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Gilbert Syndrome.
Šimáková, Eva ; Kuklík, Miloslav (advisor) ; Kovář, Jan (referee)
This thesis focuses on finding a possible link between genotype typical for Gilberts syndrome and specific diseases. It nvestigates a possible protective effect of the 7TA allele. It explains the origin, symptoms, pathology of this syndrome and its consequences for clinical medicine. Possible protective effect of this polymorphic mutation including reduced incidence of vascular diseases (myocardial infarction, stroke, atherosclerosis, etc.). is discussed. Reduced oxidative stress in hyperbilirubinaemia can be the mechanism behind. The work was carried out in the co-operation with the GENVIA laborator.
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Role of the mitochondrial pathway in apoptosis induction by taxanes in breast cancer cells
Schmiedlová, Martina ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee)
Apoptosis represents one of the cell death mechanisms which is realized after the application of taxanes in breast cancer cell lines. Apoptosis induction can be principally triggered either by outer or inner pathway. The aim of the diploma thesis is to contribute to the elucidation of role and mechanisms of the inner mitochondrial pathway of apoptosis induction after taxane application (paclitaxel and SB-T-1216) employing a model of breast carcinoma cell lines SK- BR-3 (nonfunctional p53, functional capase-3) and MCF-7 (functional p53, nonfunctional caspase-3). Specifically, we tested the effect of both employed taxanes on mitochondrial membrane potential, ROS level and the expression and localization of proteins regulating inner mitochondrial pathway. Taxane application resulted in mitochondrial membrane dissipation in SK-BR-3 cell line. However, this was not shown in MCF-7 cell line. We found no changes in Bax and Smac/DIABLO expression after taxane application in both tested cell lines. There was a decrease of Bid expression after taxane application in SK-BR-3 line, but not in MCF-7 line. Taxane application did not lead to the translocation of Bax and Bid (tBid) proteins from cytosol to mitochondria in both tested cell lines. Similarly, there was no Smac/DIABLO release from mitochondria to...
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Molecular mechanisms of apoptosis induced by photodynamic activation in cancer cells
Moserová, Irena ; Králová, Jarmila (advisor) ; Kuželová, Kateřina (referee) ; Kovář, Jan (referee)
Photodynamic therapy (PDT) is a treatment modality for cancer. It combines selective accumulation of chemical compounds, called photosensitizers (PS), with light to irreversibly damage cancer cells via oxidative stress. The main goal of this thesis was to study photosensitizers represented by a unique group of newly synthesized porphyrin derivatives with glycol chain substitution. Glycol-functionalized porphyrins containing one to four low molecular weight glycol chains that are linked via ether bonds to the meta-phenyl positions of meso-tetraphenylporphyrin (mTPP(EG)1-4) were compared with fluorinated (pTPPF(EG)4) and nonfluorinated (TPP(EG)4) derivatives having glycol chains in para-phenyl positions. The cellular uptake and photodynamic activity was significantly dependent on terminal groups of the glycol substituent. Hydroxy glycol porphyrins, in contrast with methoxy glycol porphyrins, exhibited efficient intracellular transport and high induction of apoptosis in tumor cell lines in vitro. After initial testing effective prototype hydroxy ethylene glycol derivatives were selected and analyzed in detail. Para derivatives pTPP(EG)4 and pTPPF(EG)4 accumulated mainly in lysosomes whereas meta derivatives mTPP(EG)1-4 in the endoplasmic reticulum (ER). Position of ethylene glycol chain on the...
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Mechanisms of invasiveness and transcription regulation in cancer cells
Tolde, Ondřej ; Folk, Petr (advisor) ; Kovář, Jan (referee) ; Brdička, Tomáš (referee)
The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...
