoxic models led to decrease in the number of β1-adrenergic receptors (β1-ARs) in the RV. There were no significant changes in β-ARs in LV preparations. Although adenylyl cyclase (AC) activity stimulated through Gs proteins was decreased in the RV and increased in the LV after adaptation to CNH and INH, there were no significant changes in the expression of dominant AC 5/6 isoforms. Expression of Gs proteins was decreased in RV in both hypoxic models. These results suggest that chronic normobaric hypoxia may have a strong effect on myocardial β-adrenergic signaling without differences between...
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Transcriptomic Characterization Using RNA-Seq Data Analysis
Abo Khayal, Layal ; Babula, Petr (referee) ; Lexa,, Matej (referee) ; Provazník, Ivo (advisor)
Vysoce výkonné sekvenční technologie produkují obrovské množství dat, která mohou odhalit nové geny, identifikovat splice varianty a kvantifikovat genovou expresi v celém genomu. Objem a složitost dat z RNA-seq experimentů vyžadují škálovatelné metody matematické analýzy založené na robustníchstatistických modelech. Je náročné navrhnout integrované pracovní postupy, které zahrnují různé postupy analýzy. Konkrétně jsou to srovnávací testy transkriptů, které jsou komplikovány několika zdroji variability měření a představují řadu statistických problémů. V tomto výzkumu byla sestavena integrovaná transkripční profilová pipeline k produkci nových reprodukovatelných kódů pro získání biologicky interpretovovatelných výsledků. Počínaje anotací údajů RNA-seq a hodnocení kvality je navržen soubor kódů, který slouží pro vizualizaci hodnocení kvality, potřebné pro zajištění RNA-Seq experimentu s analýzou dat. Dále je provedena komplexní diferenciální analýza genových expresí, která poskytuje popisné metody pro testované RNA-Seq data. Pro implementaci analýzy alternativního sestřihu a diferenciálních exonů jsme zlepšili výkon DEXSeq definováním otevřeného čtecího rámce exonového regionu, který se používá alternativně. Dále je popsána nová metodologie pro analýzu diferenciálně exprimované dlouhé nekódující RNA nalezením funkční korelace této RNA se sousedícími diferenciálně exprimovanými geny kódujícími proteiny. Takto je získán jasnější pohled na regulační mechanismus a poskytnuta hypotéza o úloze dlouhé nekódující RNA v regulaci genové exprese.
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Transport mechanisms of secondary metabolites across membranes of plant cells
Kubeš, Jan ; Tůmová, Lenka (advisor) ; Babula, Petr (referee) ; Obložinský, Marek (referee)
Faculty of Pharmacy in Hradec Králové, Charles University Department of pharmacognosy Candidate: Mgr. Jan Kubeš Supervisor: PharmDr. Lenka Tůmová, CSc. Title of Doctoral thesis: Transport mechanisms of secondary metabolites across membranes of plant cells An attention was focused on the metabolism of isoflavones in recent years, including their transport within plants and cultures in vitro. A research is focused on the transport mechanism identification across cell or vacuolar membrane. There are localized different transporters as or the transport of some metabolites can take place through membrane vesicules. The release of five isoflavones (genistin, genistein, biochanin A, daidzein and formononetin) into nutrient medium was observed in suspension culture of Genista tinctoria L and Trifolium pratense L. These cultures were treated with NH4VO3 solutions (1 a 10 M concentration) when isoflavon content in medium was higher after 24 hours. This increased volume of isoflavones was supressed by different transport mechanism inhibitors. The transport of isoflavones in T. pratense was influenced by ABC inhibitors from MRP subfamily, but genistein concentration in medium was lower after treatment with MDR subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased concentration of...
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Transport mechanisms of secondary metabolites across membranes of plant cells
Kubeš, Jan ; Tůmová, Lenka (advisor) ; Babula, Petr (referee) ; Obložinský, Marek (referee)
Faculty of Pharmacy in Hradec Králové, Charles University Department of pharmacognosy Candidate: Mgr. Jan Kubeš Supervisor: PharmDr. Lenka Tůmová, CSc. Title of Doctoral thesis: Transport mechanisms of secondary metabolites across membranes of plant cells An attention was focused on the metabolism of isoflavones in recent years, including their transport within plants and cultures in vitro. A research is focused on the transport mechanism identification across cell or vacuolar membrane. There are localized different transporters as or the transport of some metabolites can take place through membrane vesicules. The release of five isoflavones (genistin, genistein, biochanin A, daidzein and formononetin) into nutrient medium was observed in suspension culture of Genista tinctoria L and Trifolium pratense L. These cultures were treated with NH4VO3 solutions (1 a 10 M concentration) when isoflavon content in medium was higher after 24 hours. This increased volume of isoflavones was supressed by different transport mechanism inhibitors. The transport of isoflavones in T. pratense was influenced by ABC inhibitors from MRP subfamily, but genistein concentration in medium was lower after treatment with MDR subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased concentration of...
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Optical Measurement of Electromechanical Characteristics of Heart Cells
Čmiel, Vratislav ; Križanová,, Olga (referee) ; Babula, Petr (referee) ; Provazník, Ivo (advisor)
Dissertation is focused on the application of optical measurement methods using techniques of optical microscopy and fluorescence microscopy in measurements of electromechanical characteristics of isolated cardiac cells and clusters of differentiated cardiomyocytes. The first proposed method uses a practical combination of fluorescence microscopy equipped with fluorescent fast and high-resolution camera and atomic force microscopy for simultaneous measurement of calcium transients and contraction of cardiomyocyte clusters. The signals obtained undergoes filtration, processing and analysis. Result function parameters obtained by analyzing signals after application of caffeine are evaluated by comparison with functional parameters obtained during the control measurement. The second proposed method is applied to the cardiomyocyte clusters for the purpose of cardiomyocyte contraction signals measurement. The signals obtained by optical methods are analyzed and compared with the reference signal obtained using atomic force microscopy. Optical measurement method of cell contractins based on detection of cell ends using adjusting of microscopy images by re-sharpening and fluorescence method for cardiomyocyte contractions measurements were designed to increase realiability in simultaneous measurement of cell contractions simultaneously with calcium transients in isolated cardiomyocytes experiments.
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