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Reorganization of in vitro reconstituted actin-based networks
Sabó, Ján ; Lánský, Zdeněk (advisor) ; Varga, Vladimír (referee) ; Cifra, Michal (referee)
The cytoskeleton provides living organisms with machinery to move. On the molecular scale, the same cytoskeletal components undergo constant reorganization to contribute to distinct cellular processes, such as the navigation of neuronal growth cones in brain development or the creation of chiral actin flows during cellular division. During brain development, neuronal growth cones navigate by employing cytoskeletal crosstalk between actin filament networks and microtubules. Crosstalk in the growth cones between these cytoskeletal components was linked with known microtubule polymerase human cytoskeleton associated protein 5 (CKAP5; homolog of XMAP215, MSPS, Zyg9), while the details of the direct molecular mechanism of CKAP5-provided remodeling of both microtubules and actin networks are lacking. Therefore, we used total internal reflection fluorescence (TIRF) microscopy together with state- of-the-art in vitro reconstituted assays combining microtubules, actin filament networks, and recombinant proteins to understand cytoskeleton-provided molecular mechanisms underlying cellular motion. In this thesis, we show that CKAP5 alone bundles both supposedly randomly oriented and parallel actin filaments, crosslinks actin filaments to microtubules regardless of their polarity, positions prevailing actin...
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Ultra-weak photon emission from biological samples
Vahalová, Petra ; Cifra, Michal (advisor) ; Rác, Marek (referee) ; Šachl, Radek (referee)
Oxidative processes are an inseparable part of the life of almost all living organisms. They help to maintain homeostasis but they can also stand behind various dysfunctions or diseases. Therefore, an effective method for monitoring oxidative processes in biosamples is an essential tool for medicine, agriculture, and food industry. In this thesis, an overview of available methods for monitoring oxidation in biosamples with a special focus on biological autoluminescence (BAL) is provided. This thesis uses the term BAL to encapsulate various synonyms including the commonly used term ultra-weak photon emission. BAL correlations with various physical, chemical, and biological factors (as original research and an overview from other authors) are also presented. Specifically, the relationships between spontaneous BAL of yeast Saccharomyces cerevisiae and the selected physical and chemical parameters (pH, oxygen partial pressure, and cell concentration) during cell growth were established. Additionally, the correlation of BAL intensity from yeast cells or protein bovine serum albumin (BSA) and the number of reactive oxygen species (ROS) that originated as a result of the Fenton reaction were measured. Physical enhancement of BAL from BSA by pulsed electric field was also studied and a basic reaction...
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Electronic effects at the interface between biomolecules, cells and diamond
Krátká, Marie ; Rezek, Bohuslav (advisor) ; Cifra, Michal (referee) ; Skládal, Petr (referee)
Understanding and control of interactions between biological environment (cells, proteins, tissues, membranes, electrolytes, etc.) and solid-state surfaces is fundamental for biomedical applications such as bio-sensors, bio-electronics, tissue engineering and implant materials as well as for environmental monitoring, security and other fields. Diamond can provide unique combination of semiconducting, chemical, optical, biocompatible and other properties for this purpose. In this thesis we characterize electronic properties of protein-diamond interface by employing a solution-gated field-effect transistor (SGFET) based on hydrogen-terminated diamond, surface of which is exposed to biological media. We elucidate the role of adsorbed protein layer on the electronic response of the diamond transistor. We investigate effects of cells (using mainly osteoblast cells as model) on diamond SGFETs transfer characteristics and gate currents. We employ nanocrystalline diamond (NCD) thin films of different grain sizes (80 - 250 nm) to characterize and discuss influence of grain boundaries and sp2 phase on bio- electronic function of SGFETs. We investigate effects of gamma irradiation on function and stability of hydrogen-terminated diamond SGFETs interfaced with proteins and cells, showing feasibility of...
