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Structure and function relationships of model hemoproteins
Lengálová, Alžběta ; Martínková, Markéta (advisor) ; Hudeček, Jiří (referee) ; Muchová, Lucie (referee)
Heme is one of the most important and most studied cofactors that are essential for proper function of many proteins. Heme-containing proteins comprise of a large group of biologically important molecules that are involved in many physiological processes. The presented dissertation is focused on two groups of heme sensor proteins, namely prokaryotic heme-based gas sensors and eukaryotic heme-responsive sensors. Heme-based gas sensors play an important role in regulation of many bacterial processes and consist usually of two domains, a sensor domain and a functional domain. The dissertation thesis aims at the study of two model bacterial heme-based gas sensors, histidine kinase AfGcHK and diguanylate cyclase YddV, in order to elucidate their mechanism of interdomain signal transduction. Using X-ray crystallography and hydrogen-deuterium exchange coupled to mass spectrometry approaches, significant differences in the structure of the AfGcHK protein between the active and inactive forms were described. The signal detection by the AfGcHK sensor domain affects the structural properties of the protein, and these conformational changes then have indirect impact on the enzyme activity of the functional domain. Further, the dissertation pays more attention to the effect of a sensor domain dimerization...
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Assessment of anthropogenic modification of the river network using hydromorphological survey as an example of the upper Metuje River
Kavalír, Václav ; Matoušková, Milada (advisor) ; Kujanová, Kateřina (referee)
Human modifications and related pressure on watercourses of its ecosystem became greatly discussed problematic in the latest dry years. The translated bachelor's thesis deals with these encroachments on watercourses as well as with its impact on hydromorphological quality. In recherché there are discussed basic hydromorphological knowledge and modifications of watercourses with its negative and positive effects on the watercourse along with its surrounding. For the evaluation of the hydromorphological condition was used HEM methodology (Langhammer 2014) applied on upper course of the Metuje River. Overall, based on the outcome of the HEM methodology the upper stream of the Metuje River is evaluated as a weakly modified. Less disturbed sections can be found in the area of the National Nature Reserve Adršpašsko-Teplické skály. The largest modifications can be seen in the build-up areas of Teplice nad Metují, where these modifications come particularly from the period of the First Republic. The area of the floodplain, is the most affected area. Trace of the rivercourse was overall shorted by 1 569 meters, therefore 7,5 % since mid-19th century, chiefly in the surrounding of Teplice nad Metují. The greatest concentration of diverse modifications is in the build-up zones. Key Words: Hydromorphology,...
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Biochemical characterization of a model heme sensor protein
Tajovská, Eva ; Martínková, Markéta (advisor) ; Jeřábek, Petr (referee)
Hemoproteins play a lot of important roles within the living organism. One of these being the sensor function - heme sensor proteins are able to detect the changing concentration of heme in organisms. Heme itself serves as a signalling molecule for these proteins. Heme-based gas sensor proteins use a biatomic gas molecule as a signalling molecule. Signal transduction begins once the gas molecule is bound to the heme molecule which is already tightly bound in the protein sensing domain. Both these types of signalling regulate a number of physiological processes in the prokaryotic and eukaryotic organisms. The theoretical part of the bachelor thesis summarizes recent scientific studies on heme sensor proteins, while the experimental part focuses on the properties of a specific model sensor hemoprotein. A direct oxygen sensor from E. coli (EcDOS) was selected as a model protein. Furthermore, the thesis deals with a truncated form of the EcDOS protein, an isolated sensor domain with a PAS structure (EcDOS-PAS). The experimental part aimed at expressing and isolating of both mentioned proteins from E. coli BL-21 (DE3) cells. Finally, both the EcDOS and EcDOS-PAS proteins were preliminarily characterised and their properties compared. The yield of the EcDOS-PAS was several times higher than the yield of...
