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Analysis of Thick Film Amperometrical Sensors Signal and Its Usage for Measurement and Characterization of Enzymes
Ondruch, Vít ; Kizek, René (referee) ; Masojídek,, Jiří (referee) ; Vrba, Radimír (advisor)
V práci je popsán princip synchronní detekce (SD), který byl uplatněn při měření s biosenzory. Metoda SD umožňuje dosažení výrazně lepšího poměru signálu k šumu, vyššího limitu detekce a celkové zlepšení robustnosti měření. Uplatnění SD při měření s biosenzory umožní zlepšit analýzu jeho odezvy a umožní odstranění nežádoucích interferencí nebo šumů, které mohou být způsobeny například mícháním roztoku, elektromagnetickými vlivy nebo parazitními proudy. SD také umožňuje rozložit získaný signál na odezvu stimulace a na dlouhodobý signál jiného procesu, a dále také identifikovat jevy druhého řádu. Pro identifikaci stimulačního signálu ve výstupním signálu měření byl na základě lineárního statistického modelu vyvinut specializovaný software. SD byla ověřena na modelovém případu výstupního signálu biosenzoru s aplikovaným komplexem fotosystému II (PSII) a jeho odezvě na stimulaci světlem. Odezva PSII se řídí kinetikou prvního řádu a může být také ovlivněna inhibitory. Kinetické konstanty vazby herbicidu na PSII závisí lineárně na koncentraci herbicidu. To umožňuje jejich měření také při nízkých koncentracích herbicidu.
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Biogenesis of the cyanobacterial photosystem II complex: involvement of selected accessory factors with emphasis on the novel Psb35 protein
PASCUAL AZNAR, Guillem Miquel
In the last twenty years, intensive work has been devoted to elucidating the structure and function of a number of protein factors involved in the assembly and repair of Photosystem II (PSII). These proteins are not found in the crystal structure of the active PSII but transiently interact with one or more of the assembly/repair intermediates in order to facilitate the PSII assembly/repair or protect their components. The principal aim of this thesis consists in the description of a novel PSII assembly factor that we termed Psb35. Additionally, research on the previously identified Ycf48 and RubA assembly factors is also presented. Based on these results their structure and function are further clarified.
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Function of the Type IV pili proteins in the cyanobacterium Synechocystis sp. PCC 6803.
LINHARTOVÁ, Markéta
The thesis is focused on the function of the Type IV pili machinery in the cyanobacterium Synechocystis sp. PCC. 6803. In my work, I provided evidence that in phototrophs, some pilin proteins have acquired a novel function related to metal transport and assembly of photosynthetic complexes. The tight connection between the synthesis of the major pilin PilA1 and the biogenesis of photosynthetic complexes has been demonstrated in the pilD mutant lacking prepilins peptidase. I isolated and characterized pilD suppressor strains, which revealed the important role of the minor pilin PilA2 in the glycosylation of PilA1 and the restricted mobility of PilA1 prepilin in the membrane. My unpublished data indicate that the retraction mode of pili, driven by the PilT1 molecular motor, facilitates manganese uptake and the activity of this machine is critically important during the regulation from the exponential to the linear growth phase.
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Characterisation of Arabidopsis thaliana mutants psbo1 and psbo2
Nykles, Ondřej ; Duchoslav, Miloš (advisor) ; Hála, Michal (referee)
The PsbO protein is necessary for the function of the electron-transport chain of the thylakoid membrane in higher plants. In most of the angiosperms, including Arabidopsis thaliana, this protein has two isoforms termed as PsbO1 and PsbO2. Many authors tried to reveal the fundamental difference between the PsbO1 and PsbO2 with the help of the mutant lines which lack one of the isoforms. The problem is that the mutants in psbO isoforms do not possess the same level of PsbO as WT does. So we made psbo1isoL mutants. These lines contain only one isoform but their level of the whole PsbO is comparable to the level of the whole PsbO of WT. Results from these experiments suggest that if a psbo1isoL plant has the same amount of PsbO as WT does, there is no observable phenotype difference. Thus we were not able to identify, in the usual cultivation conditions, if there are any functional differences between PsbO1 and PsbO2 Following the above mentioned results, we would like to know conditions (if there are any) in which T-DNA insertion mutants psbo2 (respectively psbo2cr which are made with the use of the CRISPR/Cas9), which have only PsbO1 isoform, could be phenotypically distinguished from WT. With the use of usual cultivation conditions, we are unable to tell apart the psbo2 and psbo2cr from WT by the...
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Function of PsbO isoforms
Duchoslav, Miloš ; Fischer, Lukáš (advisor) ; Špunda, Vladimír (referee) ; Sobotka, Roman (referee)
(English version) Oxygenic photosynthesis is crucial for most forms of the life on the Earth. The splitting of water and evolution of oxygen is conducted by photosystem II (PSII), a multi-subunit pigment- protein complex embedded in the thylakoid membrane. PsbO is an indispensable subunit of PSII, bound to its transmembrane subunits from the luminal side. The main function of PsbO is to stabilise and protect Mn4CaO5 cluster where the water splitting occurs. However, it has probably also some auxiliary functions. These additional functions might be different for isoforms of PsbO proteins, as suggested for Arabidopsis thaliana, which expresses two genes encoding protein isoforms PsbO1 and PsbO2. This thesis studies auxiliary functions of PsbO with a focus on functional differences between PsbO isoforms. We found that besides Arabidopsis thaliana, also many other plant species express two psbO genes. Interestingly, the duplication of psbO gene occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the PsbO isoforms differ at similar sites in the protein structure, suggesting that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. Biochemical characterisation of PsbO from green alga Chlamydomonas reinhardtii and...
