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Semiconductor microstructures for cellular photostimulation
Tvrdoňová, Anna ; Weiter, Martin (referee) ; Glowacki, Eric Daniel (advisor)
Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2.
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The metabolic role of pulmonary arterial fibroblasts in the activation of immune system during development of pulmonary arterial hypertension
Křivonosková, Monika ; Plecitá, Lydie (advisor) ; Zadražil, Zdeněk (referee)
The development of inflammation in the small distal pulmonary vessels plays an important role in the development of pulmonary arterial hypertension (PAH). One of the cell types found in the pulmonary vessels are fibroblasts, which, according to the "outside-in" theory, may be the first to respond to stimuli in the vessel, modulating remodeling of the pulmonary vessels toward the inner layers of the vessel and attracting other immune cells to the site. In addition to inflammation, the so- called Warburg effect also occurs in vessels affected by PAH, in which metabolism shifts toward glycolysis and lactate production. Among other changes, a pro-oxidative state is induced within the cell by mitochondrial metabolism and NADPH oxidase, leading to an imbalance in reactive oxygen species production. We therefore wanted to test whether calf lung fibroblasts with hypoxia-induced PAH have an active inflammasome, with which they would produce mature interleukin 1β (IL-1β) and to clarify the effect of a pro-oxidant environment on this expression. In our in vitro model we have confirmed IL-1β mRNA expression, but we were unable to detect its expression at the protein level. This was consistent with the inability to detect inflammasome activity. We believe that the inability to detect the protein form of IL-1β...
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Mitochondrial production of reactive oxygen species and its role in physiological regulations
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The production of mitochondrial reactive oxygen species and the resulting oxidative stress is an important phenomenon driving long-lasting research and intense discussions. Knowledge of exact mechanisms of reactive oxygen species production and pathways leading to their formation could help us to directly affect their production, a task with potential terapeutic implications. The molecular nature of the production of reactive oxygen species by some enzymes has already been well documented, but others still remain controversial and current theories are obviously far from the truth. Much more interesting is the question of physiological importace of this production. The reactive oxygen species were considered harmful factors clearly distorting the integrity of the organism for a long time. However, recent research suggest that their existence can also be beneficial and effective. Evidently they can serve as a signaling molecules in several metabolic and regulatory pathways occurring in the organism. This bachelor thesis offers insight into the current state of knowledge. It focuses on the most detailed description of the reactive oxygen species production by mitochondrial respiratory chain enzymes. Furthermore, it deals with some signaling cascades, where involvement of mitochondrially generated...
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Diversity of methods used for characterization of molluscan hemocytes
Jindrová, Zuzana ; Horák, Petr (advisor) ; Skála, Vladimír (referee)
Hemocytes are the main immune cells of invertebrates; therefore they can be found in molluscs, too. They differ both in morphology and function. The two generally accep- ted morphological types, granulocytes and hyalinocytes, vary in the level of phagocy- tosis and encapsulation, production of reactive oxygen species and nitrogen oxide, and presence of some enzymes. There is an array of methods by means of which hemocy- tes can be characterized. Microscopy serves particularly for study of morphology. An- tigens localized on the surface can be determined by monoclonal antibodies or lectin probes. Hemocytes can be divided on the basis of cell size and granularity using gra- dient centrifugation or flow cytometry. Production of nitrogen oxide and reactive oxy- gen species is monitored by adding appropriate substrate which changes its proper- ties after reaction with the radical. It may become fluorescent, change absorbance of the solution or form a visible precipitate. Another possibility is the use of chemilu- miniscence. The objective of hemocyte research is to explain mollusc-pathogen inter- action. 1
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Proteins demaged by oxidative stress, their role in physiology and ageing of yest cells
Mikešová, Jana ; Palková, Zdena (advisor) ; Borčin, Kateřina (referee)
Reactive oxygen species (ROS) are regularly produced in cells as a by-product of aerobic metabolism. Hence, organisms developed various defence mechanisms, which are able to avoid molecular damages caused by ROS under physiological conditions. In stress conditions, however, such defence mechanisms are not sufficient to avoid molecular damages. Accumulation of oxidized proteins is supposed to be a reason for ageing and many diseases including Friedreich's ataxia, Amylotrophic lateral sclerosis, Alzheimer's disease and many others. During oxidative stress, reactive oxygen species are reflected in oxidation of cystein residues in transcription factors, regulation proteins and active canters centers of enzymes. Oxidative modifications however could lead also to changes in transcription factor activity and activation of specific pathways, including changes in gene expression, cell cycle and proteolysis. This work shows defence mechanisms, ROS and proteins altered by reactive oxygen species that may function as important signalling molecules, which are essential for many cellular processes.
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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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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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Targeting mitochondria to overcome resistance of breast cancer to therapy
Rohlenová, Kateřina ; Neužil, Jiří (advisor) ; Špíšek, Radek (referee) ; Vítek, Libor (referee)
(EN) Tumours are heterogeneous and consist of multiple populations of cells. The population of cells with tumour-initiating capability is known as cancer stem cells (CSC). Cells with increased stemness properties and elevated resistance to anti-cancer treatment have been shown to be highly affected upon decline of mitochondrial respiration, linking the concept of CSCs to deregulated bioenergetics. Consistently, functional electron transport chain (ETC) is crucial in tumorigenesis. Expression of HER2 oncogene, associated with resistance to treatment in breast cancer, has been connected with regulation of mitochondrial function. We therefore investigated the possibility that manipulation of mitochondrial bioenergetics via disruption of ETC eliminates the conventional therapy-resistant populations of tumour, such as CSCs and HER2high cells. We demonstrate that HER2high cells and tumours have increased complex I-driven respiration and increased assembly of respiratory supercomplexes (SC). These cells are highly sensitive to MitoTam, a novel mitochondria-targeted derivative of tamoxifen, acting as a CI inhibitor and SC disruptor. MitoTam was able to overcome resistance to tamoxifen, and to reduce the metastatic potential of HER2high cells. Higher sensitivity of HER2high cells to MitoTam is dependent on...
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Myocardial tolerance to ischemia/reperfusion injury - possible protective mechanisms
Alánová, Petra ; Neckář, Jan (advisor) ; Nováková, Olga (referee) ; Vaněčková, Ivana (referee)
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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