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Use of capillary electrophoresis with UV photometric and electrospray ionization mass spectrometric detection for the study of interaction of compounds
Konášová, Renáta ; Jaklová Dytrtová, Jana (advisor) ; Mikšík, Ivan (referee) ; Tůma, Petr (referee)
Capillary electrophoresis (CE) is highly efficient separation method based on the different migration velocity of ions in liquid media in electric field. It is commonly used in analytical laboratories and due to the different separation principle it is applied as a complementary method to the chromatographic methods (HPLC and UHPLC). Beside the applicability of CE for quantitative/qualitative analysis, the method can be used also for physico-chemical characterization of compounds (e.g. determination of acid dissociation constants of weak electrolytes or stability constants of complexes). This work is focused on the applicability of CE methods for determination of physico- chemical characteristic of compounds (acid dissociation constants of triazole fungicides and stability constants of dibenzo-18-crown-6, benzo-18-crown-6 and 18-crown-6 ether complexes with metal ions in hydro-organic solvent mixtures) and on the possibility to use affinity CE (ACE) with electrospray ionization-mass spectrometric detection (ACE- ESI/MS) for the study of non-covalent interactions of compounds. For the online hyphenation of CE and ESI/MS, two highly sensitive CE-ESI/MS interfaces were tested: i) porous tip and ii) nano-sheath liquid flow. The ability of the CE-ESI/MS interfaces to effectively decouple spray and...
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Study of peptide digestion kinetics by trypsin and chiral separations of biologically active compounds by HPLC
Šlechtová, Tereza ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Mikšík, Ivan (referee)
This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....
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Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
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Posttranslational modification of proteins - their analysis and physiological aspects
Mikulíková, Kateřina ; Mikšík, Ivan (advisor) ; Bílková, Zuzana (referee) ; Glatz, Zdeněk (referee)
This thesi s is eoneerned with the deteetion of posttranslational modifieation of proteins. These proteins are identified using liquid ehromatography and eapillary eleetrophoresis, their eoupling and eoupling to mass speetrometry. The first part of the theoretieal ehapter is devoted to the strueture, classifieation and metabolits ehanges of proteins in the human body. Major attention is foeused on eollagen, its strueture and individual eollagen types. The seeond part eonsiders the problems of nonenzymatie glyeation and its influenee on the body and engage in produets of posttranslational modifieation. The third part summarizes the individual separation proeedures and analysis of posttranslationaly modified proteins. The seeond ehapter deseribes experimental parameters, including ehemieals, instrumentation, materials and methods. The preparation of individual samples is also deseribed. All results and findings are summarized and diseussed in the last ehapter. This ehapter is divided in two parts. The first part foeuses on problems of in vivo experiments. The seeond part foeuses on problems of in vitro experiments. Powered by TCPDF (www.tcpdf.org)
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Interaction of Proteins with Low-molecular Substances in vitro. Effect of Glycation, Substances of Plant Origin, and Combination of these Factors on Function and Spectral Properties of Selected Proteins.
Trnková, Lucie ; Dršata, Jaroslav (advisor) ; Patočka, Jiří (referee) ; Mikšík, Ivan (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Ing. Lucie Trnková Supervisor Prof. MUDr. Jaroslav Dršata, CSc. Title of Doctoral Thesis Interaction of proteins with low-molecular substances in vitro. Effect of glycation, substances of plant origin, and combination of these factors on function and spectral properties of selected proteins. Plant polyphenolic compounds naturally occurring in human diet possess a wide range of biological and pharmacological properties. They can interact with various proteins including enzymes. Their interaction with serum albumin has a great significance because of its ability to bind, transport and store many endogenous and exogenous low-molecular compounds present in blood circulation. One of the objectives of this thesis was to study chemical structure-binding affinity relationships of two important groups of polyphenolic substances, namely hydroxycinnamic acids and catechins (flavanols), with the molecule of serum albumin in the in vitro models using spectroscopic (UV-Vis absorption spectroscopy, fluorescence quenching method) and electrophoretic (native and SDS PAGE) methods. Some polyphenols caused the changes in protein conformation and the relationships between their structures and obtained...
