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Caspase inhibitors and regulation of the IL-1 family cytokines
Brabcová, Eva ; Stříž, Ilja (advisor) ; Holáň, Vladimír (referee)
Proinflammatory cytokines from IL-1 family play a key role in immune and immunopathological reactions and are involved also in initial phases of rejection mechanisms. One of their multiple functions is the induction of chemokines attracting immune cells into the site of injury. The aim of our study was to assess the effect of proinflammatory cytokines on the chemokine release and mRNA induction in epithelial cell lines of renal and lung origin. Furthermore, we tested the effect of caspase-1 inhibitors isolated from Streptomyces nodus (manumycin, asukamycin, MM273) on the release of IL-1 beta and IL-18 from human macrophages. These cytokines are cleaved into their active form by caspase-1 and inihibtion of the enzyme activity might be a perspective therapeutical approach to reduce inflammatory reaction. In our experimental model, we used cell line derived from renal adenocarcinoma (RA), A549 cell line (alveolar type II-like cells), and THP-1 monocyte/macrophage cell line. Chemokine mRNA induction was evaluated by an oligoarray and chemokine and cytokine levels were measured by Luminex or ELISA technology. Our data have showed that both epithelial cell lines produced constitutively mainly CXC chemokines attracting neutrophils (CXCL5/ENA-78, CXCL8/IL-8) and some of the CC chemokines characteristic for the...
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The characterization of adenosine signal pathway in \kur{Drosophila} imaginal disc cells
TICHÝ, Vlastimil
The aim of this work was to characterise the influence of adenosine on imaginal disc cell line Cl8+ of Drosophila. I prepared stable cell lines with the overexpression or RNA interference of genes coding adenosine receptor AdoR (CG9753) and adenosine transporter DmENT2 (CG11045) in D. melanogaster. These cell lines were subsequently used to test their response to extracellular adenosine signal by the measurement of cell viability and level of second messengers cAMP and Ca2+ in Cl8+ cells.
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Doxorubicin cytotoxicity and metabolism in MCF7 cell line
Hanušová, Veronika ; Šimůnek, Tomáš (referee) ; Skálová, Lenka (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Bc. Veronika Hanušová Title, Name, Surname of tutor: Doc. RNDr. Lenka Skálová, Ph.D. Title of a diploma work: Doxorubicin cytotoxicity and metabolism in MCF-7 cell line. Doxorubicin (DOX) is ranked among anthracycline chemoterapeutics, significantly participant in the treatment of solid tumors, inclusive breast carcinoma and haematologic malignancies. DOX is metabolized to 13-OH-doxorubicinol (DOX-OL) produced by carbonyl reductase 1 (CBR1). DOX-OL is lower antineoplastic, but more cardiotoxic compared to the parent drug. Aim of this work was research of reduction DOX and testing possible increasing cytotoxicity of DOX through inhibition his reductase. For experiments has been selected human breast cancer cell line MCF-7. Initially has been elicit optimal composition of cultural medium and optimalized process on sample preparation for HPLC. In the study of DOX metabolism in cytosol the MCF-7 cells act oracin (potentional chemoterapeutic) as significantly kompetition's inhibitor of DOX reductases. In cytotoxicity tests acted DOX more toxic in cells with bovine serum in medium, oracin was again more toxic in cells without bovine serum. Combination DOX with oracin...
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Transport of ion channel blockers across the blood-brain barrier in vitro
Leblochová, Hana ; Opletalová, Veronika (advisor) ; Štaud, František (referee)
Transport of ion channel blockers across the blood-brain barrier in vitro Diploma thesis, 2010 Hana Leblochová Abstract Six different substances blocking ion channels were chosen (verapamil, diltiazem, nifedipine, phenobarbital, memantine, amantadine) for the purpose of this work. All these substances are routinely used in clinical practice and it is well known that adverse effects on the central nervous system can occur during therapy with them. Therefore, both single and group transport studies were carried out to investigate and compare the transport abilities of chosen ion channel antagonists to penetrate the BBB and to find out the interference between them. The required data for each substance used in single and group studies were determinated using the Transwell BBB in vitro model based on EVC304 cell line. Diazepam and carboxyfluorescein were used as internal standards for normalization of permeability data. According to the obtained data from single studies nifedipine as drug acting in periphery passes the BBB faster than internal standard diazepam (acting in CNS) and much more faster than other ion channel blockers acting in periphery, too. In clinical practice it can mean that nifedipine used for concrete clinical problem can have more CNS adverse effects in comparison to verapamil or diltiazem....
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DNA damage induced with sulphur mustard
Říhová, Helena ; Vopršalová, Marie (advisor) ; Melicharová, Ludmila (referee)
In our study, we used the comet assay to assess the sort and range of DNA damage induced by sulfur mustard. Sulfur mustard belongs to chemical warfare agents and is embedded in the blistering agent category. It reacts with a wide range of macromolecules within cells (e.g. DNA, RNA and proteins) and induces for example alkylation of DNA. The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA, ultraviolet (UV)-induced pyrimidine dimers, oxidized bases, and alkylation damage. The aim of the present study was to find out, how can be relaxed loops related to the amount of present strand breaks within DNA molecule. We were also interested in the mechanism of DNA damage caused by sulfur mustard. The cell lines employed were HeLa, UV-20, A549 and AA8 cells. Initially, we assessed the influence between cross-links caused by sulfur mustard and strand breaks caused by styrene oxide. We found sulfur mustard to be highly potent to form cross-links even in low doses and to prevent relaxation of DNA loop caused by strand brakes. Using selected enzymes, we have identified the sort of DNA damage induced by sulfur mustard. First we used endonuclease III, a protein from E. coli which acts both as N-glycosylase and a AP-lyase. It causes strand brakes in apurinic or...
