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Mitochondrial cytochrome c oxidase: cyanide inhibition and role of assembly factor Surf1 defect
Nůsková, Hana ; Kalous, Martin (referee) ; Drahota, Zdeněk (advisor)
The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....
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Effect of CO2 on free-radical damage
Dušková, Eliška ; Kalous, Martin (referee) ; Wilhelm, Jiří (advisor)
CO2, known for its role in acid-base regulations, is a mediator of peroxynitrite-induced oxidative damage and it also enhances Cu,Zn-SOD's antioxidant activity. These two means, by which CO₂ affects free-radical damage, are well-explored, but there are many other mechanisms. When CO2 joins in free radical reactions, carbonate radical is often produced. The carbonate radical specifically damages substrates, but it is not possible to generalize, if the effect is pro-oxidant or antioxidant. A protective role of CO2 has been observed during lipid peroxidation and during peroxynitrite-induced oxidation of DNA, when the carbonate radical caused injury to specific bases, but in the same time it prevented DNA strand breaks. Similarly, CO2 prevented peroxynitrite- induced protein fragmentation as well as it caused injury to specific aminoacids. These observations are mostly based on experiments in a chemical system, which means under simplified conditions. In vivo, CO2 exerts much more mechanisms to affect free radical reactions. Under more complex conditions, as cell culture is, there was an increase of oxidative stress after CO2 exposure. Increased concentration of CO2 causes a change in erythrocyte's function and an increase of oxidative stress on the organism's level.
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Complex I of mitochondrial respiratory chain a its disorders.
Rodinová, Marie ; Kalous, Martin (referee) ; Hansíková, Hana (advisor)
NADH: ubiquinone oxidoreductase (Complex I) is a multisubunit protein complex of inner mitochondrial membrane. Complex I is the biggest and most complicated part of oxidative phosphorylation system, which is responsible for the cell ATP production. It consists of 45 subunits. 7 subunits are mitochondrial encoded, remainder 38 are nuclear encoded. NADH: ubiquinone oxidoreductase has L-shaped structure, which is built of two arms: membrane arm and matrix located peripheral arm. Complex I oxidize the NADH molecule. The electron transport is coupled with proton pumping across the inner mitochondrial membrane to intermembrane space, where proton gradient developed and which is used by ATP synthase to ATP synthesis. Deficiencies of NADH: ubiquinone oxidoreductase represent extensive, clinically and genetic heterogeneous group of mitochondrial diseases. Decrease of activity and amount of complex I, decrease of ATP production, changes of membrane potential, mitochondrial morphology and mitochondrial network and increasing of production of reactive oxygen species are found in cells with defects of NADH: ubiquinone oxidoreductase. Combination of this features lead to serious illnesses, which are almost fatal and we still haven't any useful therapy. Aim of this study is to summarize present knowledge about...
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The study of the changes of hepatocyte energy metabolism: the effect of oxidative stress and triiodthyronine
Endlicher, René ; Červinková, Zuzana (advisor) ; Kalous, Martin (referee) ; Rauchová, Hana (referee)
Changes of energy metabolism of hepatocytes: The effect of oxidative stress and triiodothyronine Liver is a vital organ performing numerous essential functions. Due to its position in the blood circulation, liver is the first organ incessantly exposed to a great number of toxic substances. Respiratory chain located in mitochondria is a frequent target of toxic action of these substances. There are various mechanisms that participate in hepatocyte damage, nevertheless the most important mechanism of hepatotoxic effect is oxidative stress induced by increased production of free radicals. Impact of oxidative stress on hepatocytes is very complex and still not fully elucidated. The aim of my thesis was to investigate the effect of oxidative stress on energy metabolism of rat hepatocytes using isolated hepatocytes and isolated mitochondria. We evaluated the effect of oxidative stress on the activity of mitochondrial enzymes and function of mitochondrial permeability transition pore (MPTP), respectively. Opening of this pore induces activation of apoptotic and necrotic processes. Our results document selective action of oxidative stress on the activity of various mitochondrial enzymes. Tert-butylhydroperoxide (t-BHP) causes significant inhibition of NADH-dependent substrates, while oxidation of...
