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Development of ultrastructural methods and their application in studies on the cell nucleus
Filimonenko, Anatoly ; Hozák, Pavel (advisor) ; Nebesářová, Jana (referee) ; Lanctôt, Christian (referee)
Despite the capabilities of molecular-biological methods in deciphering the interplay of different biological molecules and molecular complexes, the understanding of respective functions in living cells requires application of in situ methods. Obviously, these methods should provide maximal resolution and the best possible preservation of the biological object in a native state, as well as correct statistical evaluation of the spatial characteristics of detected molecular players. Transmission electron microscopy provides the highest possible resolution for analysis of biological samples. The simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. To analyze this, we have developed a complex stereological method for statistical evaluation of immunogold clustering and colocalization patterns of antigens on ultrathin sections, including a user-friendly interface. Functional microarchitecture of DNA replication and transcription sites has been successfully characterized using the developed stereological tools. Our data demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view...
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Development of surface and body musculature of the bird schistosome Trichobilharzia regenti
Bulantová, Jana ; Horák, Petr (advisor) ; Nebesářová, Jana (referee) ; Toledo, Rafael (referee)
Description of Trichobilharzia regenti as a new species of nasal bird schistosome in 1998 was only the first step in our knowledge of this extraordinary parasite. Natural definitive hosts of T. regeni are anseriform birds, but infective larvae - cercariae - are able to penetrate also into mammalian hosts including humans. There they are causative agents of hypersensitive skin immune reaction called cercarial dermatitis or swimmer's itch. Contrary to other schistosomes, miracidia of T. regenti hatch directly inside the definitive host tissue. Schistosomula migrate through the nervous system of vertebrates and, together with adult worms, they have predominantly extravascular localization in definitive hosts. Adult worms have a short lifespan and low degree of sexual dimorphism, connected with lower dependence of adult females on long-term contact with males. During the life cycle, T. regenti can be found within three different environments (freshwater, tissue of intermediate molluscan host and tissue of vertebrate host). Each of the seven developmental stages has a different role in the life cycle which corresponds with different organization of various organ systems. The introductory part of the thesis is focused entirely on ontogenetic changes of surface ultrastructure and body musculature of...
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Electron cryo-microscopy techniques in biological research and nanotechnologies
Mistríková, Veronika ; Bednár, Jan (advisor) ; Nebesářová, Jana (referee) ; Benada, Oldřich (referee)
Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...
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The Students themselves - Student Center Poříčí
Nebesářová, Jana ; Pecka, Lukáš (referee) ; Sochor, Jan (advisor)
Project of student housing, which is interesting mainly for its rotation, which improves solar conditions of residential space, and creation of two separate objects, separated as in appearence as in function. This leads also to new city street, which is also a visible axis connecting st. Leopold Church and Táborského waterfront. The street is partly glazed and it forms an atrium, which is a center of life of whole building and a center of all ways through building. In atrium there are a little bridges, which connects both objects and also creates community spaces for accommodated students. Above atrium there are community spaces at hanging boxes. In the opposite of lower spaces, it provides more peace and beautiful views.
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Comparison of freeze fracture images of mixed bacterial/yeast biofilm in cryo-SEM with high pressure freezing fixation
Hrubanová, Kamila ; Nebesářová, Jana ; Růžička, F. ; Krzyžánek, Vladislav
Microscopic organisms include bacteria and yeasts have been studied in this project. Besides the planktonic way of living, microbes are able to adhere to surfaces or interfaces and to form organized communities, a so-called biofilm, which are embedded in a matrix of extracellular polymeric substances that they produce; visualization and quantification of this microscopic formation is the main goal of this study. In medicine the biofilm formation allows microorganisms to colonize the surface of implants and it also protects the microbial cells from attacks by the immunity system as well as from the effect of antibiotics. Therefore, the biofilm is considered to be important virulence factor in these microorganisms. The characteristic features of the biofilm infections, especially high resistance to antifungal agents, complicate therapy. Understanding of the biofilm structure can contribute to understanding the biofilmformation and basic biochemical mechanisms underlying this process. It may help to develop more efficient treatment strategy for biofilm infection.
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Very low energy STEM for biology
Frank, Luděk ; Nebesářová, Jana ; Vancová, Marie ; Paták, Aleš ; Müllerová, Ilona
Examination of tissue sections with transmitted electrons has been performed at energies of hundreds and tens of eV with thicknesses of sections of 10 nm or less. This was possible by employing the cathode lens principle working without lowest energy limitations with the help of biasing the sample to a high negative potential. The reflected and transmitted electrons were attracted with the same electric field to earthed detectors situated above and below the sample. Very high image contrasts have been obtained even for samples free of any heavy metal salts for contrast enhancement.
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