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Molecular events associated with resistance to tyrosine kinase inhibitors in leukemia cells.
Hrdinová, Tereza ; Vyoral, Daniel (advisor) ; Klener, Pavel (referee) ; Holoubek, Aleš (referee)
Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disease characterized by the expression of BCR-ABL oncoprotein with constitutive tyrosine kinase activity. Although the development of tyrosine kinase inhibitors (TKI) such as imatinib dramatically improved the treatment of CML, a certain subset of patients develops resistance to TKI drugs. The most common cause of TKI resistance are point mutations in the BCR-ABL1 gene, followed by other mutation-independent mechanisms. Survival and proliferation of CML cells in the presence of TKI drugs are accompanied by adaptive changes in their metabolism. Drug resistance can be maintained by extrinsic signals, among which exosomes, small vesicles released by (drug-resistant) cells, have been shown to play an important role. The aim of this thesis was to characterize two CML cell lines sensitive and resistant to imatinib, as well as the exosomes derived from imatinib-resistant CML cells by proteomic approaches. Identification of metabolic vulnerabilities in drug-resistant cells enables their targeting by clinically available drugs, thus offering potential therapeutic targets for their selective elimination. Analysis of exosomes derived from imatinib-resistant cells can identify specific membrane surface proteins exploitable as clinically relevant...
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Proteomic analysis in hematology: Identification of alfa2-macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the of leukemic K562 cell differentiation induced by sodium butyrate.
Pešlová, Gabriela ; Vyoral, Daniel (advisor) ; Krijt, Jan (referee) ; Suttnar, Jiří (referee)
The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate
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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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Proteomic analysis in hematology: Identification of alfa2-macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the of leukemic K562 cell differentiation induced by sodium butyrate.
Pešlová, Gabriela ; Vyoral, Daniel (advisor) ; Krijt, Jan (referee) ; Suttnar, Jiří (referee)
The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate
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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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Iron-sulfur proteins and the role of iron in the gene expression of Trichomonas vaginalis
Horváthová, Lenka ; Tachezy, Jan (advisor) ; Rasoloson, Dominique (referee) ; Vyoral, Daniel (referee)
Iron is an essential nutrient for the parasitic protist Trichomonas vaginalis as a component of iron-sulfur (FeS) proteins that are indispensable for energy metabolism of the parasite. The FeS clusters are formed by FeS cluster (ISC) assembly machinery that resides, together with a number of FeS proteins, in Trichomonas hydrogenosomes. These double-membrane bound organelles, which are related to mitochondria, metabolize malate and pyruvate and produce ATP and molecular hydrogen. To obtain more complete information about hydrogenosomal pathways with particular focus on FeS proteins and ISC machinery, we participated on analysis of T. vaginalis genome sequence. To predict hydrogenosomal localization of putative gene products, we developed an application "Hunter" for the in silico searching for N-terminal presequences that are required for protein targeting into the hydrogenosomes. This approach substantially contributed to annotate genes coding for hydrogenosomal proteins that provided base for construction of novel map of hydrogenosomal metabolism as well as for following proteomic studies. Investigation of hydrogenosomal proteins led to identification of three members of Hyd machinery that is required for the maturation of the specific FeS cluster of hydrogenases named H cluster. T. vaginalis is,...
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The Biochemistry of Zine and Iron Proteomic Studies
Babušiak, Marek ; Vyoral, Daniel (advisor) ; Hrkal, Zbyněk (referee) ; Černý, Jan (referee)
This thesis, The Biochemistry of Zinc and Iron - Proteomic Studies, consists of five sections. We were studying the biochemistry of two different metal elements - iron and zinc, therefore the thesis has two main aims (iron and zinc related) and each of the experimental sections are divided into two logical subsections A and B. The first section - Introduction includes a general introduction reviewing the current knowledge about the biochemistry of iron and zinc and it also specifies the objectives of this study. The first part of the Introduction is a review of iron biochemistry. It has following seven chapters: General properties of iron; Iron absorption, storage and toxicity in human; Iron transport and homeostasis; Iron regulatory proteins - IRP/IRE system; Mitochondrial iron metabolism; Liver and iron and Iron disorders. In the second part of the Introduction, the biochemistry of zinc is reviewed in these eight chapters: Zinc - general properties; Zinc transporters; Zinc finger proteins; The role of zinc fingers in leukemogenesis; Functions of zinc in cellular signalling; Regulatory functions of zinc in cell proliferation; Role for Zinc in cell differentiation and apoptosis; Zinc - remarks and perspectives. The third part of the Introduction introduces the specific objectives of this thesis. It gives a...
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