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The role of stress granules and 4E-BP in heat-stressed cells of S. cerevisiae
Kolářová, Věra ; Hašek, Jiří (advisor) ; Zimmermannová, Olga (referee)
The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...
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Function of phospholipases D and lipid phosphate phosphatases in the regulation of plant cell morphogenesis
Bezvoda, Radek ; Žárský, Viktor (advisor) ; Hašek, Jiří (referee) ; Vaňková, Radomíra (referee)
of the thesis The presented work explores the function and regulation of intracellular signaling that utilizes phospholipase D (PLD) and phosphatidic acid (PA), especially in the context of cellular morphogenesis of plants. PLDs cleave membrane phospholipids to phosphatidic acid, which has important biophysical and signaling role in many contexts, such as stress response, regulation of cytoskeletal dynamics and vesicular transport. Vesicular transport is essential in focused tip growth of plant pollen tubes and root hairs. Part of the work deals with NADPH oxidases, that are an emerging counterpart of PLD/PA signaling. Tobacco pollen tubes served as the main experimental model, as it enables assessing of changes in secretory pathway after pharmacological or genetic treatments. A technique utilizing antisense oligonucleotides was used for selective knock-down of PLD isoforms, NADPH oxidase and newly studied family of lipid phosphate phosphatases (LPPs) in pollen tubes. This enabled to assess functions of individual isoforms. For studying of selected gene families, various bioinformatic tool were utilized, such as dendrogram construction, analysis of available expression data and creating of virtual proteome. These tools together enabled to select potentially important genes for further experimental...
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Phenotypes of Saccharomyces cerevisiae cells lacking Isw2 chromatin remodelling factor
Frýdlová, Ivana ; Hašek, Jiří (advisor) ; Janderová, Blanka (referee) ; Sychrová, Hana (referee)
8 SUMMARY nd invasive growth. We showed that this phenotype is caused by de-repression that ncentrations of pheromone. Detailed analysis however uncovered some differences birth scar, rea that is commonly prohibited for budding. Based on our microarray data and double eletion mutant analyses, we identified one gene that is responsible for this phenotype. w2Δ cells as well as rth-scar phenotype as well as CWI pathway activation. These data prove, that de-regulation of expression of the daughter cell-specific gene DSE1 is The non-essential Saccharomyces cerevisiae protein Isw2 is a subunit of the ATP- dependent chromatin remodelling complex Isw2 that regulates the structure of chromatin, and thus plays an important role in regulation of transcription. Absence of the Isw2p induces the α-mating type-specific aberrant "shmoo-like" morphology a of a-specific genes in MATα cells and production of inappropriate a-factor which in turn activates the pheromone response pathway of isw2Δ MATα cells. Our results showed isw2Δ MATα cells can express the MATa-specific genes at the level which enables them to mate with other MATα cells. Invasion of isw2Δ MATα cells depends neither on the cell surface protein Flo11, nor on the invasive growth-specific transcription factor Tec1. On the other hand it requires components of the...
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Regulatory mechanisms of centrosomal microtubule nucleation
Klebanovych, Anastasiya ; Dráber, Pavel (advisor) ; Hašek, Jiří (referee) ; Vomastek, Tomáš (referee)
The spatio-temporal organization and dynamic behavior of microtubules accurately react to cellular needs during intracellular transport, signal transduction, growth, division, and differentiation. The cell generates centrosomal microtubules de novo with the help of γ-tubulin complexes (γTuRCs). The post-translational modifications fine-tune microtubule nucleation by targeting the proteins, interacting with γTuRCs. However, the exact signaling pathways, regulating centrosomal microtubule nucleation, remain mostly unknown. In the presented thesis, we functionally characterized protein tyrosine phosphatase SHP-1 and E3 UFM-protein ligase 1 (UFL1) with its interacting protein CDK5RAP3 (C53) in the regulation of centrosomal microtubule nucleation. We also elucidated the role of actin regulatory protein profilin 1 in this process. We found that SHP-1 formed complexes with γTuRC proteins and negatively regulated microtubule nucleation by modulating the amount of γ-tubulin/γTuRC at the centrosomes in bone marrow-derived mast cells (BMMCs). We suggested a novel mechanism with centrosomal tyrosine-phosphorylated Syk kinase, targeted by SHP-1 during Ag-induced BMMCs activation, regulating microtubules. We showed for the first time that UFL1/C53 protein complex is involved in the regulation of microtubule...
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Risk Analysis in Transport Infrastructure Projects
Hašek, Jiří ; Holá, Michaela (referee) ; Hromádka, Vít (advisor)
The subject of the master’s thesis is a risk analysis in transport infrastructure projects. In the theoretical part, I deal with public sector, life cycle of the project, evaluation of public projects, conception of risk, clasification of risk, risk analysis and valuation of the risk. In the practical section I process risk analysis of the project in transport infrastucture.
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Regulation of translation iniitiation in yeast saccharomyces cerevisiae
Mašek, Tomáš ; Pospíšek, Martin (advisor) ; Krásný, Libor (referee) ; Hašek, Jiří (referee)
IV. Shrnutí výsledků 63 64 Denaturační RNA elektroforéza v TAE agarózových gelech 60% koncentrace formamidu postačuje k dostatečné denaturaci RNA pro elektroforetickou separaci. Denaturační RNA elektroforéza v TAE pufru vykazuje stejné separační rozlišení RNA molekul jako nejčastěji používaná RNA elektroforéza v MOPSovém pufru a navíc je rychlejší. Denaturační RNA elektroforéza v TAE pufru je použitelná nejen k separaci čisté směsi RNA molekul, ale i směsných vzorků obsahujících také DNA a proteiny (např. buněčných lyzátů). Denaturační RNA elektroforézu v TAE pufru lze kombinovat s kapilárním přenosem a následnou hybridizací (pro blotování lze použít jak konveční 10xSSC pufr, tak levnější 8 mM NaOH). Tento elektroforetický protokol poskytuje levnější a rychlejší alternativu k RNA elektroforéze v MOPSovém pufru, snižuje expozici laboratorních pracovníků toxickým látkám a je vhodný i pro laboratoře, které s RNA běžně nepracují. Rck2 se zapojuje do reprogramování ribozómů během oxidativního stresu Oxidativní stres inhibuje translaci stejnou měrou jako stres osmotický. Lze pozorovat pokles množství aktivně translatujících ribozómů. Tento pokles je proporcionální ke zvětšení ploch 40S, 60S a 80S "vrcholů" v polyzomálním profilu. Aplikace t-BOOH vede k vyšší disociaci polyzomálních komplexů u rck2Δ...
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