Accessibility controls selective degradation of photosystem II subunits by FtsH protease

The oxygen-evolving photosystem II (PSII) complex located in chloroplasts and cyanobacteria is sensitive to light-induced damage1 that unless repaired causes reduction in photosynthetic capacity and growth. Although a potential target for crop improvement, the mechanism of PSII repair remains unclear. The D1 reaction center protein is the main target for photodamage2, with repair involving the selective degradation of the damaged protein by FtsH protease3. How a single damaged PSII subunit is recognized for replacement is unknown. Here, we have tested the dark stability of PSII subunits in strains of the cyanobacterium Synechocystis PCC 6803 blocked at specific stages of assembly. We have found that when D1, which is normally shielded by the CP43 subunit, becomes exposed in a photochemically active PSII complex lacking CP43, it is selectively degraded by FtsH even in the dark. Removal of the CP47 subunit, which increases accessibility of FtsH to the D2 subunit, induced dark degradation of D2 at a faster rate than that of D1. In contrast, CP47 and CP43 are resistant to degradation in the dark. Our results indicate that protease accessibility induced by PSII disassembly is an important determinant in the selection of the D1 and D2 subunits to be degraded by FtsH

Conformal prediction

The main objective of this work is to formalize the concept of conformal prediction. This robust, nonparametric method allows the construction of an accurate prediction interval at a specified level, for which it is sufficient to assume that the input data are independent, equally distributed. In the context of random sampling from a one- dimensional continuous distribution, we expose the theoretical foundations of the method. Subsequently, we define the key concept of the degree of nonconformance and present the algorithmic design, first for random sampling and then in the context of regression ana- lysis. At the end of the work, we compare the reliability and effectiveness of conformal prediction with a specific frequency method on randomly generated data. 1Hlavním cílem této práce je formalizovat koncept konformní predikce. Tato robustní, neparametrická metoda umožňuje konstrukci přesného predikčního intervalu na stanovené hladině, k čemuž stačí předpokládat, že vstupní data jsou nezávislá, stejně rozdělená. V kontextu náhodného výběru z jednorozměrného spojitého rozdělení vystavíme teoretické základy metody. Následně definujeme klíčový pojem míra nekonformity a prezentujeme algoritmické provedení, nejprve pro náhodný výběr, poté v kontextu regresní analýzy. V závěru práce porovnáváme na náhodně generovaných datech spolehlivost a efektivitu konformní predikce s konkrétní frekventistickou metodou. 1Department of Probability and Mathematical StatisticsKatedra pravděpodobnosti a matematické statistikyFaculty of Mathematics and PhysicsMatematicko-fyzikální fakult

FtsH4 protease controls the biogenesis of PSII complex by dual regulation of high light-inducible proteins

FtsH proteases are membrane-embedded proteolytic complexes important for protein quality control and regulation of various physiological processes in bacteria, mitochondria, and chloroplasts. Like most cyanobacteria, the model species Synechocystis sp. PCC 6803 contains four FtsH homologs, FtsH1-FtsH4. FtsH1-3 form two hetero-oligomeric complexes, FtsH1/3 and FtsH2/3, that play a pivotal role in acclimation to nutrient deficiency and photosystem II quality control, respectively. FtsH4 differs from the other three homologs by the formation of a homo-oligomeric complex and together with Arabidopsis thaliana AtFtsH7/9 orthologs, it has been assigned to another phylogenetic group of unknown function. Our results exclude the possibility that Synechocystis FtsH4 structurally or functionally substitutes for the missing or non-functional FtsH2 subunit in the FtsH2/3 complex. On the other hand, we demonstrate that FtsH4 is involved in the biogenesis of photosystem II by dual regulation of high light-inducible proteins (Hlips). FtsH4 positively regulates expression of Hlips shortly after high light exposure but is also responsible for Hlips removal under conditions when their elevated levels are no longer needed. We provide experimental support for Hlips as proteolytic substrates of FtsH4. The fluorescent labeling of FtsH4 allowed us to assess its localization using advanced microscopic techniques. Results show that FtsH4 complexes are concentrated in well-defined membrane regions at the inner and outer periphery of the thylakoid system. Based on the identification of proteins that co-purified with the tagged FtsH4 we speculate that FtsH4 concentrates in special compartments in which the biogenesis of photosynthetic complexes takes place

Sub-cellular location of FtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid membranes

Marie Curie Initial Training Networks. Grant Number: FP7-PEOPLE-ITN-2008 Biotechnology and Biological Sciences Research Council. Grant Numbers: BB/G021856, BB/F020554/

The Transcriptional Landscape of the Photosynthetic Model Cyanobacterium Synechocystis sp. PCC6803.

