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In vitro assessment of antiproliferative effects of novel intracellular iron chelators
Macková, Eliška ; Kvasničková, Eva (referee) ; Šimůnek, Tomáš (advisor)
Charles rnáversáty án mrague caculty of mharmacy án eradec hrálové aeéartment of Báochemácal pcáences CandádateW bláška jacková puéervásorW aocK mharmarK Tomáš ŠámůnekI mhKaK Tátle of dáéloma thesásW In vitro assessment of antáéroláferatáve effects of novel ántracellular áron chelators Aroylhydrazones are trádentate láéofálác áron chelators wáth very good entry to cellsK fn our éreváous exéerámentsI salácylaldehyde ásonácotánoyl hydrazone EpfeFI has shown very ánterestáng antáoxádant and antáéroláferatáve effácáencáes K eoweverI áts major dásadvantage ás low stabáláty caused by a fast hydrolysás of hydrazone bondK ThereforeI the seráes of náne new analogues of pfe deráved from aromatác ketones has been synthetázed at the aeéartment of fnorganác and lrganác Chemástry of our facultyK lur recent study extends our éreváous exéeráments wáth these substances and examánes éotentáal use of these new aroylhydrazones án antátumour treatmentK The breast adenocarcánoma cell láne jCc-T and human éromyelocytác leukemáa cell láne eiSM were usedK mroláferatáon tests took TO hoursI when cells were exéosed to actáon of substances or theár combánatáons ptatástácally ságnáfácant antáéroláferatáve actáváty was noted án all new chelators at the both cell lánesK te coméared the fC5M values of each chelator to cancer cell...
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Relation between alkali-metal-cation homeostasis and resistance to antimycotics in Candida species
Elicharová, Hana ; Buchta, Vladimír (referee) ; Šimůnek, Tomáš (advisor)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences Candidate: Hana Elicharová Supervisor: PharmDr. Tomáš Šimůnek, Ph.D.; Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague Supervisor specialist: RNDr. Hana Sychrová, DrSc.; Department of Membrane Transport, Institute of Physiology AS CR, v.v.i. Title of diploma thesis: Relation between alkali-metal-cation homeostasis and resistance to antimycotics in Candida species The family of Candida species, normally a harmless human commensal of the gastrointestinal and genitourinary tract, can become a human pathogen under certain circumstances. Mainly in immunocompromised patients, Candida cause a wide range of infections and are the most prevalent pathogenic yeast. In this work we have focused on the most common Candida species (C. albicans, C. parapsilosis and C. glabrata) and we included C. dubliniensis as well. Candida species differ in their resistance to azole-antimycotics and their halotolerance. Azoles form the most important group of drugs with antifungal activity. They destabilise plasma membrane of Candida cells. The essential function of plasma membrane is regulation of cation homeostasis. The combination of subinhibition concentrations of...
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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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Proteomic characterization of the host cell after interaction with intracellular pathogen Francisella tularensis
Římalová, Pavla ; Klimentová, Jana (referee) ; Šimůnek, Tomáš (advisor)
Diploma Thesis Proteomic characterization of host cell after interaction with intracellular pathogen Francisella tularensis Pavla Římalová Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences 2009 Francisella tularensis (F. tularensis) is an intracellular gramnegative bacterium, causative agent of the serious human disease tularemia. It is a typical bacterium with unknown mechanisms of pathogenesis. A proteome characterization of host-pathogen interaction could significantly contribute to their elucidation. The aim of this diploma thesis was to reveal the protein expression changes in macrophage mouse cell line J774.2 after infection with F. tularensis using the quantitative "shotgun" proteome technology. The proteomes of host cells infected with F. tularensis for three various time intervals (6, 12 and 24 hours) were compared with the proteome of non-infected cells. Four subcellular fractions were prepared using ProteoExtract Subcellular Extraction Kit (S-PEK, Calbiochem). Fraction enriched for cytosolic proteins was further analyzed and quantified by iTRAQ-HPLC-MS/MS technology. This approach led to the successful identification of 248 proteins, the production of 37 proteins was then observed to be different compared to the non-infected cells....
