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The role of tubulin transport between the nucleus and the cytoplasm
Bokvaj, Pavel ; Schwarzerová, Kateřina (advisor) ; Zažímalová, Eva (referee)
Tubulin is the basic building block of microtubules which ensure intracellular transport, morphological changes, the connection of proteins of metabolic pathways, the segregation of chromosomes during mitosis and many other essential processes in plant cells. The localization of tubulin regarding nucleus is strictly controlled and during the interphase, tubulin is usually not present in the nucleus. As we previously showed, one of the exceptions of the presence of tubulin in the nucleus is the cold stress condition. Also, we discovered several plant-specific nuclear export sequences (NES) in tubulin molecules: one in α-tubulin and two in β-tubulin. In this work I found that double mutation of both functional β-tubulin NESes cause slight accumulation of the protein in the nuclei. However, the phenotype of the transgenic plants expressing β-tubulin with mutations is probably not affected by the presence of the mutated protein. The effect of the expression of the mutated β-tubulins was observed in Nicotiana tabacum BY-2 cells as well. The expression of β-tubulin carrying a single NES3 mutation or double mutation of both NESes bring changes of the division activity of the cells, while the NES2 mutation does not have any effect. Furthermore, it was discovered that the 0řC cold tr eatment does not cause...
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The proteomic study of abiotic stress of plants.
Barabášová, Kamila ; Podlipná, Radka (advisor) ; Smrček, Stanislav (referee)
Keywords: Arabidopsis thaliana, phytoremediation, abiotic stress, ibuprofene, doxorubicin, two-dimensional electrophoresis Nowadays, develop of the pharmaceutical industry is very fast. Reason of this trend is ever-increasing number of diseases, lifestyle and still increasing demand for the drugs. With this trend growing interest in the analysis of the residues of pharmaceuticals in the environment which is result of incomplete wastewater treatment. This diploma thesis is studying effect of cytostatic drugs, specifically doxorubicin and one of the most widely used analgesics - ibuprofen, at the proteome level of the model plant Arabidopsis thaliana. Proteins isolated from plants exposed to the drugs were separated by two-dimensional electrophoresis. Comparing of protein maps by PDQest program (Bio-Rad, USA) was found several proteins whose expression was affected by the presence of drugs in the culture medium. Selected proteins were identified by LC - MS / MS.
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Vliv růstových regulátorů na proteom rostlin v průběhu klíčení
Habánová, Hana
Seed germination is a complex process during which an embryonic plant contained within a seed transforms into a seedling. The majority of species, including model plant Arabidopsis thaliana produce dry seeds that will fully re-establish metabolism after imbibition. This early phase of a plant's life is highly important for its survival and its progress depends on both internal and external stimuli. This thesis entitled "Effects of plant growth regulators on proteome dynamics during seed germination" is focused on effects of light and plant growth regulators on germination. To get an insight into molecular mechanism of germination, proteome dynamics in germinating Arabidopsis were investigated. Seeds were imbibed in presence of plant growth regulators in continuous light or dark and material for LC-MS proteome profiling was harvested after 24 and 48 hours. Altogether, abundances of more than 1100 proteins were followed. PCA analysis and a detailed pair-wise comparison of mock-treated seeds provided evidence that the modern proteome-wide analysis is a promising tool for plant germination research.
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The role of AGC protein kinases in the regulation of auxin transport
Martincová, Marie ; Petrášek, Jan (advisor) ; Opatrný, Zdeněk (referee)
There are several members of the subfamily of plant AGC kinases (AGCVIII) suggested to play a role in the regulation of auxin transport, protein kinases PID, WAG1, WAG2 and D6. They all have been shown to perform regulatory phosphorylation of PIN auxin efflux carriers. It is the asymmetrical subcellular localization of PIN proteins that enables the auxin molecules to be transported through a tissue in a polar manner. Regulation of their expression, localization or activity can therefore affect the quantity and directionality of auxin transport. This thesis is focused on better understanding of the PID-mediated regulation of auxin transport. The auxin accumulation as well as the localization of PIN and PID proteins has been studied using stable and transient expression of Arabidopsis thaliana PID in tobacco cell line BY-2. As shown here, the activity of PID does not enhance the activity of PINs, but still it has a positive effect on auxin efflux by increasing the amount of PIN proteins on the plasma membrane. Results presented here suggest that PID-mediated phosphorylation of PIN proteins most likely promotes their exocytosis from endosomal compartments towards the plasma membrane. Using transient co-expression of PID kinase mutated in its ATP-binding site and PIN1-RFP it was shown that functional...
