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Energy metabolism and apoptotsis markers in cold heart aclimated rat.
Pospíšilová, Barbora ; Horníková, Daniela (advisor) ; Knytl, Martin (referee)
Cold adaptation and her effects has been known for many decates. Positive or negative impact depends especially on its length and strength. The lower temperature can very often cause the stress for organism. On the other hand in expreriment with long-term adapatation were found positive consequences on cardiovascular system. We found the lack of studies devoted to the energy metabolism and apoptosis in heart tissue during long-term cold adaptation. In this work we used a model with milder conditions of the adaptation (10žC±1), so there wouldn't be damage of the experimental animals. We compared the resuls betwen control, cold and regressive group of rats. In this expreriment we used methods of electrophoresis and Western blot. The target of the work was found if we can find any differences betwen chosen HIF targeted genes. The next goal was to detect the differences betwen chosen pro-apoptotic and anti-apoptotic markers. Keywords: cold adaptation, heart, energetic metabolism, HIF, apoptosis
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Apoptosis of tumor cells : role of TRAIL and caspase 10
Truxová, Iva ; Živný, Jan (advisor) ; Anděra, Ladislav (referee)
One of the key features of cancer cells is the ability to escape programmed cell death (apoptosis). As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro-apoptotic genes have been reported in many cancers. Caspase 10 is an initiator caspase whose physiological function remains poorly understood. Also the ability of caspase 10 to substitute for caspase 8 in the death receptors apoptotic pathway is still controversial. However, the fact that some of the mutations found in CASP10 gene was associated with apoptosis defects (79, 81) suggest that caspase 10 could be also important in apoptosis initiation. In our lab, there was found a heterozygous mutation in CASP10 gene of Jurkat (human T-acute lymphoblastic leukemia) clone resistant to TRAIL (J-TR1). This mutation influence the amino acid composition close to the active site of the enzyme. The aim of this thesis was to confirm the mutation by ARMS-PCR and to determine if an overexpression of normal (unmutated) or mutated caspase 10 D in TRAIL sensitive and/or TRAIL resistant Jurkat cells (J-WT and/or J-TR1) will influence TRAIL induced apoptosis. Mutation was confirmed. We created J-WT and J-TR1 stable clones transfected by vector with unmutated or mutated CASP10 D (CASP10 D WT or CASP10 D MUT). CASP10 D MUT overexpression in J-WT...
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Gene expression of enzymes involved in the regulation of apoptosis in rat moycardium - effect of chronic and acute hypoxia
Blahová, Tereza ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee)
Adaptation to chronic hypoxia provides myocardial protection against ischemia - reperfusion injury (IR). Cardioprotective effect of adaptation depends on the degree and duration of hypoxic exposure and daily regime of adaptation. Certain protective regimes of adaptations to hypoxia have been reported to activate proapoptotic signaling pathways and bioactive sphingolipids were recently shown to play important role in the regulation of apoptosis in the heart. We aimed to determine the mRNA level of selected genes related to apoptotic pathways and to sphingolipid metabolism in two models of hypoxic adaptation, continous normobaric hypoxia (CNH 10% O2) with different exposures (4h, 48h, 120h, 21days) and intermitent hypobaric hypoxia (IHH 7000 m, 8h/day). Both ventricles, LV and RV, were analysed after adaptation to CNH and only LV was analysed after IHH adaptation. Our results show that both types of adaptation increased mRNA of proapoptotic genes, CNH mainly in RV and IHH in LV. Furthermore, increased expressions of proapoptotic genes were accompanied by the increase of expression of enzymes producing predominantly protective kinds of sphingolipids. The exact role of apoptosis and sphingolipid signaling molecules in endogenous myocardial protection requires further research. Key words: Apoptosis,...
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Cell death as a result of iron-induced cellular damage
Běhounek, Matěj ; Balušíková, Kamila (advisor) ; Truksa, Jaroslav (referee)
Iron is an essential trace element for almost all living organisms. Iron overload in cells and tissues, however, leads to their disruption. Most oftenly damaged are parenchymatic organs such as the liver, pancreas and heart. The aim of this thesis was to create cellular in vitro models for the investigation of effects of excess iron on hepatocytes and pancreatic beta cells and on these models to investigate cellular processes which lead to cellular damage during iron overload. We focused on examining the presence of oxidative and endoplasmic reticulum stress and the activation of apoptotic cell death. For our experiments, we used HEP-G2 cell line which represents human hepatocytes and NES2Y cell line which represents human pancreatic beta cells. To study the mechanisms of cellular damage during iron overload, we used two approaches by which we observed both acute and long-term effects of high levels of iron on damage of the tested cell lines. When studying the acute effect of excess iron on the cells, we applied high doses of iron (using 15 mM ferric citrate in medium) that led to the activation of cell death in hours. Long-term effects of iron overload were tested on cells regularly cultivated in the presence of 50 μM and 100 μM ferric citrate over a period of several months. Iron concentrations...
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Canonical and non-canonical signalling triggered by activated TRAIL receptors in human cells
Nahácka, Zuzana ; Anděra, Ladislav (advisor) ; Rudolf, Emil (referee) ; Vondráček, Jan (referee)
TRAIL ligand can trigger apoptosis of permissive human cells via engagement of its two pro- apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Its ability to induce apoptosis independently on p53 status and to selectively kill cancer cells in vitro and in vivo made this ligand an attractive target in cancer research. However, acquired resistance of primary cancer cells, unsatisfactory outcome of clinical trials and recent studies arguing that TRAIL might under specific conditions promote cancer progression, opened new plethora of questions, which need to be addressed. Though both receptors DR4 and DR5 are ubiquitously expressed, different types of tumours show preference for either of the receptors. The relative participation of DR4 and DR5 in TRAIL- induced signalling is still largely unknown. To analyse TRAIL receptor-specific signalling, I prepared Strep-tagged, trimerised variants of recombinant human TRAIL ligands with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, I examined a contribution of individual pro-apoptotic receptors to TRAIL-induced signalling pathways. I found that in TRAIL resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeded comparably in both DR4- and...
