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Deconvolution fluorescence microscopy of yeast cells
Štec, Tomáš ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
Title: Deconvolution fluorescence microscopy of yeast cells Author: Tomáš Štec Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jarmoír Plášek, CSc., Institute of Physics of Charles Uni- versity Abstract: Fluorescence microscopy presents an fast and cheap alternative to more advanced imaging methods like confocal and electron microscopy, even though it is subject to heavy image distortion. It is possible to recover most of the original distortion-free image using deconvolution in computer image processing. This al- lows reconstruction of 3D structure of studied objects. Deconvolution procedure of NIS Elements AR program undergoes an thorough inspection in this diploma the- sis. It is then applied on restoration of 3D structure of calcofluor stained cell wall of budding yeast Saccharomyces cerevisiae. Changes of the structure of the cell wall during cell ageing are being examined. Cell wall of aged cells shows increased surface roughness and even ruptures at the end of cell life. Keywords: fluorescence, microscopy, deconvolution, NIS Elements AR, calcofluor, yeast, cell wall, ageing
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Conventional and Novel Functions of the Exocyst Complex in Plants
Kulich, Ivan ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.
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Role of cytoskeleton in plant cell morphogenesis
Miklánková, Pavlína ; Schwarzerová, Kateřina (advisor) ; Sekereš, Juraj (referee)
The cells are able to acquire variety of shapes, in which cytoskeleton plays an important role. Cytoskeleton influences deposition of cell wall materials, regulates vesicle movement in cell, participates in exocytosis and endocytosis. Cortical microtubules affect celulose accumulation in cell wall and determine direction of cell expansion, although the exact connection between microtubules and cellulose remains unclear. Actin promotes growth and contributes to its spatial regulation in both tip ang diffuse growing cells. Actin is important for secretion in expanding cells but its exact functions in cell growth regulation are not explained yet. Analysis of mutants, spectroscopic methods, cytoskeletal drugs, fluorescence proteins and other methods are used to better understand how actin and microtubule cytoskeleton are integrated during plant cell morphogenesis. Epidermal and trichome cells of Arabidopsis thaliana are a good model of research and they are used for most studies.
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Specificity of selected exocyst subunits in trichome development
Glanc, Matouš ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy
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The effect of desiccation on streptophyte algae - mechanisms of stress resistance
Pošmourný, Martin ; Pichrtová, Martina (advisor) ; Vosolsobě, Stanislav (referee)
In this thesis I dealt streptophyta algae resistance against desiccation. Even though the area previously devoted only a few people. Considerable amount of work in recent years has been published on the subject. They were found interesting information and discovered new facts. Research continues on and on, and it would be useful to look at what was observed. I believe that understanding this phenomenon is the key to understanding some of the events in the evolution of nature and realizing how tough life can be on the very border of its possible occurrence. I tried to sort out the current knowledge about the mechanisms of stress resistance streptophyta algae and hope that I obtained an overview will help me understand better this issues. So far, it has been observed several approaches to defend against drying. Preventing drying, adaptation to water shortage and tolerance to desiccation. Among the preventive methods of defense include creating clusters of cells, multi-layered mats or secretion mucilage. As an adaptation to the lack of water algae evolved more complex answers in the form of changes in ultrastructure, or regulation of physiological processes. Klebsormidium is capable of half an hour to start the production of significant quantities callose and incorporate it into the cell wall. This...
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Secretory pathway in plant cell wall polysaccharide biosynthesis and FT-IR spectroscopy methods in plant cell wall composition analysis
Glanc, Matouš ; Žárský, Viktor (advisor) ; Votrubová, Olga (referee)
All plant cells are encapsulated in a cell wall that determines the cells' shape and size and is essential to many of their vital processes. The cell wall of streptophyte plants is composed mainly of polysaccharides of high molecular weight. Cellulose, the main constituent of the plant cell wall, is synthesized by protein complexes bound to the PM, while hemicelluloses and pectins are synthesized in the Golgi apparatus. Several proteins that participate in the synthesis of cell wall polysaccharides have been identified, but hundreds of them remain to be discovered. Secretory pathway plays an important role in plant cell wall biogenesis as it transports cellulose synthase complexes and noncellulosic polysaccharide molecules to the cell surface. Some regulatory mechanisms that might be involved in cell wall material secretion, such as actin cytoskeleton, Ca2+ gradient or PIP kinases have been proposed; however, the regulation of this process is very complex and far from being understood. FT-IR spectroscopy is a method that can detect molecular vibrations and provide information about chemical composition of virtually all substances. It has been used successfully in screens for cell wall mutants, the study of interactions between cell wall polymers, as well as other areas of cell wall biology. FT-IR...
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Mechanisms of epidermal cells polarization in plants.