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Molecular mechanisms of apoptosis induction in tumor cells
Koc, Michal ; Kovář, Jan (advisor) ; Červinka, Miroslav (referee) ; Haškovec, Cedrick (referee)
V. Závěr Tato práce se zaměřila na studium procesů uplatňujících se v indukci apoptosy různými faktory. V našem případě jsme indukovali buněčnou smrt u nádorových buněk deprivací železa, klinicky používanými taxany (paclitaxel, docetaxel) a nově syntetisovanými fotosensitivními látkami. Z presentovaných publikací jsme získali tyto závěry. (1) Studie týkající se indukce apoptosy deprivací železa prokázaly účast odlišných signálních drah vedoucí k indukci apoptosy u myších a lidských nádorových buněk. V obou případech byla použita B lymfomová buněčná linie citlivá k deprivaci železa. U myších buněk jsme popsali významnou část apoptotické signální dráhy u 38C13 buněk sensitivních k indukci apoptosy deprivací železa. Deprivace železa u těchto buněk vede k translokaci proapoptotického proteinu Bax z cytosolu na mitochondrie, která je následována zhroucením mitochondriálního membránového potenciálu v důsledku porušení integrity vnější mitochondriální membrány, uvolněním cytochromu c z mitochondrií, aktivací kaspasy 9 a následnou aktivací kaspasy 3. U lidských buněk jsme taktéž zaznamenali indukci apoptosy deprivací železa, avšak charakterem mechanismu odlišnou od apoptosy popsané u myších 38C13 buněk. V případě lidských Raji buněk sensitivních k indukci apoptosy deprivací železa docházelo pouze k aktivaci kaspasy...
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Mechanisms of apoptosis induction and inhibition by fatty acids in pancreatic β-cells
Šrámek, Jan ; Kovář, Jan (advisor) ; Brunerová, Ludmila (referee) ; Šeda, Ondřej (referee)
Recently, diabetes mellitus type 2 (DMT2) represents one of the most important metabolic diseases according to its incidence and economic impacts. One of the main reasons of this diesease is loss of function and viability of pancreatic β-cells due to the effect of increased levels of saturated fatty acids (FAs). Unsaturated FAs are better tolerated by β-cells. They are even capable of inhibiting detrimental effects of saturated FAs. Molecular mechanisms of apoptosis induction in pancreatic β-cells by saturated FAs as well as mechanisms of inhibition of this induction by unsaturated FAs are not completely elucidated. The main aim of this study was to contribute to elucidation of these mechanisms. Concerning human pancreatic β-cell line NES2Y we demonstarted: (1) Activation of caspase-2 by stearic acid (SA), in apoptosis inducing concentration (1 mM), is not crucial for the process of apoptosis induction. However, this caspase modulates SA-induced endoplasmic reticulum (ER) stress pathways. (2) SA (1 mM) activates the p38 MAPK signaling pathway and inhibits the ERK signaling pathway. Inhibition of the ERK signaling pathway is probably a consequance of the p38 MAPK pathway activation. However, p38 MAPK is not very likely crutial for the apoptosis induction by SA. Unsaturated oleic acid (OA, 0.2 mM) is able to...
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Molecular mechanisms of apoptosis induction by taxanes in breast cancer cells
Jelínek, Michael ; Kovář, Jan (advisor) ; Brábek, Jan (referee) ; Reiniš, Milan (referee)
Taxanes are cytostatic routinely used for the treatment of solid breast, ovarian, prostate, head and neck tumors and other types of tumors. Resistance of tumor cells to the effect of taxanes represents serious obstacle for the employment of taxanes in the treatment of tumors. This resistance can be associated, among other things, with lower rate of apoptosis induction in cancer cells or also with increased level of transporters transporting taxanes out of the cell. In this PhD thesis we tried: (1) to contribute to elucidation of the role of molecular mechanisms of apoptosis induction by taxanes in cells of human breast cancer. Specifically, it meant to contribute to elucidation of the role of initiator caspase -8 a - 9 and mainly of initiator caspase-2. Next, to contribute to elucidation of the role of executioner caspase -3 - 6, and -7 and selected proteins of the Bcl-2 family. (2) To contribute to elucidation of molecular mechanisms of resistance of human breast cancer cells to taxanes. Specifically, it meant to describe the role of selected functional groups in taxane structure in bringing about and overcoming resistance to taxane and next to contribute to elucidation of the role of P-glycoprotein (ABCB1 transporter) in the resistance to individual taxanes. 1) We found that caspase-2 represents...
