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Cell inclusions in microscopic eukaryotes
Pilátová, Jana
This thesis reports, for the first time, a systematic study identifying various cell inclusions across the broad diversity of all major eukaryotic supergroups. Raman microspectroscopy technique was employed as a powerful technique, which is becoming the method of the first choice for such studies. In Chapter 1, the spatial reconstruction of chloroplasts of the unicellular zygnematophyte Cylindrocystis sp. (Streptophyta) revealed "empty spaces" inside. Subsequent analysis showed massive accumulations of polyphosphate, which is not located in the cytoplasm as in other species, but inside the chloroplasts. This newly discovered ability of the zygnematophytes, a sister group of terrestrial plants, to accumulate polyphosphate leads us to speculate about a possible adaptation to extreme Arctic conditions or even a preadaptation leading to plant terrestrialization. Chapter 2 summarizes the serendipitous discovery of purine crystalline inclusions in dinoflagellates and other microalgae. Investigated dinoflagellate species comprised zooxanthellae, the endosymbionts of reef-building corals, and the species causing toxic algal blooms. Purine crystals were further shown to act as dynamic high-capacity nitrogen storage. To show how widespread purine inclusions are, a revision of crystalline inclusions in all...
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Cell inclusions in microscopic eukaryotes
Pilátová, Jana ; Schwarzerová, Kateřina (advisor) ; Gierlinger, Notburga (referee) ; Prášil, Ondřej (referee)
This thesis reports, for the first time, a systematic study identifying various cell inclusions across the broad diversity of all major eukaryotic supergroups. Raman microspectroscopy technique was employed as a powerful technique, which is becoming the method of the first choice for such studies. In Chapter 1, the spatial reconstruction of chloroplasts of the unicellular zygnematophyte Cylindrocystis sp. (Streptophyta) revealed "empty spaces" inside. Subsequent analysis showed massive accumulations of polyphosphate, which is not located in the cytoplasm as in other species, but inside the chloroplasts. This newly discovered ability of the zygnematophytes, a sister group of terrestrial plants, to accumulate polyphosphate leads us to speculate about a possible adaptation to extreme Arctic conditions or even a preadaptation leading to plant terrestrialization. Chapter 2 summarizes the serendipitous discovery of purine crystalline inclusions in dinoflagellates and other microalgae. Investigated dinoflagellate species comprised zooxanthellae, the endosymbionts of reef-building corals, and the species causing toxic algal blooms. Purine crystals were further shown to act as dynamic high-capacity nitrogen storage. To show how widespread purine inclusions are, a revision of crystalline inclusions in all...
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Crosstalks between nitrogen metabolism, the TOR pathway and the metabolism of lipids
Grulyová, Michaela ; Převorovský, Martin (advisor) ; Maršíková, Jana (referee)
Cells coordinate their metabolism based on various factors, for example nitrogen availability. The TOR pathway is an important regulator of nitrogen metabolism, it has a role in sensing intracellular amino acids status, and it controls especially cell growth, protein synthesis, proliferation and cell survival. However, it has been shown that the TOR pathway also controls lipid biosynthesis and lipid accumulation through various mechanisms in response to nitrogen availability. Although the TOR pathway is well conserved among the eukaryotic organisms, its outcomes differ diametrically when it comes to the lipid accumulation. This essay provides some insides into the mechanisms of regulation of the lipid metabolism by the TOR pathway.
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Inducible RNAi against essential genes of nitrogen metabolism as a tool for control of GM plants
Kobercová, Eliška ; Fischer, Lukáš (advisor) ; Tylová, Edita (referee)
Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...
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Glutamine synthetases and their role in nitrogen metabolism in plant shoot
Kobercová, Eliška ; Fischer, Lukáš (advisor) ; Tylová, Edita (referee)
Glutamine synthetase is a key enzyme for ammonium assimilation and glutamine biosynthesis in plants. Ammonium ions are important intermediates in nitrogen metabolism, but their elevated concentration is toxic to plants. Correct function of glutamine synthetase is essential to plant life - glufosinate, a specific inhibitor of glutamine synthetase acts as a total herbicide. Glutamine synthetase is involved in a number of important metabolic processes: primary assimilation of nitrogen nutrients, in reassimilation of ammonium ions released during photorespiration or metabolism of phenylpropanoids and in nitrogen remobilization in developing seeds, during germination or senescence. According to localization in the cell glutamine synthetases in angiosperms are divided into the plastid form (GS2), which is typically encoded by a single gene, and the cytosolic form (GS1), which is encoded by a small multigene family. The various isoforms of glutamine synthetases have different location within plant organs and tissues, ways of regulation and role in nitrogen metabolism. Keywords: plastid and cytosolic glutamine synthetase, ammonium assimilation, nitrogen metabolism, photorespiration
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Candida parapsilosis secreted aspartic proteinases: processing and secretion
Vinterová, Zuzana ; Heidingsfeld, Olga (advisor) ; Hodek, Petr (referee) ; Szotáková, Barbora (referee)
Candida parapsilosis is an emerging human opportunistic pathogen causing a wide spectrum of potentially life-threatening infections in immunocompromised hosts. One of the most important virulence factors of Candida spp. is a production of secreted aspartic proteinases (Saps). Presented thesis is mainly focused on the study of secreted aspartic proteinase 1 (Sapp1p) of C. parapsilosis, its processing and secretion under variable conditions and by use of various experimental models. Sapp1p is secreted by C. parapsilosis cells into the extracellular space as a completely processed and fully active enzyme. Experiments studying the C. parapsilosis cell wall (CW) confirmed the prolonged presence of completely processed Sapp1p on the cell surface (CW- Sapp1p). Proteolytic activity assay performed with the intact cells showed that CW-Sapp1p is proteolytically active prior to its release into the extracellular space and is capable of substrate cleavage. Biotinylation experiments with consecutive MS analysis revealed that CW-Sapp1p biotinylation is incomplete but saturable process, leaving partially unlabelled molecules. The accessibility of individual lysine residues in the Sapp1p molecule varied, with exception of four residues that were labelled in all of our experiments performed. The final step of...
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The effect of biotic stress on nitrogen metabolism in tobacco plants
Fiala, Martin ; Ryšlavá, Helena (advisor) ; Müller, Karel (referee)
In this project the effect of viral infection on the metabolism of nitrogenous compounds in tobacco plants (Nicotiana tabacum L. cv. Petit Havana SR1) was studied. The tobacco plants were infected with Potato virus Y, strain NTN, ELISA confirmed the presence of the virus. Enzymes that participate in C4 plants in Hatch-Slack cycle fosfoenolpyruvate carboxylase (EC 4.1.1.31, PEPC), NADP-dependent malic enzyme (EC 1.1.1.40, NADP-ME), pyruvate, phosphate dikinase (EC 2.7.9.1, PPDK) are present also in C3 plants and are related to plant responses to stress conditions. As a result of viral infection, the activities of all these enzymes were increased. Infection caused by PVYNTN decreased activity of nitrate reductase (EC 1.7.1.1, NR), an enzyme catalyzing reduction of nitrates to nitrites. The activity of enzymes catalyzing the synthesis of glutamine from glutamate and ammonium ions: glutamine synthetase (EC 6.3.2.1, GS) and glutamate synthase (EC 1.4.1.14, GOGAT) was enhanced. In addition to this main route of nitrogen fixation the plant can still use glutamate dehydrogenase (EC 1.4.1.2, GDH). This enzyme can also catalyze the opposite reaction, deamination of glutamate. The direction of response depends on environmental conditions. In this case a significant increase of oxidative-deaminating activity...
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