Cyanobacteria exhibit a great capacity to adapt to different environmental conditions through changes in gene expression. Although this plasticity has been extensively studied in the model cyanobacterium Synechocystis sp. PCC 6803, a detailed analysis of the coordinated transcriptional adaption across varying conditions is lacking. Here, we report a meta-analysis of 756 individual microarray measurements conducted in 37 independent studies-the most comprehensive study of the Synechocystis transcriptome to date. Using stringent statistical evaluation, we characterized the coordinated adaptation of Synechocystis' gene expression on systems level. Evaluation of the data revealed that the photosynthetic apparatus is subjected to greater changes in expression than other cellular components. Nevertheless, network analyses indicated a significant degree of transcriptional coordination of photosynthesis and various metabolic processes, and revealed the tight co-regulation of components of photosystems I, II and phycobilisomes. Detailed inspection of the integrated data led to the discovery a variety of regulatory patterns and novel putative photosynthetic genes. Intriguingly, global clustering analyses suggested contrasting transcriptional response of metabolic and regulatory genes stress to conditions. The integrated Synechocystis transcriptome can be accessed and interactively analyzed via the CyanoEXpress website (http://cyanoexpress.sysbiolab.eu)

Photoactivation: The light-driven assembly of the water oxidation complex of Photosystem II

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II. The assembly of the Mn4O5Ca requires light and involves a sequential process called photoactivation. This process harnesses the charge-separation of the photochemical reaction center and the coordination environment provided by the amino acid side chains of the protein to oxidize and organize the incoming manganese ions to form the oxo-bridged metal cluster capable of H2O-oxidation. Although most aspects of this assembly process remain poorly understood, recent advances in the elucidation of the crystal structure of the fully assembled cyanobacterial PSII complex help in the interpretation of the rich history of experiments designed to understand this process. Moreover, recent insights on the structure and stability of the constituent ions of the Mn4CaO5 cluster may guide future experiments. Here we consider the literature and suggest possible models of assembly including one involving single Mn2+ oxidation site for all Mn but requiring ion relocation.Peer reviewedMicrobiology and Molecular Genetic

Conformal prediction

The main objective of this work is to formalize the concept of conformal prediction. This robust, nonparametric method allows the construction of an accurate prediction interval at a specified level, for which it is sufficient to assume that the input data are independent, equally distributed. In the context of random sampling from a one- dimensional continuous distribution, we expose the theoretical foundations of the method. Subsequently, we define the key concept of the degree of nonconformance and present the algorithmic design, first for random sampling and then in the context of regression ana- lysis. At the end of the work, we compare the reliability and effectiveness of conformal prediction with a specific frequency method on randomly generated data.

Postmodern in the Music Pedagogy

The diploma thesis called Postmodernism and its Use in Music Education looks at the theme of postmodernism both from the general and the musical point of view. Its content reflects the needs of teachers interested in the use of postmodernism in their lessons. In the first part the sociological, philosophical and historical aspects are summarized. In the second part the postmodern tendencies in literature, visual art and films are outlined. The third part of the thesis defines the term of postmodern music and describes in details its evolution, main characteristics and the compositional techniques associated with it. It focuses particularly on the polystylism and the composers who use it in their works. The last part of the thesis describes several lessons dealing with these compositions taking into consideration the contemporary innovative methods of education (for example, group work or the project method). These lesson plans may inspire teachers to use contemporary music in their lessons and contribute to increase of its use

Role of FtsH proteases in the cyanobacterium Synechocystis sp. PCC 6803

This thesis focuses on the functional and structural characterization of FtsH proteases in Synechocystis PCC 6803. One of the aims was to determine localization and subunit organization of FtsH homologues in Synechocystis cells using GST and GFP tagged FtsH derivatives. The main result of the thesis is identification of two FtsH hetero-oligomeric complexes and one homo-oligomeric complex in Synechocystis cells. The large part of the thesis is aimed at establishing the role of the first hetero-oligomeric complex, FtsH2/FtsH3, in quality control of Photosystem II and at identification of a mechanism, how its substrate proteins D1 and D2 are recognized. Another part is dedicated to characterization of the second hetero-oligomeric complex, FtsH1/FtsH3, which consists of two essential FtsH homologues and which is here identified as an important regulatory element in maintaining iron homeostasis

Postmodern in the Music Pedagogy

The diploma thesis called Postmodernism and its Use in Music Education looks at the theme of postmodernism both from the general and the musical point of view. Its content reflects the needs of teachers interested in the use of postmodernism in their lessons. In the first part the sociological, philosophical and historical aspects are summarized. In the second part the postmodern tendencies in literature, visual art and films are outlined. The third part of the thesis defines the term of postmodern music and describes in details its evolution, main characteristics and the compositional techniques associated with it. It focuses particularly on the polystylism and the composers who use it in their works. The last part of the thesis describes several lessons dealing with these compositions taking into consideration the contemporary innovative methods of education (for example, group work or the project method). These lesson plans may inspire teachers to use contemporary music in their lessons and contribute to increase of its use