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Analysis of the pathways responsible for the resistance of leukemic cells towards L-asparaginase
Šimčíková, Markéta ; Konvalinka, Jan (advisor) ; Poljaková, Jitka (referee)
Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood. Despite the very successful ALL therapy, relapses occur to 15-20 % of children. One of the possible relaps causes is the resistance to therapeutics. ALL is treated with combined chemotherapy in which cytostatic agent L-asparaginase plays the essen- tial role. L-asparaginase depletes extracellular asparagine and glutamine. Antagonist of the L-asparaginase is asparagine synthetase enzyme, which synthesizes the cellular asparagine. The specific antileukaemic effect of L-asparaginase is believed to be thanks to lower activity of the asparagine synthetase in leukaemic cells comparing to the healthy cells. The asparagine and glutamine deficiency harms the cellular proteosyn- thesis and induces apoptosis. Mechanism of the L-asparaginase cytotoxic effect and mechanism of corresponding resistance is still not fully explained. This bachelor thesis is a part of a project studying mechanisms of leukaemic cells resistance to L-aparaginase. In the model leukaemic REH cell line a deletion del(5)(q34) was discovered, which cannot be found in the resistant clone of these cells. This thesis focuses on proving different sensitivity of leukaemic cells, with or without the deletion, to L-asparaginase. The limiting dilution was used to obtain...
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Carbohydrate dimers in tumor immunotherapy
Krupová, Monika ; Bezouška, Karel (advisor) ; Ryšlavá, Helena (referee)
Carbohydrate dimers in tumor immunotherapy Monika Krupová (Department of Biochemistry, Faculty of Science, Charles University in Prague) One possible approach to tumor immunotherapy is an activation of killer lymphocytes through specific ligands for their surface receptors. CD69 is a molecule greatly widespread among various cells of haematopoietic origin. Since the physiological ligand for this receptor is still unknown, ligand mimetics are used for modulation of its activity. The mimetics tested in this work are based on monomeric or oligomeric carbohydrated attached through two different chemical groups to the central linker of varying length, giving rise to thiourea and triazole series. The ability to precipitate soluble NKR-P1 and CD69 receptors was evaluated in precipitation assays and the optimal length of the linker for NKR-P1 receptor was found to be decyl. On the other hand, cross-linking of CD69 is not so dependent on the length of the linker. The aim of this work was to describe in vitro effect of the tested compounds on cellular signalization, natural killing of leukemic cell lines and activation-induced apoptosis. Compounds of triazole series containing two disaccharides (GalNAc β1→4 GlcNAc) linked by a linker were found to have the strongest effect on the production of...
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The influence of acyclic nucleotide phosphonates PMEG and PMEDAP on p38 kinase signaling in human leukemic cells
Nejedlá, Michaela ; Entlicher, Gustav (advisor) ; Slaninová, Jiřina (referee)
PMEG [9-(2-phosphonomethoxyethyl)guanine] and PMEDAP [9-phosphonomethoxy- ethyl)-2,6-diaminopurine] are acyclic nucleoside phosphonates possessing cytotoxic properties. Antiproliferative effect of PMEG was demonstrated in various tumor cell lines in vitro. PMEG also represents an active component of some experimental prodrugs with enhanced selectivity and efficacy (such as GS-9219). PMEDAP seems to have weaker effect in vitro compared to PMEG, however it exhibited pronounced antitumor effect in SD-rats with spontaneous lymphoma. Therefore it was included in the present study as well. The aim of this study was to describe the interactions of PMEG and PMEDAP with p38 MAP kinase signaling and its relationship to the apoptosis. We investigated the influence of these compounds on the expression of four genes encoding p38 MAPK isoforms and whether this change is translated into the protein. It was found that PMEG up-regulates p38β and γ mRNA in CCRF-CEM cells and p38 β and δ in HL-60 cells. The effect of PMEDAP was less pronounced than that of PMEG. However, total p38 protein level remained unaffected by PMEG and PMEDAP. Activation of p38 MAPK cascade was also measured in the cells exposed to these agents using phospho-specific antibodies. We found that neither PMEG nor PMEDAP activated p38 kinase...
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Immunological testing of combined LELTE / saccharide dendrimers
Kádek, Alan ; Man, Petr (referee) ; Bezouška, Karel (advisor)
Immunological testing of combined LELTE / saccharide dendrimers. (in Czech) Alan Kádek (Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic) CD69 is a widespread receptor of the immune system cells. Although a physiological ligand for this receptor is still unknown, in Laboratory of protein architecture its affinity towards a range of compounds including calcium, carbohydrates and charged compounds such as carboxylated calixarenes, has been proved. Moreover, a pentapeptide sequence LELTE derived from a mycobacterial chaperonine protein hsp65 has been recently identified as a ligand for CD69 representing a "danger" signal for the immune system. However, this peptide is not immunoactive per se, but only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using bi- or polyfunctional reagents. In this thesis I evaluate for four newly synthesized compounds their in vitro immunological effects on cellular signalization, proliferation, natural killing of tumors and on induction of apoptotic cell death in immunocytes. The tested compounds present LELTE peptide through attachment to a cyclopeptidic RAFT scaffold (K-K-K-P-G)2 through the ε-amino groups of lysine residues, alone or in combination with a...
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Plant alkaloid sanguinarine and its derivatives
Tůmová, Lucie ; Hýsková, Veronika (referee) ; Stiborová, Marie (advisor)
This work is summarizing actual knowledge about sanguinarine and quaternary benzo[c]fenanthridine alkaloids. The quaternary benzo[c]fenanthridine alkaloids were found in roots plants Sanguinaria canadensis and Macleaya cordata. This plants are used in tradicional Chinese medicine for its antimycotic, antibacterial and anti-inflammatory activities since antiquity. Regarding to possibility quaternary benzo[c]fenanthridine alkaloids to induce apoptosis these investigated such as possible agents for cancer treatment. The quaternary benzo[c]fenanthridine alkaloids interact with DNA and proteins. They are able to intercalate to the DNA. The alkaloids can be used like fluorescence DNA probe. Metabolism by sanguinarine and next quaternary benzo[c]fenanthridine alkaloids has not yet completely determinated. The first step sanguinarine detoxication is its conversion to less toxic dihydrosanguinarine. Sanguinarine oxidation is mediated by cytochrome P450 1A1. Key words: Quaternary benzo[c]fenanthridine alkaloids, sanguinarine, apoptosis, intercalate, heterogenous substances, enzymes, cytochrom P450
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