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Analysis of proteins and their changes in biological tissues
Kulhavá, Lucie ; Mikšík, Ivan (advisor) ; Coufal, Pavel (referee) ; Bílková, Zuzana (referee)
(EN) Proteins are the important biologically active substances for the organism, their qualitative and quantitative composition determines thein function in the organism. Protein analysis in biological material is an important part of biological and medical research. This dissertation is focused on the optimalization of sample preparation - the biological materials - namely the pre-analytical phase of sample processing and their preparation for their own analases and also introducing new procedures in theie praparation. The conditions of individual analysis approaches were always optimized by means of one- dimensional and two-dimensional gel electrophoresis and mass spectrometry. The obtained results were statistically evaluated and other possible approaches to futher research were outlined. The main biological material studied was human saliva with regard to oral health. Comparative study of human saliva was performed (human saliva obtained from caries-free and caries-susceptible people). The saliva composition of individual fractions was investigated with regard to oral health. The study was carried out to compare differences in the abundances of proteins in the saliva of caries-free and caries-susceptible persons, based on label-free mass spectrometry quantification, and to perform a gender...
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Study of (bio)molecular interactions and enzymatic reactions by capillary electrophoretic methods
Růžička, Martin ; Hudeček, Jiří (advisor) ; Mikšík, Ivan (referee) ; Křížek, Tomáš (referee)
Non-covalent interactions participate in majority of processes in living organisms. The strength of interaction between (bio)molecules can be characterized by binding constants of respective complexes, which can be determined by variety of physico-chemical methods. From these methods, capillary electrophoresis features several advantages: (1) interactions takes place in aqueous solutions between free molecules without necessity of their immobilization, (2) short analysis time, (3) small consumption of analyzed compounds, and (4) easy automation of analyses. Within this dissertation thesis, methods of partial-filling affinity capillary electrophoresis (PF-ACE) were developed for study of interactions between double-strand DNA oligonucleotides and well characterized intercalator ethidium bromide as a model compound. Subsequently, binding constants of oligophenylene derivatives complexes with DNA oligonucleotides were determined. The PF-ACE method was optimized for study of enantioselective interactions between helquats and selected chiral acidic aromatic analytes. Several compounds whose enantiomers were separated using helquats as chiral selectors were identified. Capillary electrophoresis was applied as a separation analytical method for monitoring of peptide substrate cleavage by rhomboid...
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Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
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Use of capillary electrophoresis with UV photometric and electrospray ionization mass spectrometric detection for the study of interaction of compounds
Konášová, Renáta ; Jaklová Dytrtová, Jana (advisor) ; Mikšík, Ivan (referee) ; Tůma, Petr (referee)
Capillary electrophoresis (CE) is highly efficient separation method based on the different migration velocity of ions in liquid media in electric field. It is commonly used in analytical laboratories and due to the different separation principle it is applied as a complementary method to the chromatographic methods (HPLC and UHPLC). Beside the applicability of CE for quantitative/qualitative analysis, the method can be used also for physico-chemical characterization of compounds (e.g. determination of acid dissociation constants of weak electrolytes or stability constants of complexes). This work is focused on the applicability of CE methods for determination of physico- chemical characteristic of compounds (acid dissociation constants of triazole fungicides and stability constants of dibenzo-18-crown-6, benzo-18-crown-6 and 18-crown-6 ether complexes with metal ions in hydro-organic solvent mixtures) and on the possibility to use affinity CE (ACE) with electrospray ionization-mass spectrometric detection (ACE- ESI/MS) for the study of non-covalent interactions of compounds. For the online hyphenation of CE and ESI/MS, two highly sensitive CE-ESI/MS interfaces were tested: i) porous tip and ii) nano-sheath liquid flow. The ability of the CE-ESI/MS interfaces to effectively decouple spray and...
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