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Molecular mechanisms of iron transport across plasma membrane in mammalian cells
Balušíková, Kamila ; Kovář, Jan (advisor) ; Ehrmann, Jiří (referee) ; Krijt, Jan (referee)
Iron belongs among the trace elements and its role in humans is irreplaceable. Up to 5 g of iron can be found in adult body distributed among different compounds. Iron ions are therefore essential to all cells of our body and its homeostasis is thoroughly controlled. Iron uptake into the organism is mediated by enterocyte cells in the small intestine, where heme as well as non-heme forms of iron are absorbed. Non-heme iron is absorbed via Dcytb (duodenal cytochrome b), DMT1 (divalent metal transporter 1), ferroportin, hephaestin, and ceruloplasmin molecules. Although these molecules can also participate in non-transferrin- bound iron transport across plasma membranes within the whole organism, mechanisms of this transport are not yet fully elucidated. The aim of the present work was to contribute to our understanding of molecular mechanisms that are involved in non-transferrin-bound iron transport across the plasma membrane of mammalian cells. Our project was focused on the description of non-transferrin- bound iron transport in human cells in vitro and in vivo under conditions of iron deficiency or iron overload. Transformed cell lines, that represent the three main types of cells involved in iron homeostasis, and tissue samples of duodenal biopsies were used as experimental models. The expression...
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Expression of apoptosome pathway regulators and activation of apoptosome apparatus in non-small cell lung carcinoma
Moravčíková, Erika ; Křepela, Evžen (advisor) ; Kovář, Jan (referee) ; Kotyza, Jaromír (referee)
Background: The apoptosome pathway is interesting as potential therapeutic target because it plays an important role in the cancer chemotherapy- and biological therapy-induced apoptosis as well as in amplifying the death receptor and cytotoxic-granule-induced pathways. The functionality of apoptosome apparatus in non-small cell lung carcinoma (NSCLC) cells and tissues is often impaired due to defects in apoptosome pathway by changes in expression and/or acquired mutations and/or modifications of apoptosome components or its regulators that play a significant role in cancer cell proliferation and treatment unresponsiveness. Therefore, this thesis is aimed at the investigation of the expression status of apoptosome pathway regulators (survivin, HBXIP, XIAP, APIP, and UACA) and of the readiness of apoptosome apparatus activation in non-small cell lung carcinoma cells and tissues. Methods: Following methods were used in this thesis: isolation and quantification of total RNA, real-time RT-PCR analysis, preparation of cell-free cytosol samples and extracts from cells and tissues, gel-filtration chromatography, Western blot analysis, enzyme analyses and cell culture techniques. Results and conclusion: Non-small cell lung carcinoma has a higher predisposition to apoptosome-mediated apoptosis than normal...
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Molecular mechanisms of apoptosis regulation by fatty acids in pancreatic β-cells
Němcová, Vlasta ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
The incidence of type 2 diabetes is growing rapidly and represents a big threat for the human health care and economy system as well in the 21st century. The association of type 2 diabetes with obesity is apparent and dysfunction and apoptosis of pancreatic β-cells caused by elevated levels of fatty acids in circulation are considered as an important factor contributing to the development of this disease. However, molecular mechanisms that underlie these detrimental effects of fatty acids are only partially understood. The aim of this research project was to contribute to elucidation of mechanisms by which saturated and unsaturated fatty acids regulate viability and apoptosis induction in human pancreatic β-cells in vitro. Employing human pancreatic β-cell line NES2Y, we showed that increased levels of relevant dietary saturated fatty acids (palmitic and stearic acid) induce apoptosis of pancreatic β-cells, in contrast to relevant dietary unsaturated fatty acids (e.g. palmitoleic and oleic acid). We found that stearic acid-induced apoptosis is accompanied by significant activation of caspase-2, -6, -7, -8 and -9, but not by significant activation of caspase-3. Nevertheless, it was not associated with significant cytochrome c release, alteration in PIDD, Fas receptor and Fas ligand expression and...
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