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The role of CIDE proteins in apoptosis and mitochondrial network morphology under pathogenetic conditions
Šantorová, Jitka ; Ježek, Petr (advisor) ; Teisinger, Jan (referee) ; Kalous, Martin (referee)
Cď deďh-indrcing DFFIDNA fragmentationfactor]-likeeffector-a(CIDEa)' may initiďe 4optosis by disruptinga complex consisting of 40-kDa caspase-3-aďvďed nucleaseDFF40 md its 45.kDa inhibitor DFF45. We measured Íhe levels of mRNA CIDEa in rattus norvegicus tissues and deteďed high lwels of RNA in white aďpose tiszue. We have confirmedthepresenceof CIDEa in mitoohon&ia andimportanceof CIDE-C domain for this localization. We havealso performedimmrrnodetectionof subcellular&actionsofHela cells adapted for a tetracycline-inducible CIDEa expression. We have observed redistribution, enhancedupon treatuotrt with camptothecinor valinomycin, of CIDEa to nucleus. CIDEa contentincreasď in the nuclear fractionbut decreasedin oýosolio fraction in cells heatedto initiaÍe4optosis. We hypothesizethatCIDEa is sequesteredin mitochondriawhile transferof this pote,ntiallydangerousprotein from mitochondriainto nucleusintensifies or even initiates apoptosis. Mitochondria in nrrmerouscell types,especiďly in culturedcells, form a reticula network undergoing constant fusion and fission. The tbree dimensionď (3D) morphology of these networks however has not been stuďed in detail to our knowledge. We have investigated insďinoma INS-IE and hepatocellular oarcinoma HEP-G2 cells hansGoted \Yith mitoohondria-addressedGFP. Using 4Pi...
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Comparison advertising systems and selection of the best solution for "Pension 48"
Kalous, Martin ; Vojíř, Stanislav (advisor) ; Chudán, David (referee)
This work deals with internet advertising. Describes the basic characteristics of Internet marketing, survey errors that nascent entrepreneurs engage in formulating goals. Discusses the different types of advertising on the Internet usable in terms of functionality, payment options. It also offers an overview of advertising systems that provide these types of ads. In connection with advertising, the work deals with the problem of blocking. On show are the best known systems that block ads, but also ways to bypass such systems. The aim is to give people who want to invest money into advertising, relevant information about our services and help them choose the right type of promotion for your website. According to predefined criteria to verify the suitability of advertising and advertising system on the guesthouse accommodation in Spindleruv Mlyn. The performance is selected advertising system, its functions, a detailed procedure for creating the campaign and its optimization. The results presented show that the ad was chosen correctly. In addition, there are suggested ways to improve an existing campaign.
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Obsah a struktura webových dokumentů a jejich přístupnost
Kalous, Martin ; Pinkas, Otakar (advisor) ; Kliegr, Tomáš (referee)
Bakalářská práce se zabývá otázkou optimalizace internetových stránek pro vyhledávače a analýzou návštěvnosti. Optimalizací internetových stránek pro vyhledávače (dále jen SEO) rozumíme správné naprogramování, nastavení a situování webových stránek takovým způsobem, aby nejen neporušovaly pravidla vyhledávačů, ale zároveň, aby měly větší šanci při samotném vyhledávání. V první části práce jsou popsány základní pravidla SEO, faktory ovlivňující hodnocení stránek (on-page faktory a off-page faktory). On-page faktory jsou veškeré objekty na webové stránce, off-page faktory jsou taktiky, jak na své stránky upoutat co největší pozornost, jak je zviditelnit bez zásahu do zdrojového kódu. Kromě etických taktik existují i taktiky neetické nebo spíše zakázané. Představené metody či taktiky jsou demonstrovány na kratičkých příkladech a na ukázkovém SEO auditu vybrané firmy, který je v příloze bakalářské práce. Druhá kapitola se zabývá dalšími způsoby zvyšování návštěvnosti. Jsou zmíněny hlavně reklamní systémy, které jsou na hlavních internetových portálech k dispozici. Třetí kapitola je v podstatě teoretické představení případové studie ? analýzy návštěvnosti. Návštěvnost signalizuje úroveň úspěšnosti online reklamy. Zde jsou uvedeny některé metody měření návštěvnosti a různé statistické nástroje, kterými se dá měřit. Ve čtvrté kapitole je konkrétně provedena ? analýza návštěvnosti vybrané firmy. Pomocí různých statistických nástrojů jsou zhodnoceny konkrétní stránky z hlediska návštěvnosti.
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