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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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interaction of adhesive with structural wood
Janová, Petra ; Rovnaník, Pavel (referee) ; Vaněrek, Jan (advisor)
This labor is based on penetration of adhesives into a wood cell walls. It focuses mainly on adhesives that are currently used for construction purposes. It also contains a methodics of selecting adhesives and wood, which will be used for glued joints and summarizes the methods used for detect these adhesives in the wood cell wall. It experimentally verifies the appropriate selection of the dye-solvent combination used in the adhesive.
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The role of adhesion of the plasma membrane to the cell wall in the development and function of the root system
Králíková, Dagmar ; Tylová, Edita (advisor) ; Schwarzerová, Kateřina (referee)
Adhesion between the plasma membrane and the cell wall and the existence of the continuum of these two compartments is needed for signal transmission, e.g. under pathogen attack, during cell expansion and cell wall growth, or in response to environmental conditions. This adhesion is, in addition to the turgor, provided by physical connection of both compartments. One of the best-known examples of physical connection is found in the root system, in the Caspary strip region, where it is required to maintain apoplastic barriers of the root system, even under adverse conditions and consequent plasmolysis. There is little information about the physical interconnection and the participating macromolecules but there are candidates, which could participate in this interaction. The diploma thesis deals with arabinogalactan (AGP) proteins with fasciclin-like domain (namely FLA9 and FLA2). These proteins may play a role in the adhesion of plasma membrane and cell wall and may be involved in the growth regulation and development of the root system. Both genes are are relatively strongly expressed in the roots, especially in the elongation zone and in the cortex, including the endodermis (according to chip data). Also, to a smaller extent, the thesis deals with integrin-like At14A protein. The most interesting...
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Molecular basis for regulation of cell wall pH in Arabidopsis thaliana
Bogdan, Michal ; Fendrych, Matyáš (advisor) ; Retzer, Katarzyna (referee)
The cell wall is one of the defining parts of the plant cell. Cell walls resist turgor pressure, give plants their rigidity and still allow the cell to grow despite their stiffness. The plant cell wall is a composite material that consists of different elements; thus, the knowledge of the plant cell wall composition helps us understand the impact of the mentioned pH changes. Alterations of plant cell wall properties result in a change of plant cell growth rate. A prime example of this is the modification of wall properties by pH change, termed the acid growth. Although acid growth has been studied for a long time, we still do not fully understand the underlying mechanisms, as we have not yet identified all the agents involved in acid growth and our ability to determine apoplastic pH is limited. Local cell wall pH fluctuations arise also due to plant organs having different roles and being affected by a diverse range of stimuli. Despite the fact that new pH measurement techniques, such as genetically encoded fluorescent probes, were developed in the last two decades, there is still a need to provide a higher spatiotemporal resolution of pH analysis. Key words: Arabidopsis, apoplastic pH, cell growth, acid growth theory, cell wall, auxin, apoplastic pH measurement
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Analysis of CESA complexes dynamics in plant cytoskeletal mutants
Dubenecká, Kamila ; Schwarzerová, Kateřina (advisor) ; Malínská, Kateřina (referee)
The basis of this study are mutant plants with ARP2/3 complex lacking in one of its subunits (arpc5 and arp2). These plants also express CSC subunit CESA6 of primary cell wall tagged by YFP. Thanks to modern imaging technologies, it is possible to observe the movement of tagged cellulose synthase complexes in vivo at plasmatic membrane. Kymograph analyses was used to measure the velocity of CESA complexes. In addition to observing CESA complexes directly on the plasma membrane, experiments were made to regenerate cell walls of protoplasts of Arabidopsis thaliana plants arpc5 and WT. It was found, that observed mutants arpc5 and arp2 have reduced velocity of CESA complexes in comparison to WT and arpc5 protoplasts regenerate cellulose mesh of cell wall slower. Keywords: Cellulose synthesis, ARP2/3 complex, CESA, CSC velocity, arpc5, arp2, Arabidopsis thaliana.
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Study of adhesive penetration into the wood cell wall
Janová, Petra ; Krbila, Jaromír (referee) ; Vaněrek, Jan (advisor)
This labor is based on penetration of adhesive into a wood cell walls. It focuse on adhesives basicly used for construction purposes, especially on epoxy resins. The labor contains the use of methods for precise detection adhesive in wood and methodics for choice of adhesive and wood basically used for bonding. It describes the experimentally detected dependence of contact angle on viscosity epoxy resins.
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Functions of the exocyst complex in secretion and cell wall biogenesis
Vukašinović, Nemanja ; Synek, Lukáš (advisor) ; Růžička, Kamil (referee) ; Kost, Benedikt (referee)
The mechanical strength of plant tissues and organs can be attributed to specific properties of the cell wall. In many cases, in order to establish their final shape, cells deposit various cell wall materials in a localized manner. This is achieved by highly organized action of the endomembrane system which is essential for biosynthesis and secretion of cell wall proteins and polysaccharides. The exocyst complex is a conserved tethering complex in eukaryotes and it is involved in tethering of secretory vesicles to the sites of secretion at the plasma membrane. In this study, we address several aspects of the plant exocyst complex architecture and cell wall development using molecular biology techniques and advanced confocal microscopy. We demonstrated that two SEC10 exocyst subunits are present in Arabidopsis thaliana and share redundant functions. We also showed that the architecture of the plant exocyst complex shares several structural features with the yeast one. We demonstrated the importance of the functional EXO84b exocyst subunit for normal tracheary element development and showed that the main constituents of the secondary cell walls are deposited normally in exocyst mutants. We described a clear difference in the exocyst microtubule-independent dynamics in epidermal cells vs. cell type...
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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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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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