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Myocardial tolerance to ischemia/reperfusion injury - possible protective mechanisms
Alánová, Petra
Ischemic heart disease is the leading cause of death and disability worldwide. The effects of ischemic heart disease are usually attributable to the detrimental effects of acute myocardial ischemia/reperfusion (I/R) injury. The aim of the thesis was to contribute to current effort to clarify the basis of mechanisms that could save the heart from I/R injury. The whole thesis is based on four studies; while the first three are published, the fourth one has been under revision. In the first study, we proved the involvement of nitric oxide (NO) in the cardioprotective mechanism of chronic hypoxia (CH). We described that exogenously increased availability of NO as well as inhibition of phosphodiesterase type 5 led to increased myocardial tolerance of normoxic and chronically hypoxic rats. The effects of both interventions were not additive, suggesting that NO is included in cardioprotective signaling of CH. Second study continued in investigating molecular mechanisms underlying cardioprotection induced by CH. We showed that infarct size-limiting effect of adaptation to CH was accompanied by increased myocardial concentration of tumor-necrosis factor alpha (TNF-α) and TNF-α receptor R2. In the third study, we examined the effect of dexrazoxane (DEX), the only clinically approved drug against...
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Compartmentalization of the beta-adrenergic signaling system in cardiac cells: the effect of hypoxia
Karlovská, Ivana ; Novotný, Jiří (advisor) ; Nováková, Olga (referee)
The aim of this thesis was to study the changes that occur in cell line H9c2 after exposure to an oxygen level reduced to 2 % for 24 hours. We monitored changes in compartmentation of chosen members of β-adrenergic signaling system. We found an increase in expression of β1AR and β2AR. Only β2AR showed change in compartmentation after hypoxia, as they relocate from membrane rafts to non-rafts fractions of membrane. AC also showed an increase of expression and was located in membrane rafts. The next aim of this work was to monitore apoptotic markers to determine whether there are activated pro-apoptotic or anti-apoptotic signals under chosen conditions of hypoxia. There was an increase in expression of both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. We compare ratios of Bcl-2 to Bax and we found that there is a bigger increase in protein Bax expression. Another apoptotic marker, caspase 3, was tested and we also found that there was an increase in expression of caspase 3 in cells after hypoxia. Furthermore, we studied possible activation of kinase signaling pathways that may contribute to protective effects of hypoxia. Expression of Akt and ERK kinases was increased after hypoxia, but we did not confirm activation by phosphorylation of these kinases. Levels of phosphorylated Akt...
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Oxidative damage to cellular components after oxidative stress induction by specific herbicides
Kramná, Barbara ; Wilhelmová, Naďa (advisor) ; Ryšlavá, Helena (referee)
Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...
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The effect of heavy metals on the physiology of lichens
Hrdinová, Aneta ; Peksa, Ondřej (advisor) ; Svoboda, David (referee)
Lichens are known for their sensitivity to environmental changes, however, the tolerance to different changes may vary among particular taxa. There are lichen species extremely sensitive as well as very tolerant to pollution. The species growing in areas with a higher content of heavy metals belong to the second group. These sites may by of natural origin (ore), but very often they have arose as a result of anthropogenic activities (imissions in industrial and urban areas, various heaps etc.). The interaction between photobiont and mycobiont in the thallus can probably explain the success of lichens in environment enriched by heavy metals. The stress caused by heavy metals leads to increasing production of reactive oxygen species in cells of photobiont and mycobiont. Reactive oxygen species cause lipid peroxidation, damage of proteins and nucleic acids, and the degradation of chlorophylls. Although mycobiont provides the photobiont cells partial protection (particularly thanks to binding of metal ions to the cell wall and the formation of water-insoluble secondary metabolites), photobiont is exposed to high amounts of metals. Therefore, both symbionts have developed a range of detoxification mechanisms. The aim of this thesis was to summarize the findings on the effect of heavy metals on the...
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Changes in oxidative phosphorylation during development of cellular senescence
Zima, Michal ; Hodný, Zdeněk (advisor) ; Kašparová, Dita (referee)
Cellular senescence represents a state of permanent cell cycle arrest. It is considered to be an active response of the cell to various extrinsic and intrinsic types of stress, which are damaged and/or uncapped telomeres, activation of certain oncogenes, DNA damage and effects of several cytokines. This thesis describes current mechanisms which may result in establishment of senescence phenotype, putting those facts in association with changes in oxidative phosphorylation. In thesis are also mentioned features of senescence cells and their impact on the neighborhood. Special attention is focused on the role of reactive oxygen species in promotion of cellular senescence, mechanisms of their elevation, the role of NADPH oxidases and the inhibition of mitochondrial oxidative phosphorylation complexes by activity of cytokine signaling pathways STAT3 and TGFbeta. Key words: cellular senescence, reactive oxygen species, cytokines, mitochondria, oxidative phosphorylation chain, NADPH oxidases, Signal Transducer and Activator of Transcription 3 (STAT3), TGF-β, DNA damage response (DDR)
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The study of the influence of vitamin E analogues on malignant mesothelioma
Kovářová, Jaromíra ; Neužil, Jiří (advisor) ; Kozubík, Alois (referee) ; Vítek, Libor (referee)
Cancer is a leading cause of death in the western world and is increasing in frequency world-wide. Although diagnosis, treatment and therapeutic approaches to cancer have improved, many types of cancer are still lethal due to the lack of radical treatment. One of the fatal neoplastic disease types with poor prognosis is represented by malignant mesothelioma (MM). MM is characterised by very high mortality rate and limited therapeutic options. The etiology of the disease is mainly associated with exposure to asbestos fibres. The incidence of MM is increasing in many countries. The search for novel molecular targets, anti-cancer strategies and drugs, which would considerably improve the treatment is of great importance. Certain new drugs, especially those with specific molecular targets, show high selectivity in their action to cancer cells, and have considerably increased the cure rate in some types of cancer. Mitochondria have recently emerged as a very promising target for anti-cancer agents. A group of compounds with anti-cancer activity that induce apoptosis by way of mitochondrial destabilisation, termed 'mitocans', have been a recent focus of research. Several mitocans have been shown to selectively induce apoptosis in cancer cells and suppress the growth of many types of carcinomas in...