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Complementation of Francisella tularensis dsbA mutant strain and analysis of potential binding partners of DsbA protein
Šenitková, Iva ; Červený, Lukáš (referee) ; Šimůnek, Tomáš (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Science Candidate: Bc. Iva Šenitková Supervisor: doc. PharmDr. Tomáš Šimůnek, Ph.D. Supervisor - consultant: RNDr. Petra Špidlová, Ph.D. Title of diploma thesis: Complementation of Francisella tularensis dsbA mutant strain and analysis of potential binding partners of DsbA protein Conserved hypothetical lipoprotein FTT1103 is a virulence factor of gram- negative intracellular bacterium F. tularensis. This protein shares homology with proteins of disulfide oxidoreductase DsbA family. These proteins catalyse formation of disulfide bonds, which are essential for forming proteins conformation, for activity and stability of proteins. The aim of this study was to identify binding partners of lipoprotein FTT1103, commonly known as DsbA. The knowledge of these partners could help us in understanding a role of lipoprotein DsbA in virulence F. tularensis. We prepared fused ftt1103 gene with sequence coding FLAG® tag and cloned this fused gene into plasmid vector which can be replicated in F. tularensis. We used electroporation for introducing plasmid vector into the in frame deletion mutant F. tularensis subsp. holarctica strain FSC200/∆1103 thereby we complemented this deletion mutant in trans. Imunoprecipitation by...
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The role of hDia1 and hDia2 in melanoma cell invasion
Šťastná, Jana ; Skálová, Lenka (advisor) ; Šimůnek, Tomáš (referee)
The aim of the diploma thesis was to elucidate the role of hDia1 and hDia2 proteins in A375-M2 melanoma cell invasion. Different methods, such as siRNA transfection, western blotting, fluorescence microscopy and confocal microscopy, were used. It was revealed that both proteins really play the role in invasion behavior of A375-M2 tumor cells. Furthermore, the hypothesis arose proposing the mechanism of hDia1 and hDia2 in A375-M2 tumor cell invasion.
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The study of influence inositolhexaphosphate on proliferation and apoptosis in cells isolated out of colorectal cancer
Hašková, Pavlína ; Šimůnek, Tomáš (advisor) ; Rudolf, Emil (referee)
Introduction: Apoptosis - programmed cell death significantly participates in maintaining of tissue homeostasis. Its alteration is leading to cancer disease. Inositolhexaphosphate (IP6) is naturally occurring substance that is present in most legumes, cereals and seems. IP6 and its lower phosphorylated forms are also found in most mammalian cells, where they assist in regulatory a variety of important cellular function (including regulation of cell cycle). Methods: This work has been engaged in the study of influence IP6 on apoptosis in cells isolated out of colorectal cancer by in vitro quantification enzymatic activity of caspase 3 in cell extract. Results: It has proved by the Student's t-test that IP6 statistically significant influence apoptosis in cells isolated out of colorectal cancer.
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Gene expression profiling after experimental perinatal asphyxia and the effect of complement-derived anaphylatoxin C3a
Šourková, Hana ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
Background: The complement system is involved in neuroprotection and brain repair after brain damage. To understand the molecular mechanisms of these processes, we performed gene expression profiling using quantitative real-time polymerase chain reaction (qPCR), which is the most accurate modern strategy for gene expression analysis. Project: Our project was directly aimed at expression profiling of selected genes potentially involved in loss and rescue of neural tissue during three weeks after hypoxic-ischemic brain injury, an experimental model of perinatal asphyxia. Recent experiments have shown that over-expression of C3a under the control of the GFAP promoter (C3a/GFAP) reduced hippocampal injury after left common carotid artery ligation in neonatal mice by 50%, compared to wild type mice. Here, we assessed how the local expression of C3a/GFAP transgene affects gene expression profiles. Gene expression was measured on samples from hippocampus ipsilateral and contralateral to the injury and ipsilateral part of cortex, taken at the time of injury, 6 and 24 hours; 3, 7 and 21 days after the injury. Results: Our data showed that the regulation of gene expression after hypoxic-ischemic injury differs in timing and intensity and may also be region dependent. The analysed genes belong to families...
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Study of antioxidant and cytoprotective effects of intracellular iron chelators in catecholamine-induced cardiomyocyte injury
Koubková, Lucie ; Šimůnek, Tomáš (advisor) ; Boušová, Iva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Kralove Department of Biochemical Sciences Candidate: Lucie Koubková Supervisor: Profa . Dra . Natércia Teixeira Profa . Dra . Georgina Correia da Silva Doc. Pharm.Dr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Evaluation of anti-aromatase activity of isoflavones from biotransformed soya Breast cancer belongs to the most frequently diagnosed cancers in women. Approximately 60% of breast cancer is hormone-dependent containing estrogen receptors and requiring estrogen for tumour growth. Estrogens are able to promote transcription of genes involved in cell proliferation. Aromatase is responsible for the final step of estrogen biosynthesis and its inhibition has been considered as an important target for the treatment of estrogen-dependent breast cancer. Soya is a source of phytoestrogens, such as genistein and daidzein, which are thought to be effective in breast cancer prevention. However, the mechanisms associated with this effect are not fully understood. In this study we evaluated the effects of a soya extract containing genistein and daidzein on aromatase activity. Our results demonstrated that this extract had the ability to inhibit aromatase in a dose-dependent manner in placental microsomes and also in the MCF-7aro breast cancer...
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