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Antimicrobial lytic factors of the coelomic fluid of Earthworms
Josková, Radka ; Bilej, Martin (advisor) ; Horák, Petr (referee) ; Prokešová, Ludmila (referee)
I. SUMMARY lnvertebratesare widely distrbuted animals.They can be found in almostany krnd ofhabitat. Their successfulsurvival shategiesare basedon short life span combinedwith numerousoffspring and, more importantly,all invertebratespecieshavedevelopeda wriety ofdefensemechanismselficientlyrecognizingand respondingto non-selfsubstances. The defensemechadsmsof earthwoms were studiedover the pastfou decades.It becameappdent thattheearthworms.aswell asotherinvertebrates,lackspecificimmunoglobulins,lymphoc)4esor otherfeatures ofthe adaptiveimmunesystemdescribedin verteblates,but possessirmatedefensecomponents In this thesis,we foousedon the detaildescriptionof somedefensemoleculesinvolvedin imate immunityof earthworms. l. Lysozyme is an erzyme with strong antibacterialactivity describedin many organisms. We characterizedthelysozymeof Erieniaandrei(formerly E.fetida andrei)earthwormbothstruchmlly and functionally.Moleoularcharactefiationof lyso4rne providesa new tool for monitoringof innateimmunity in earthworms. 2. A cltol''tic effect of the coelomicfluid of E. fetida was observedin experimentswith TNF-sensitive tumorL929 cell line. Subsequentisolationof l)tic proteinsled to theidentificationof 42-kDaprotein,which wasnamedcoelomiccytolyticfactor- CCF.CCFwasshom to bepresentalsoin coelomicfluid of mother...
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Oxidative damage by organic extracts from urban air particulate matter
Hanzalová, Kateřina ; Rössner, Pavel (advisor) ; Machala, Miroslav (referee)
The aim of this master thesis was to investigate the ability of selected individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs: benzo[a]pyrene, B[a]P; dibenzo[a,l]pyrene, DB[a,l]P), an artificial mixture of c-PAHs (c-PAH mix) and extractable organic matter (EOM) from urban air particulate matter (PM) to induce oxidative damage in vitro. Two cell lines (human hepatoma cells, HepG2, and human diploid lung fibroblasts, HEL) were treated for 24 h and 48 h with various concentrations of compounds or mixtures. The studied oxidative stress markers included 8-oxodeoxyguanosine (8-oxodG) as a marker of oxidative DNA damage, 15-F2t-isoprostane (15-F2t-IsoP) as a marker of lipid peroxidation and protein carbonyl groups as a marker of oxidative damage to proteins. The response of the cell lines to the tested compounds and mixtures differed substantially. In summary the results demonstrate the ability of EOM to induce oxidative damage to DNA and lipids after 24 h of treatment and to proteins after 48 h, in HepG2 cells. The effect of c-PAHs was substantially less. The induction of oxidative damage by c- PAHs and EOM in HEL cells was weak. Since c-PAHs had lower ability to cause oxidative damage that was limited only to longer incubation periods, it is probable that other components of EOM are responsible for...
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Rozdílná jaderná lokalizace aberantních a normálních homologů chromosomu 8 v intaktních nebo NaBt diferencovaných buňkách HT-29
Harničarová, Andrea ; Bártová, Eva ; Kozubek, Stanislav
An accumulation of numerous chromosomal structural aberrations is a typical feature of the human colon adenocarcinoma cell line HT-29. In these cells the chromosome 8 territories were visualized using fluorescence in situ hybridisation (FISH), which enabled to distinguish the aberrant chromosome 8 homologue from the normal one. Analyses of nuclear parameters revealed differences in radial positioning between chromosome homologues analysed. In comparison with an normal chromosome 8, the aberrant chromosome 8 territory was positioned closer to the nuclear periphery. Under standard conditions HT-29 cells displayed relatively undifferentiated phenotype but incubation of these cells in the presence of sodium butyrate (5mM) induced biochemical and morphological changes that are typical of well-differentiated phenotype of enterocytes.
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Effect of flavonoids on the metabolism of xenobiotics
Smetanová, Anna ; Szotáková, Barbora (advisor) ; Boušová, Iva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Mgr. Anna Smetanová Title, Name, Surname of tutor: Doc. Ing. Barbora Szotáková, Ph.D. Title of a rigorous work: The influence of flavonoids on the metabolism of xenobiotics Flavonoids are wide spread plant secondary metabolites with the protective effect against the cancer. One of the protective mechanisms of action is the modulation of the biotransformation enzymes. Xenobiotics - heterocyclic aromatic amines are presumably carcinogenic compounds generated during the ordinary cooking of meat and fish. The first metabolic step of the heterocyclic aromatic amines is the procarcinogen activation to the active N-hydroxy metabolites by cytochrome P4501A2 (CYP1A2). It would be desirable if the flavonoids in the appropriate manner modulate the enzymatic activity to eliminate the undesirable effects of the heterocyclic aromatic amines. The aim of this study was to investigate the influence of the rutin and quercetin flavonoids and heterocyclic aromatic amines 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) on CYP1A activity in the cell line LS174T and HCT-8. Rutin, quercetin, IQ and MeIQx were not toxic for cells up...
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