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Assessment of runoff conditions on the river in the Morava River Basin
Serafin, Petr ; Niklová, Věra (referee) ; Uhmannová, Hana (advisor)
The aim of the diploma thesis was to assess the curent state of the Svratka river and propose appropriate measures in accordance with the plans of Povodí Moravy, s.p. Locality of interest is located in the built-up area and non-urban area part of Herálec at river kilometer 163,9809-164,5189 and extends over the cadastral areas of Český Herálec, Herálec na Moravě and Svratka. The assessment of the river was performed on the flow series using the 1D mathematical model HEC – RAS version 5.0.6. On the basis of the results of the flow rate, was the locality od interest divided into four parts. For parts which are located in the urban, was designed a flood protection against of the 20 – year flow. For parts which are located in the non-urban area, was designed revitalization with the possibility of water retention in the landscape. The final design was verified by calculation in the same program HEC – RAS. The diploma thesis contains text part, calculation part and drawing part documenting the proposed measures.
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Study of mechanism of signal transduction in case of two model heme-containing sensor proteins
Mihalčin, Peter ; Martínková, Markéta (advisor) ; Kavan, Daniel (referee)
Heme-based gas sensing proteins belong to a group of proteins that are present in signalling pathways of bacteria. A precise regulation of physiological functions, such as intercellular communication or biofilm production, is essential for the survival of these bacteria and their adaptation to the changing surrounding conditions. Heme-based gas sensors are able to detect the concentration of gas molecules in the local environment via their sensory domain (which contains a heme molecule as the intrinsic detection site) and transmit the signal to the functional domain helping to regulate the adaptation of many processes. These, often pathogenic, processes contribute to extended resistance of bacteria against antibiotics. Heme-based sensors are thus potentially a new therapeutic object of interest in antimicrobial treatment. In order to provide this type of treatment, it is crucial to understand the exact mechanism of intramolecular signal transduction facilitated by heme-based sensors. One of the approaches to unravel these mechanisms is further study of model sensory proteins. This thesis focuses on the analysis of a signal transduction performed by two model globin-coupled heme-based oxygen sensors.
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Characterization of selected properties of model heme-containing sensor proteins
Fojtík, Lukáš ; Martínková, Markéta (advisor) ; Svášková, Dagmar (referee)
Heme sensor proteins are the fourth group of hemoproteins. In this group of hemoproteins heme plays an important role in signalization. Dissociation and/or association of heme detecting proteins serves as an important physiological function in regulation of enzyme activity or gene expression. In this bachelor thesis all the actual knowledge about selected forms of eukaryotic heme sensor proteins previously published in scientific articles are summarized. The experimental part of this bachelor thesis is focused on preparation of recombinant protein heme regulated inhibitor (HRI) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). Firstly the preparation of the plasmids with genes HRI and eIF2α was conducted. In the next step these proteins were prepared in prokaryotic system formed by E. coli BL-21(DE3). The final sample of HRI (7,7 μM in total volume 400 µl and 60 % of homogenity) and the final sample of eIF2α (51,3 μM in total volume 400 µl and 80 % of homogenity) were obtained by the purification process. The study of thermal stability of these samples provided important informations on appropiate storage and manipulation with them in further experiments. Key words: heme-base sensors, heme, kinase, tranduction of signal, isolation of plasmids, prokaryotic expresion,...
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Assessment of runoff conditions on flow in the cadastral area of Dašice
Janovská, Michaela ; Vysloužilová, Lucie (referee) ; Uhmannová, Hana (advisor)
This thesis is aimed at assessment of drainage conditions at the river Loucna in the cadastral area of Dasice. The examined section is 3,88 km long and runs through out of the city and in the city of Dasice. On the left bank of the river there are several historic buildings that are potentially at risk for 100 years of flow. Capacity assessment is performed using the 1D mathematical program HEC - RAS in which the flow model is created and the calculation of the flow rates for individual N - year flows. On the basis of the results of the program is conducted conceptual desing of flood protection measure. At the same time is conducted conceptual desing of fish ladder on the weir and a sluice closure on the intake for the race.
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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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