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ALAS2 gene sequencing
HOLOUBKOVÁ, Renata
In my bachelor thesis I dealt with the detection of mutations in the ALAS2. In the theoretical part, I dealt with the introduction to genetic concepts and the field of genetics itself. The work begins from the cell itself, to the chromosomes, through mutations. I also dealt directly with the ALAS2 gene. In the following chapters, I focused on heme, because the ALAS2 gene encodes an enzyme that is responsible for heme production. Last but not least, I have described the disease erythropoietic protoporphyria and sideroblastic anemia, which arise precisely due to mutations in this gene. In the practical part, I examined the ALAS2 gene of exon 11. I examined 25 anonymized samples, from which I isolated DNA. I then amplified the desired section of the gene by PCR. I prepared the samples for the sequencing method, which took place at GenSeq s.r.o. In the last part of my bachelor's thesis, I dealt with the evaluation of sequences and processing of results, but also with the evaluation of sequences from children from Karviná and České Budějovice, which I received. No mutations were found from the whole examined group and the group to be evaluated. For evaluation from a larger data set, the results of the mutations found in the research were added.
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Proteins involved in the tetrapyrrole pathway in Synechocystis sp. PCC 6803 and their localization in the proximity of PSII biogenesis
SKOTNICOVÁ, Petra
The goal of the thesis was to enhance our understanding of the tetrapyrrole pathway in cyanobacteria by a study of selected proteins involved in the pathway. During the project I have revealed functional connection between protoporphyrinogen IX oxidase HemJ and preceding enzyme in the pathway by complementation of protoporphyrinogen IX oxidase deletion mutant by its analog HemG from Escherichia coli. Heme b was identified as a cofactor of HemJ. Another protein deeply influencing tetrapyrrole accumulation, BtpA was found to form a complex with GluTR, the enzyme at the beginning of the tetrapyrrole pathway. Lastly, CurT protein, the component of the structures anticipated to function in PSII assembly and/or repair localized at plasma/thylakoid membrane interface, was co isolated with the enzymes of the tetrapyrrole pathway suggesting that the specific CurT containing membranes could be a place of both photosystem II assembly/repair and chlorophyll delivery.
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Structural characterization of a model heme-containing oxygen sensor
Tajovská, Eva ; Martínková, Markéta (advisor) ; Ryšlavá, Helena (referee)
One subgroup of hemoproteins are heme-based gas sensors, which are able to detect biatomic gas molecules in their immediate surroundings. Upon binding of a gas molecule to the heme iron in a sensor domain of these proteins or, conversely, upon its dissociation from the heme iron, the signal is then transmitted from the sensor domain to a functional domain and subsequent regulation of important cellular functions occurs. Understanding the regulatory mechanism of gas sensors is key to potentially manipulating their function. Such knowledge would then allow the use of heme-based gas sensors as therapeutic targets for the development of next-generation antibiotics, if we take into account their presence in pathological bacteria. The diploma thesis focuses on a model heme-based gas sensor, the oxygen sensor EcDOS from E. coli, and its apoform, EcDOS His77Ala. Both proteins were prepared by recombinant expression and purification, and subsequently spectrophotometrically characterized. Using gel permeation chromatography, the oligomeric states of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS His77Ala were determined under different conditions (different temperatures of protein incubation, presence of c-di-GMP substrate etc.). Furthermore, the structural dynamics of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS...
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Specific heme interaction modulates the conformational dynamics and function of p53
Sergunin, Artur ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Tumor suppressor p53 is one of the most studied proteins in terms of cancer and the mechanism of its formation. The general function of p53 is based on the transcriptional regulation of various genes, which can differently influence numerous cellular processes. Recent studies revealed a relationship between p53 and iron homeostasis within the cell. In particular, p53 was shown to interact with a molecule of heme, and this interaction ultimately disrupts the DNA-binding ability of p53 and promotes its proteasomal degra- dation. This work focuses on a detailed description of heme binding to the p53. For this purpose, we isolated two forms of p53, heme-free and heme-bound. We discovered that conformational dynamics of heme-free and heme-bound p53 differ, with the latter exhibi- ting a higher degree of flexibility. We also confirmed previous reports that heme indeed interacts with a cysteine residue in a specific manner. However, heme binding does not disrupt the oligomeric state of p53 or its native zinc binding ability. Finally, we showed that heme-bound p53 exhibits severely impaired DNA-binding ability as opposed to the heme-free form. Keywords: heme, sensor proteins, p53 protein, transcription factor, intrinsically disor- dered proteins
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Ecological survey and assessment of streams within European Standards. Application in the Rolava River basin.
Beranová, Zuzana ; Matoušková, Milada (advisor) ; Kliment, Zdeněk (referee)
This thesis deals with implementation of Water Framework Directive (2000/60/EC) in the Czech Republic. The study area was the Rolava River basin in the Ore Mountains (northwest of Bohemia). The ecological status assessment was applied to both - the main stream and relevant tributaries. Available hydrobiological and hydrochemical data enhanced with the field hydromorphological survey using the Hydroecological monitoring (HEM, Langhammer, 2007) were applied in this thesis. Results of the field survey were compared with two ecomorphological methods: LAWA-FS (LAWA, 2000) and EcoRivHab (Matoušková, 2003, 2007). As a final result, it was found out the water body on upper course of the Rolava River achieved good ecological status. Water body on lower course of the Rolava River and of the tributary the Nejdecký Brook were classified as not achieving good ecological status. The Rolava catchment has very specific conditions and huge potential to achieve the good status in the future.