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The role of fatty acids in apoptosis induction in pancreatic beta cells
Žigová, Ivana ; Němcová, Vlasta (advisor) ; Libusová, Lenka (referee)
Type 2 diabetes belongs to lifestyle diseases. To its development contributes also the decrease in functional cell mass caused particularly by long-term action of increased blood levels of fatty acids and glucose. Experiments from recent years demonstrated that apoptosis of pancreatic cells is induced especially by saturated fatty acids, in contrast to unsaturated fatty acids which do not exert significant cytotoxic effect and they are even able to inhibit the proapoptotic effect of saturated fatty acids. Despite intensive research, the mechanisms of fatty acid-induced apoptosis of pancreatic cells are not convincingly explained. Understanding of the processes that lead to destruction of functional cells could enable development of new therapies for type 2 diabetes treatment that would be targeted directly at one of the causes of this disease. The aim of this work is to summarize the recent knowledge about mechanisms by which fatty acids induce and regulate apoptosis of pancreatic cells.
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The role of autophagy in apoptosis induction by fatty acids in pancreatic beta cells.
Žigová, Ivana ; Němcová, Vlasta (advisor) ; Truksa, Jaroslav (referee)
Type 2 diabetes mellitus represents a metabolic disease reaching epidemic dimensions in the 21st century. Fatty acid-induced apoptosis of pancreatic β-cells significantly contributes to its pathogenesis. Saturated fatty acids (FAs) are strongly cytotoxic for β-cells, whereas unsaturated FAs are well tolerable by β-cells, they are even able to inhibit proapoptotic effects of saturated FAs when co-incubated. According to recent studies, FAs-induced apoptosis in pancreatic β-cells is partly regulated by autophagy, a catabolic process involved in the degradation and recyclation of cell components in lysosomes. The aim of this diploma thesis was to contribute to the clarification of the role of autophagy in FAs-induced apoptosis regulation. We induced apoptosis in human pancreatic β- cell line NES2Y by 1 mM stearic acid (SA) and inhibited it with 0.2 mM oleic acid (OA) co- incubated with SA. We revealed, that the saturated SA used in apoptosis-inducing concentration simultaneously inhibits the autophagic flux in pancreatic NES2Y cell line. When SA is co- incubated with unsaturated OA in concentration sufficient for inhibition of proapoptotic effect of SA, OA is also able to inhibit the block of autophagy induced by the effect of SA. Application of unsaturated OA alone in this concentration did not...
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Application of Mass Spectrometry for Analysis of Biologically Active and Clinically Significant Compounds.
Štícha, Martin ; Jelínek, Ivan (advisor) ; Smrček, Stanislav (referee) ; Tůma, Petr (referee)
- 8 - ABSTRACT (EN) The thesis is submitted as a commented set of reviewed publications documenting and depicting the possibilities of mass spectrometry in the field of chemical, biological and pharmaceutical research; namely for the purposes of structure elucidation of selected organometallic complexes, analyses of drugs and their metabolites, monitoring of important biological markers. In course of experimental work, the following objectives were studied and solved: Proposal and realization of micro-scale preparation of selected rhenium complexes with aromatic ligands, utilizing tetrabutyammonium tetrachlorooxorhenate as a starting material; preparation and structure characterization of oxorhenium(V) complexes with 1,2-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2,3- dihydroxynaphtalene as ligands by means of ESI/MS, APPI/MS and LDI-MS; ESI/MS and UV/Vis study of kinetic behavior of complexes arising from the reaction of tetrabutylamonnium tetrachlooxorhenate with pyrogallol and catechol as ligands. Special aim was devoted to the study of subsequent chemical transformation of primarily formed Re(V) complexes; structure characterization of selected ferrocene complexes with copper, gold and silver by means of ESI/MS. Proposal of methodology of structure characterization and quantification of the...
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Resenzitalizace leukemických a lymfomavých buněk k trailerem indukované apoptóze
Molinský, Jan ; Klener, Pavel (advisor) ; Hyršlová Vaculová, Alena (referee) ; Vyoral, Daniel (referee)
Apoptosis serves as a natural barrier to cancer development, and the resistance to apoptosis represents one of the key capabilities acquired during tumor development or progression. Impairment of the intrinsic apoptotic pathway exemplifies one of the established mechanisms of constitutive or acquired drug resistance. As most of the currently used cytotoxic drugs initiate tumor cell death by direct or indirect triggering of the intrinsic apoptotic pathway, impairment of the intrinsic pathway is associated with therapy failure. Targeting of the death receptors, however, enables induction of apoptosis even in the chemotherapy resistant cancer cells. TRAIL is a death ligand belonging to the TNFα superfamily that specifically kills tumor cells while sparing healthy tissues. Much enthusiasm has been generated for TRAIL as a highly promising targeted anti-cancer agent. However, many primary tumors have been shown to be TRAIL resistant. In attempt to overcome such an intrinsic TRAIL resistance a wide array of agents have been shown to sensitize tumor cells to TRAIL. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. In this study we analyzed the sensitivity of diverse hematologic malignancies to TRAIL-induced apoptosis and measured the...
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