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Enzyme activity analysis of function domains belonging to model heme-containing sensor proteins
Prošková, Veronika ; Martínková, Markéta (advisor) ; Macek, Tomáš (referee) ; Vítek, Libor (referee)
EN This Ph.D. thesis focuses on the heme containing gas sensor proteins. These proteins are predominantly present in bacteria, in which play an important role in processes like, sporulation, antibiotic resistance and so on. Heme containing sensor proteins composed of two domains. First one is a globin domain, which contains the heme molecule. Interaction of heme with gas molecule acts as a signal for the activation/inactivation of the second functional domain. Part of this thesis is formed by a review, which summarized the current knowledge about heme containing sensor proteins. In the next part of this thesis we focused on three representatives from the group of oxygen sensor proteins - histidine kinase AfGcHK, diguanylate cyclase YddV and phosphodiesterase EcDOS. The main aim of this thesis was to solve the mechanism of interdomain/intraprotein signal transduction in two oxygen sensor proteins with globin fold of their sensor domain (AfGcHK, YddV). For this purpose, we used the kinetic analysis of their functional domain activity and the methods of structural biology. We also studied the mechanism of interprotein signal transduction in AfGcHK and its cognate partner RR protein. It was also tested, how the presence of sodium disulfide affects the functional properties of oxygen sensor proteins...
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Examples of good practice in teaching English grammar through the inductive metod
MARTÍNKOVÁ, Markéta
The master thesis describes in its introduction the deductive and inductive approach to teaching English grammar, compares them, and also lists the pros and cons of each approach. The main content of the theoretical part is the presentation of key concepts and principles of inductive teaching of English grammar. The practical part converts theoretical knowledge to real grammar teaching. Pupils learned how to express past events within four lessons. These sample lessons are examples of good practice of teaching grammar inductively. The reflection of these lessons is provided at the end of this part.
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Preparation of a plasmid, its expression and preliminary isolation of MafK protein - the interacting partner of heme sensor Bach1
Mihalčin, Peter ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
Bakalárska práca Abstract Heme-sensing proteins are heme proteins to which heme serves as a signalling molecule. Association or dissociation of heme moiety and heme-sensing proteins influences various physiological functions, such as enzyme activity or gene expression regulated by these heme-sensing proteins. The main object of this thesis is heme-sensing protein Bach1 and its interaction partner, transcription factor MafK. Bach1 refers to the group of transcription factors involved in repression of gene expression. The target genes of Bach1 regulation are hemeoxygenase genes. Hemeoxygenase controls the excess free heme degradation. Due to the excess of free heme in the cell, Bach1-heme interaction inactivates Bach1 controlled repression of hemeoxygenase resulting in the free heme degradation. In the state of physiological free heme concentration, Bach1-heme interaction does not occur and activated Bach1 represses the hemeoxygenase expression via binding to the target gene enhancers. Bach1 is incapable of making efficient Bach1-DNA bonding by itself, therefore the transcription factor MafK is essential. Protein MafK modulates the Bach1-DNA binding by making the heterodimer formation Bach1-MafK, which binds to DNA. The first aim of this thesis is to summarize the recent knowledge about transcriptional...
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Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
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Molecular mechanisms of signal transduction in model heme-containing oxygen sensor proteins
Stráňava, Martin ; Martínková, Markéta (advisor) ; Obšil, Tomáš (referee) ; Macek, Tomáš (referee)
EN Heme containing gas sensor proteins play important role in bacterial physiology in regulating many processes such as cell differentiation, virulence, biofilm formation or intercellular communication. For their structure, typical modular architecture is characteristic where various sensor domains (usually at the N-terminus) regulate the activity of the catalytic or functional domains (usually at the C-terminus). In this dissertation thesis, we focused on three representatives from the group of oxygen sensing proteins, namely histidine kinase AfGcHK, diguanylate cyclase YddV, phosphodiesterase EcDOS and also on protein RR, which is the interaction partner of AfGcHK. The main aim of the thesis was to study intra-protein/inter-domain signal transduction in two representatives of heme sensor proteins with a globin fold of the sensor domain (AfGcHK, YddV) and in one representative with PAS fold of the sensor domain (EcDOS). Another objective was to describe inter-protein signal transduction in the two component signaling system AfGcHK-RR and structurally characterize these two interacting partners. Emphasis was also placed on the study of the interaction between model sensor domains and different signaling molecules and also on function of individual amino acids involved in the binding of these...
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Study of interactions between the FOXO3-DNA binding domain and small-molecular compounds
Dolejš, Vojtěch ; Obšilová, Veronika (advisor) ; Martínková, Markéta (referee)
This diploma thesis is part of a project aiming for the development of low molecular compounds which would be capable to inhibit the interaction between human transcription factor FOXO3 and DNA. Main goal of this thesis is the characterization of the interaction between the low molecular inhibitor S9 and the DNA-binding domain of FOXO3 protein (FOXO3-DBD) by using variety of biophysical methods such as NMR, microscale thermophoresis and native electrophoresis. FOXO transcription factors are important and evolutionary conserved regulatory proteins, which are involved in many crucial cellular processes. The activity of FOXO proteins is regulated by posttranslational modifications, out of which the most important are phosphorylation, acetylation and ubiquitination. Forkhead transcription factors participate in a variety of different cellular functions and are part of several signaling pathways. Its expression might be tissue specific. They contain approximately 100 amino acids long DNA- binding domain composed of several parts. Among its main functions belong the regulation of cell cycle and apoptosis, proliferation and cell differentiation, metabolism control and stress- response regulation. Tumor cells of lymfoblastome have developed resistance against chemotherapy by increasing activity of FOXO3...
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The study of properties of anticancer drugs ellipticine, etoposide and doxorubicin in the forms of nanocarriers
Lengálová, Alžběta ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...
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