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Proposal of restoration measures in Slubice River basin based on ecohydromorphological survey
Šmerousová, Kateřina ; Matoušková, Milada (advisor) ; Just, Tomáš (referee)
This diploma thesis deals with ecohydrological state of the streams as an answer river habitat quality to exploitation, regulation and otherwise modification (flood-defence engineering, impoundment, river restoration, climate change or the spread of alien species), and with revitalization of water bodies for the purpose of elimination and mitigation of the negative consequences river ecosystem modification and improvement of ecological function river ecosystems in landscape. Focus is on the Slubice river basin in Landscape Protected Area Žďárské vrchy where field survey ecomorphological methods EcoRivHab a HEM was applied. On the basis of the obtained outputs antropogenic transformation of the river network, water quality, hydrological condition, definition of the reference state and assessment of the river patterns and changes of the length on the basis of the historical maps and ecomorphological survey outputs in the area of interest strongly antropogenic changed watercourse reaches was identified which require restoration and suitable restoration measures was proposed to the purpose of WFD goals attaining.
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The role of iron in the metabolism of the amoeba Naegleria gruberi
Ženíšková, Kateřina ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
Iron is a biogenic trace element that is vital for all organisms on the planet Earth. This element occurs in biological systems in the form of Fe3+ and Fe2+. These two forms are often incorporated in heme structures or iron-sulfur clusters. Proteins containing iron ions have a wide range of functions in organisms. The main functions include the transport of electrons in the respiratory chain (Rieske's proteins, cytochromes), DNA synthesis (ribonucleotide reductase) and the participation in the Krebs' cycle (aconitase, succinate dehydrogenase). Naegleria gruberi is a nonpathogenic amoeba known for its pathogenic relative Naegleria fowleri. This organism causes the primary amoebic meningoencephalitis. An interesting fact about Naegleria gruberi genome is that it contains genes for both aerobic and anaerobic metabolisms. The purpose of my bachelor work was to investigate the effect of availability of iron ions on metabolism in Naegleria gruberi. Changes in the activities of enzymes from different metabolic pathways were studied including lactate dehydrogenase, isocitrate dehydrogenase, Fe- hydrogenase, aconitase and fumarase. The most significant changes were observed in the activities of alcohol dehydrogenase and Fe-hydrogenase. Key words: Iron, heme, iron-sulfur clusters, availability of iron ions,...
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Hydromorphological survey of the Bilina River
Kyselka, Jan ; Matoušková, Milada (advisor) ; Langhammer, Jakub (referee)
The main aim of this thesis is assessment and presentation of results of the hydromorphological survey of the Bílina river, which belongs to rivers with the biggest anthropogenic impact in the Czech Republic. The method Hydroecological monitoring (HEM, Langhammer, 2007) was used for the survey. This method is compatible with the Water Framework Directive 2000/60/EC. 53 parts of the river with the lenght 31,99 km were assessed. The thesis also includes assessement of the rainoff regime and information about protection of water ecosystems and sources in the Bílina river basin. Keywords: hydromorphology, HEM, Bílina river
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Preparation and preliminary characterization of the eukaryotic initiation factor 2α and its heme regulated kinase
Ovad, Tomáš ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
Heme sensor proteins perform a variety of important functions in both prokaryotic and eukaryotic organisms. Heme-regulated inhibitor (HRI) is an example of a eukaryotic heme-sensor protein, which catalyzes the phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α). In this bachelor thesis, the pET-21c(+)/eIF2α plasmid was amplified and its authenticity for the eIF2α expression was verified with the use of two independent methods. Next, HRI and eIF2α were produced using the recombinant expression in E. coli BL-21(DE3) cells transformed with the pET- 21c(+)/eIF2α and pET-21c(+)/HRI plasmid, respectively. Both proteins were then isolated from the cells and purified with the use of affinity chromatography and gel permeation chromatography. eIF2α was obtained in sufficient yield (560 μg out of 1 l of TB medium) and purity (90%). A lower yield (25 μg out of 1 l of TB medium) and purity (20%) was reached in the case of HRI. On the other hand, the authenticity of the HRI product was confirmed using spectrophotometric characterization and its enzyme activity was verified as well. Pilot experiments showed that GTP may replace ATP in the process of eIF2α phosphorylation, while UTP and CTP may not.
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Heme sensor proteins sensing both heme and CO
Andrlová, Dominika ; Martínková, Markéta (advisor) ; Libus, Jiří (referee)
Heme, a protoporhyrin IX iron complex, is an important component of many proteins necessary for oxygen transfer, storage and activation, as well as for electron transfer. Another group of hemoproteins includes heme sensor proteins. They are either capable of detecting heme itself, which can regulate in turn the sensor function (heme-responsive sensors) or heme forms a binding site for small gas molecules (O2, CO and NO) and the heme-based gas sensors are regulated by these diatomic gases. However, in the case of some proteins their classification is not clear showing a properties of both heme sensor proteins families. Their functions are regulated by heme interaction and a further change in their function after binding of a gas molecule to heme was observed. This summary search is focused on specific representatives of heme-responsive sensors (which function is regulated by heme binding), in which the further influence of the CO molecule on their functions have recently been observed. It is discussed whether some heme-responsive sensors are also heme-based CO sensors aiming the most recent findings about the selected specific heme sensors representatives. Key words: heme, heme sensor proteins, heme-based gas sensors, CO sensors, heme-responsive sensors, heme redox sensors
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