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The role of acetylation in the RNA recognition motif of SRSF5 protein
Icha, Jaroslav ; Staněk, David (advisor) ; Šenigl, Filip (referee)
Acetylation is emerging as an important posttranslational modification, which is found in thousands of proteins in eukaryotes, as well as prokaryotes. Global proteomic studies implicated acetylation in regulation of various processes like metabolism, gene expression, cell cycle or aging to name a few. In this work I set out to investigate the role of acetylation of a splicing regulatory protein SRSF5 by creating mutations in its acetylation site. I tested the hypothesis that acetylation influences SRSF5 interaction with RNA. I expressed acetylation-mimicking (Q) or non-acetylable (R) mutant of SRSF5 in HeLa cells and measured their interaction with RNA by RNA immunoprecipitation or in vitro by fluorescence anisotropy. Both approaches agreed that mutants interact with RNA less than the wild type protein and Q mutant bound RNA weaker than R mutant. I did not detect further difference in localization or dynamics among the proteins in vivo, which suggests that difference caused by weakened interaction of mutants with RNA was outweighed by other factors influencing SRSF5 behaviour, probably protein-protein interactions. I also found out that mutant SRSF5 proteins do not have a dominant effect on splicing of fibronectin alternative EDB exon. The data obtained give an indirect evidence for the hypothesis that...
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Effect of tapeworm (Hymenolepis diminuta) on bioaccumulation of zinc in the host (Rattus norvegicus)
Sloup, Vladislav ; Jankovská, Ivana (advisor)
This research investigates the ability of rat tapeworm (Hymenolepis diminuta) to accumulate zinc in tissue and the influence of its intake and excretion by its host (laboratory rat). The host was fed by food with zinc in two forms: 1) a mixture of standard food ST1 with hyperaccumulator plant Arabidopsis halleri, 2) ST1 mixture with zinc lactate, which is ordinarily used as a feed supplement for increasing zinc content and it is often included in human diet supplements. Rat control group fed by ST1 only was included in the experiment for verification of the difference. Rats were divided to six groups (OO, OT, RT, RO, MO a MT). Three rat groups were infected by rat tapeworm (OT, RT, MT) and three rat groups were not infected (OO, RO, MO). The control groups OO and OT were fed by ST1 only. RO and RT groups were fed by ST1 with admixture of Arabidopsis halleri and groups MO and MT were fed by ST1 with zinc lactate. Urine and excrements of the rats were collected twice a week during the experiment and their amounts were measured every day. The rats were weighted every week. At the end of the balance phase of the experiment rats were euthanized and seven selected tissues were removed (liver, kidney, spleen, small intestine, testis, muscle and bone). Rat tapeworm was removed from the infected rats. Blood was drawn from the rats. The results show that rat groups infected by the rat tapeworm had lower concentration of zinc in almost all analyzed tissues except for spleen, where the concentration of zinc was the same as in groups without the rat tapeworm. Based on the results the rat tapeworm also has an influence on the excretion of feces and urine.
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Ovlivnění odpovědi rostlin na teplotní stres modulovanými hladinami cytokininů - fenomická a protemická analýza
Vícha, Daniel
Cytokinins are important group of phytohormones regulating many physiological processes ranging from cell division to programmed cell death. This thesis is focused on effects of cytokinin levels in response to heat stress in Arabidopsis thaliana. Analysis of transgenic plants with regulated expression of ipt and HvCKX showed that cytokinins and their optimal levels play important role in the morphological alterations induced by heat stress. Seedlings with increased and decreased levels of cytokinins exhibit inhibition of petioles growth, decreased length of blades of true leaves and reduced leaf area. To obtain insights into molecular events underlying early response to heat stress LC-MS analysis of whole proteom was performed. Analysis revealed 57 differentialy regulated proteins in response to heat stress in Columbia ecotype. On the cellular level, most of the proteins were located in cytosol (47 %) nebo plastids (32 %). Coparative analysis between wild-type seedlings and seedlings with decreased level of cytokinins confirmed 31 proteinInfluencing plant responses to temperature stress modulating cytokinin levels - fenomic and proteomic analysiss regulated by cytokinins in response to heat stress. Among these proteins, desarurase 7 and Tudor SN1 protein were previously found as important factors in response to heat stress.
