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Development and implementation of new approaches for proteomic characterization of bone tissues in oral surgery
Michalus, Iva ; Hynek, Radovan (advisor) ; Šebela, Marek (referee) ; Hrabal, Richard (referee)
1 Abstract Proteomics is a booming field with application in many areas of medicine, including dentistry. Nevertheless, proteomic characterization of bone tissues in oral surgery is not still commonly used. The main reason is involvement of demanding analytical approaches due to insoluble chatacter of bone tissues. The goal of this work was to develop and apply straightforward methodology that could lead to the routine use of proteomics in this area as well. Using porcine jawbones as a model samples, a technique was developed allowing identifying about hundreds of proteins thanks to their trypsin digestion directly in bone tissues ("in-bone digestion") followed by analysis using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This technique was subsequently applied to the analysis of human maxillary and mandibular bone tissues obtained during surgical procedures. In both maxillary and mandibular bone samples, it was possible to identify a considerable amount of proteins using the "in-bone digestion" technique. Additionaly, the mathematical analysis of the obtained data was able to distinguish between the inflammatory and healthy tissues. The approach based on direct cleavage was subsequently successfully extended to the analysis of in vitro models of human bone tissues. Direct...
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Proteomic analysis of engineered hyaline cartilage
Kaňovská, Zuzana ; Vičarová,, Petra (referee) ; doc. RNDr. Irena Koutná, Ph.D (advisor)
The aim of this thesis was to observe the expansion of chondrocytes, specialized cartilage cells, in vitro in culture medium containing platelet rich plasma, to evaluate the effect of the addition of growth factors TGF-1 and IGF-1 to the medium and to explore the possibility of proteomic analysis application in the development of cartilage joint replacements. Chondrocytes were isolated form articular hyaline cartilage samples acquired in collaboration with the FN Brno and expanded in media. The resulting constructs were subsequently analyzed by immunocytochemical method, flow cytometry and proteomic analysis using LC-MS/MS.
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Molecular events associated with resistance to tyrosine kinase inhibitors in leukemia cells.
Hrdinová, Tereza
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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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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Changes of membrane-bound and soluble proteins of frontal rat brain cortex induced by morphine
Ujčíková, Hana
The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....
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Oxidace proteinů a lipidů u ryb: dráhy, kinetika a produkty
HEMATYAR, Nima
Fish muscle contains omega-3 fatty acids and high quality protein and other valuable nutrients. On the other hand, due to the high content of water, neutral pH, presence of PUFA and sensitive amino acids, fish muscle is a very perishable food. The progress of lipid and protein oxidation as well as interaction between the oxidation products could have a negative role during storage time. Negative effect of oxidation on the fish fillet quality has been reported by several authors, but still the pathways and products of protein oxidation owning to the diversity of the protein oxidation products is not clear. Pathways of lipid and protein interaction, target amino acids, storage conditions and also, final impact on the fish muscle quality have been investigated in this study. We examined the quality of fish muscle in common carp and perch during different storage conditions (+4°C and -20°C). Also, we compared some quality parameters in perch muscle from two different rearing systems to identify the pathways and products of oxidation. The present study investigated the oxidation progress with respect to TBARS and carbonyls as markers of lipid and protein oxidation respectively as well as contribution of PUFA on the lipid oxidation development. Further, the effect of storage conditions and oxidation on the textural and sensorial parameters were examined. Possible changes of pH, rigor index and biogenic amines, during post mortem were studied. Additionally, we performed proteomic analysis in order to investigate more deeply the protein alteration and find a correlation between protein structures and fish muscle quality. Our results showed a good stability in common carp (Cyprinus carpio) fillet during 6 months storage at -20oC. According to our results, we observed a significant decrease in the firmness of common carp muscle after 1 week and then it was constant while liquid loss increased significantly with storage time. Regarding the sensory parameters, we could not find any differences in the examined parameters in both stored raw and cooked fillets compared to the fresh one. Although, both lipid oxidation parameters (TBARS and PV) were increased significantly but it did not reach the critical level for human consumption. On the other hand, protein oxidation which is involved in WHC deterioration was increased significantly. In addition, we investigated the muscle quality of Eurasian perch (Perca fluviatilis L.) from two rearing systems (RAS and pond). During frozen and refrigerated storage, we observed a significant increase in the amount of TBARS and carbonyls in both rearing systems. Comparison between both rearing systems did not show any differences in the amount of TBARS while carbonyl was significantly higher in the RAS fillets rather than pond fillets particularly, until 4 month storage at -20oC. Also, we found a significant increase in liquid loss and considerable decrease in firmness in the fillets from both rearing systems. Our results showed higher firmness and liquid loss in the RAS fillets compared to the pond one. Proteomic analysis on the fillets from both rearing systems during frozen storage showed more stability in the RAS fillet however, western blot revealed more oxidized carbonyls in the RAS fillets rather than pond fillet specially, at time 0 and 4. Probably, formation of ice crystals and accumulation of ROS have key roles on the development of oxidation during frozen storage while enzymatic activity follow by protein degradation are important during refrigerated storage.
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Changes of membrane-bound and soluble proteins of frontal rat brain cortex induced by morphine
Ujčíková, Hana ; Svoboda, Petr (advisor) ; Mikšík, Ivan (referee) ; Farghali, Hassan (referee)
The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....
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Changes of membrane-bound and soluble proteins of frontal rat brain cortex induced by morphine
Ujčíková, Hana
The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....
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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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