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NITRIC OXIDE AND REACTIVE OXYGEN SPECIES IN ACUTE CORONARY SYNDROME
Šnorek, Michal ; Herget, Jan (advisor) ; Ošťádal, Petr (referee) ; Al-Hiti, Hikmet (referee)
In cardiology, there are different conditions associated with the release of free radicals in some forms of hypoxia, such as ventilatory hypoxia or reduced perfusion. The role of free radicals during hypoxia in cardiology is the key point of our interest. In presented thesis, we have focused on hypoxia-induced pulmonary vasoconstriction and acute myocardial ischemia. Hypoxic pulmonary vasoconstriction (HPV), an important physiological mechanism, is regulated by changes in the production of and interactions among reactive oxygen species (ROS). There is controversy, however, over whether HPV is mediated by an increase or a decrease in ROS production. Also, the role of nitric oxide (NO) in HPV remains unclear. Our results indicate that inhibition of HPV by the superoxide dismutase mimetic tempol does not depend on either NO production or a decrease in basal vascular tone. The effect of three-day fasting on cardiac ischemic tolerance was investigated in another experimental model. Short-term fasting conclusively decreases ROS production. Three-day fasting effectively protected rat hearts against major endpoints of acute ischemia-reperfusion injury. It prevented severe ventricular arrhythmias and reduced the extent of myocardial infarction. These beneficial effects can be linked to altered mitochondrial redox...
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The role of Hippo Signalling pathway in tumor cell metabolism
Lettlová, Sandra ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Vitamine E analogues α-tocopheryl succinate (α-TOS) and mitochondrially targeted vitamine E succinate (MitoVES) are anti-cancer agents from the group of "mitocans", the compounds acting via mitochondria which present a promising invariant target for cancer cell therapy. α-TOS and MitoVES induce apoptosis selectively in various cancer cell types involving generation of reactive oxygen species (ROS). Generated superoxid anion radicals in response to α-TOS and MitoVES are believed to be converted into hydrogen peroxide that is known to activate Mammalian sterile 20-like kinase (Mst1), the central component of Hippo signalling pathway, that presents an universal size control mechanism in all metazoans and its deregulation is linked to tumourigenesis. MitoVES and α-TOS were both reported to activate Mst1 that phosphorylates Forkhead box O1 (FoxO1) transcription factor resulting in its transport to nucleus where induce the expression of pro-apoptotic genes, including NOXA, and thus promote apoptosis. The target of Hippo signalling pathway is transcriptional co- activator Yes-associated protein (Yap) which was found in Drosophila melanogaster to regulate the expression of transcription factor c-Myc which is known as the most prominent human oncogene. This thesis focused on involvement of Hippo signalling...
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Mitochondrial subpopulations in rat myocardium - effect of chronic hypoxia
Kovalčíková, Jana ; Nováková, Olga (advisor) ; Žurmanová, Jitka (referee)
Adaptation to chronic hypoxia induces endogenous cardioprotection and increases the heart resistance to ischemia/reperfusion injury. The heart mitochondria, which produce reactive oxygen species (ROS) in addition to ATP, play an important role in these processes. During ischemia/reperfusion, ROS are produced in excessive amounts and damage the cells. However, in lower concentrations, ROS are involved in the signalling pathway of cardioprotection induced by adaptation to chronic hypoxia. In the heart, two mitochondrial subpopulations have been observed, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IMFM), which differ in cell localization as well as in morphological and biochemical properties. The aim of this work was to introduce the method of SSM and IMFM isolation in our laboratory and to analyse their antioxidative capacity after adaptation to chronic hypoxia. Adult male Wistar rats were kept either under normoxic conditions or exposed to intermittent high-altitude hypoxia (IHA; 7000 m, 5 days a week/8 hours a day, totally 25 exposures). Mitochondrial subpopulations were isolated from heart left ventricle and their functionality was verified by measuring oxygen consumption and enzyme activities. The IMFM had higher oxygen consumption in comparison with SSM and activities...
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