Vojtíková, Zdeňka ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
Plant epidermal cells form contact area of the plant, they protect it from impacts of surrounding environment and they mediate the communication with its neighbourhood. In the epidermis there are evenly distributed several cell types with quite specialized morphology (pavement cells, trichomes and guard cells) due to the polarization mechanisms. The cytoskeleton and signal molecules of ROP GTPase family promote the polarized growth. Thanks to polarized growth the cells reach their shapes. The cytoskeleton responds to the signal by expanding the cell, helps with targeting of the secretion to the sites of active growth and mediates polarized formation of the cell wall. On the upper side of the epidermis the cuticle and layer of epicuticular waxex is secreted. The secretion of cuticular components is baso-apicaly polarized. This work summarizes the mechanisms of the polarization in plant epidermal cells discoverd untill now.
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Visualization of root apoplastic pH in plants
Wernerová, Daša ; Fendrych, Matyáš (advisor) ; Paris, Nadine (referee)
Plant oriented movements, or tropisms allow the plant to actively respond to environmental stimuli to get more light, better access to nutrients and to grow roots deeper into the soil. Gravitropism drives the growth of roots along the gravity vector. Perception of gravity is triggered by the sedimentation of statoliths in columella root cap, but the exact signalling pathway behind this process is not known. Perception of gravity results in an unequal redistribution of the phytohormone auxin in the outer cell layers which leads to different rate of growth on the root's upper and lower side and bending of the root. The changes in auxin redistribution are accompanied by changes in apoplastic pH. Knowing an exact pattern of these pH changes could shed light on the mechanisms laying behind the gravitropic response pathway. While microelectrodes can be used to measure pH precisely, they are not suitable for the long-term imaging of growing roots. In the past few years, several pH sensitive dyes and genetically encoded sensors emerged. These can be used for long-term live in vivo imaging of pH changes in growing roots. In this thesis, I analysed the performance of several published pH sensitive genetically encoded sensors and available dyes in the roots of Arabidopsis thaliana. I observed that dyes varied...
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FUNCTIONAL ANAYSIS OF SELECTED EXO70 EXOCYST SUBUNITS IN PLANTS
Kubátová, Zdeňka ; Kulich, Ivan (advisor) ; Grossmann, Guido (referee) ; Lichtscheidl-Schultz, Irene (referee)
Arabidopsis thaliana trichomes are large unicellular epidermal outgrowths with a specific development and intriguing shape, which makes them an excellent cell type for our research of cell polarization mecha- nisms. Cell polarity is essential for plant development and the exocyst complex is one of its key regulators. It is an octameric protein complex that mediates polarized exocytosis and growth by targeted tethering of secretory vesicles to the plasma membrane. Its EXO70 subunit functions as a landmark for exocytosis site and physically binds the target membrane through interaction with phospholipids. A remarkable multipli- cation of EXO70 subunit paralogs in land plant genomes is well documented, but the functional diversity of these paralogs remains to be described. In trichomes we revealed the specific role of the EXO70H4 paralog in secondary cell wall deposi- tion, especially in callose synthase delivery. We documented formation of a thick secondary cell wall during the maturation phase of wild type trichome development and a lack of it in the exo70H4 mutant. Moreover, we showed evidence for silica deposition dependency on callose synthesis. Further, we unveiled the formation of apical and basal plasma membrane domains, which differ in their phospholipid compo- sition and ability to bind...
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Study of yeasts transglycosylases
Čurillová, Natália ; Ing.Hana Schusterová, Ph.D. (referee) ; Stratilová, Eva (advisor)
This study is interested in properties of fungal transglycosylases, specifically Phr1, Phr2 and Crh2. These enzymes are involved in the remodelling of yeast cell walls due to their cleavage of structural donor polysaccharides and transfer of their fragments to the other acceptor (poly)saccharide molecules. The mammalian cells do not contain cell walls, nor cell wall transglycosylases, that´s why these enzymes are possible targets for antifungal agents. In this diploma thesis the effect of 67 commercially available inhibitors on Phr1 and Phr2 enzymes was studied by rapid screening. In the case of the Phr1 enzyme, two inhibitors showed a potential effect which was subsequently tested by size exclusion chromatography column incorporated into HPLC device. None of the inhibitors were found to have an inhibitory effect on Phr1 or Phr2 enzymes in contrast to DMSO in which all inhibitors were dissolved. The mode of action of Phr enzymes was also studied by thin layer chromatography and high performance liquid chromatography. The first method allowed to monitor the formation of products only in the later stages of the reaction, but more sensitive size exclusion chromatography showed the product formation at the beginning of the reaction. Phr1 cleaved the donor substrate near the non-reducing end and forms small fragments that are transfered to labeled acceptors during the whole reaction. Phr2 utilized random action pattern, thus creating products with higher molecular weight from the beginning of reaction. The effect of the polymerization degree of acceptor on it´s affinity with the Crh2 was also studied. The Michaelis-Menten constants showed no effect of acceptor lenght on the affinity between enzyme and substrate.
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