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The effect of cadmium on oxidative stress in plants
Zemanová, Veronika ; Pavlíková, Daniela (advisor) ; Tomáš, Tomáš (referee)
The two ecotypes of Noccaea caerulescens originating from two differently contaminated sites (Redlschlag, Austria; Ganges, France), N. praecox (Mežica, Slovenia), Arabidopsis halleri and Spinacia oleracea were studied to compare Cd accumulation and tolerance. After 30, 60, 90 and 120 days of cultivation in Cd contaminated soil (30, 60 and 90 mg Cd/kg soil) for hyperaccumulators and 25, 40, 55 and 75 days of cultivation in Cd contaminated soil (30, 60 and 90 mg Cd/kg soil) for spinach gas-exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration), fatty acids, macro- and microelements were determined as well as N utilization by plants. Noccaea species and A. halleri showed similar changes in yield of biomass, Cd content and total content of amino acids. These species confirmed differences in the profile and contents of individual free amino acids. The comparison between Noccaea species showed that Cd stress resulted in similar changes of gas-exchange parameters. Contrasting responses of plants to Cd contamination were confirmed in macro- and microelements contents, fatty acids and amino acids metabolism. Spinach showed contrasting response to cadmium compared to hyperaccumulators, especially in profile of free amino acids and fatty acids.
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Efekt inhibice proteasomu na proteom Arabidopsis thaliana
Dufek, Martin
Proteasome is highly conserved in its structure among all eukaryotes. The main function of this multi-protein complex is to facilitate protein turnover and degrade misfolded, altered, aged or unneeded proteins. Proteasome-dependant degradation predominantly operates in cooperation with a complex system that conjugates proteins destined for degradation to ubiquitin. Emerging evidence suggests that the proteasome-dependant degradation is crucially involved in plant hormone signalling and as such could play a central role in plant growth and development. This bachelor thesis entitled "Effects of proteasome inhibition on Arabidopsis thaliana proteome" reviews proteasome-ubiquitin pathway with the focus on plant hormone signalling. In the experimental part, the effects of proteasome inhibition were investigated via LC-MS profiling. Altogether, abundances of more than 1400 proteins were followed. PCA analysis and a detailed pair-wise comparison of MG-132 and mock treated seedlings provided an insight into the molecular mechanism behind processes in the response to proteasome inhibition.
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The role of DNA damage in cellular senescence
Dušková, Pavlína ; Vondrejs, Vladimír (referee) ; Hodný, Zdeněk (advisor)
Cells grown in vitro may be introduced into terminal growth arrest termed cellular senescence. In normal cells, it always arises when they reach the end of their proliferative capacity. However, this process may be also caused by various means anytime during the cultivation of both normal and at some circumstances also in immortal cells. Current view of cellular senescence places this phenomenon into direct relationship with DNA damage. The first aim of this study is to elucidate the role of DNA damage in chemically induced senescence. Until recently, DNA damage was not studied in this particular model. All chemicals used in this work are routinely used to unveil unstable parts of the human genome, so called fragile sites, which are inherently prone to DNA breakage. The second aim of this study is to asses the possibility that these sensitive regions of DNA might be also involved in senescent phenotype. Two of the three chemicals used in this study, 5-bromodeoxyuridine and distamycin A, were commonly used to accomplish senescence, and their effect on main senescence-associated changes in cellular proteome is thus relatively well defined. The changes caused by the third compound, thymidine, were so far poorly investigated. Therefore, the last aim of this work is to find out, if the changes of protein...
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