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Role of PsbO isoforms in Arabidopsis thaliana
Svoboda, Václav ; Duchoslav, Miloš (advisor) ; Knoppová, Jana (referee)
Role of PsbO isoforms in Arabidopsis thaliana Abstract Photosystem II (PSII) uses sunlight to catalyze water oxidation and reduce plastoquinone. Water oxidation takes place in oxygen evolving complex (OEC). OEC is stabilized by extrinsic subunits of PSII. The largest and most important of them is PsbO, manganese-stabilizing protein which can be found in all known oxygenic photosynthetic organisms. Model plant Arabidopsis thaliana expresses two isoforms of psbO gene, namely PsbO1and PsbO2.Mutants psbo1 and psbo2 lacking PsbO1 and PsbO2, respectively, recently brought new findings on the particular roles of isoforms in maintaining photosynthesis. PsbO1 is commonly considered as the main isoform facilitating water splitting, whereas PsbO2 is believed to be involved in PSII repair process (replacement of photodamaged D1 subunit). This work focuses on particular roles of Arabidopsis PsbO isoforms in maintaining photosynthesis with special focus on response to light stress. Mutants psbo1, psbo2 and wild type plants Col-0 were used for extensive biochemical investigation. Our aim was to find out what is the impact on overall thylakoid structure and composition in mutants. Furthermore, to investigate response to light stress in wild type regarding to yields of particular subcompartments, changes in photosystem II...
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Proteomic and functional characterization of PsbO isoforms
Duchoslav, Miloš ; Fischer, Lukáš (advisor) ; Hála, Michal (referee)
PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...
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The role of manganese-stabilizing protein of photosystem II
Duchoslav, Miloš ; Rothová, Olga (referee) ; Fischer, Lukáš (advisor)
Miloš Duchoslav The role of manganese-stabilizing protein of photosystem II Abstract The appearance of oxygenic photosynthesis was a key event in the evolution of life on the Earth. All molecular oxygen in the atmosphere likely comes from a water-splitting reaction catalysed by the oxygen-evolving center of photosystem II. Photosystem II - a multisubunit protein-cofactor complex with a phylogeneticaly highly conserved structure - is embedded in the thylakoid membrane of chloroplasts and cyanobacteria. The mechanism of the photosynthetic water-splitting reaction, which occurs on the manganese cluster, has been widely investigated; however, it has not yet been fully understood. An essential role in the stabilization of the manganese cluster and in the facilitation of oxygen evolution is played by photosystem II extrinsic proteins that occur in thylakoid lumen. The most important among them is a manganese-stabilizing protein (MSP) that is present in all known oxyphototrophs. This protein is believed to have many functions: besides stabilizing the manganese cluster, it is also carbonic anhydrase activity, GTPase activity and regulation of the turnover of the D1 protein. The functions of the MSP are probably regulated through changes in its dynamic structure. The MSP is likely to take part in the regulation of...
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Role of Psb28 proteins in the biogenesis of the Photosystem II complex in the cyanobacterium Synechocystis sp. PCC 6803
BEČKOVÁ, Martina
The thesis focuses on the role of Psb28 proteins, namely the Psb28-1 and its homolog Psb28-2, in the biogenesis of the Photosystem II complex (PSII) in the cyanobacterium Synechocystis PCC 6803. The aims of this work were to localize the proteins within the cells, and to determine their function. A fraction of both Psb28 proteins was identified in the monomeric PSII core complexes but most proteins were found in the unassembled protein fraction associated with thylakoid membranes. Psb28-1 was mostly detected as a dimer while Psb28-2 as a monomer. Psb28-1 also differed from Psb28-2 by its higher affinity to the PSII core complex lacking CP43 antenna. Characterization of Psb28-less mutants suggested regulatory function of the proteins in PSII biogenesis in connection with chlorophyll biosynthetic pathway. Analysis of preparations isolated using FLAG-tagged versions of Psb28 proteins showed their association with Photosystem II - Photosystem I supercomplexes, especially under increased irradiance, and supported a role of Photosystem I in the PSII biogenesis.
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Biogenesis of Photosystem II in the Model Cyanobacterium Synechocystis sp. PCC 6803 - The Role of Selected Auxiliary Protein Factors and Subcellular Localisation
KNOPPOVÁ, Jana
This thesis explores localisations and roles of three auxiliary protein factors involved in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 and contributes to subcellular localisation of the initial steps of PSII biogenesis and repair-related D1 synthesis. The main results consist in i) identification of a functional interaction of the protein factor Psb27 with a lumenal domain of the Photosystem II subunit CP43, ii) discovery of a novel pigment binding complex formed by the Ycf39 protein and high-light-inducible proteins implicated in photoprotection and delivery of recycled chlorophyll to newly synthesized D1 protein during the PSII reaction centre formation, iii) providing evidence that the early steps of PSII assembly and the repair-related D1 synthesis occur in the thylakoid membrane of Synechocystis, and iv) revealing that the cyanobacterial PsbP orthologue, CyanoP, assists in the early phase of PSII biogenesis as an assembly factor facilitating the association of D2 and D1 assembly modules.
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