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Regulation of microtubule dynamics revealed by single-molecule TIRF and IRM microscopy
Zhernov, Ilia ; Lánský, Zdeněk (advisor) ; Cifra, Michal (referee) ; Varga, Vladimír (referee)
The microtubular cytoskeleton is a ubiquitous and highly diverse biopolymer network present in all eukaryotic cells. Microtubules stochastically alternate between phases of growth and shrinkage. Cells take advantage of this dynamicity to generate forces for essential processes, such as cell division, motility or morphogenesis. Regulating the microtubule dynamics enables cells to adaptively respond to a wide range of tasks and conditions. Molecular mechanisms underpinning the regulation are not fully understood. Using a bottom-up approach and the combination of single molecule total internal reflection fluorescence (TIRF) microscopy and interference reflection microscopy (IRM), we here reconstituted and explored two dynamic cytoskeletal systems. (i) Microtubule doublets, comprising incomplete B-microtubule on the surface of a complete A- microtubule, provide an essential structural scaffold for flagella. Despite the fundamental role of microtubule doublets, the molecular mechanism governing their formation is unknown. We here demonstrate an inhibitory role of tubulin C-terminus in microtubule doublet assembly. By partial enzymatic digestion of polymerized microtubules followed by the addition of free tubulin in the presence of a stabilizing agent, we assembled microtubule doublets and revealed the B-...
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Electronic effects at the interface between biomolecules, cells and diamond
Krátká, Marie ; Rezek, Bohuslav (advisor) ; Cifra, Michal (referee) ; Skládal, Petr (referee)
Understanding and control of interactions between biological environment (cells, proteins, tissues, membranes, electrolytes, etc.) and solid-state surfaces is fundamental for biomedical applications such as bio-sensors, bio-electronics, tissue engineering and implant materials as well as for environmental monitoring, security and other fields. Diamond can provide unique combination of semiconducting, chemical, optical, biocompatible and other properties for this purpose. In this thesis we characterize electronic properties of protein-diamond interface by employing a solution-gated field-effect transistor (SGFET) based on hydrogen-terminated diamond, surface of which is exposed to biological media. We elucidate the role of adsorbed protein layer on the electronic response of the diamond transistor. We investigate effects of cells (using mainly osteoblast cells as model) on diamond SGFETs transfer characteristics and gate currents. We employ nanocrystalline diamond (NCD) thin films of different grain sizes (80 - 250 nm) to characterize and discuss influence of grain boundaries and sp2 phase on bio- electronic function of SGFETs. We investigate effects of gamma irradiation on function and stability of hydrogen-terminated diamond SGFETs interfaced with proteins and cells, showing feasibility of...
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Monitoring the impact of exposure to electromagnetic fields in yeast colonies
Hájková, Renata ; Vorlíček, Jaroslav (advisor) ; Cifra, Michal (referee)
Identifikační záznam: HÁJKOVÁ, Renata. Sledování vlivu expozice elektromagnetickým polem na kolonii kvasinek [Monitoring the impact of exposure to electromagnetic fields in yeast colonies]. Praha, 2011, rok vydání. 74s., 2. příl. Diplomová práce (Mgr.). Univerzita Karlova v Praze, 1. Lékařská fakulta, Fakulta elektrotechnická, Katedra elektromagnetického pole, ČVUT v Praze. Vedoucí závěrečné práce Vorlíček, Jaroslav, Ing. Abstract: Current development of mobile telecommunication device increases interest of public to the question of influence of electromagnetic field to the living organisms. Results of this work contribute to better knowledge of interaction between electromagnetic field and cell structures. Beginning of the thesis is review of morphology and physiology of the yeast cell that was used in this work as a tested organism. Second chapter describe the basic facts about electromagnetic fiend and wireless energy transfer. Goal of the practical part of this master thesis is describe influence of electromagnetic field on population of yeast during their proliferation. We constructed special box connected with wireless energy generator that was source of exactly defined electromagnetic field. Cells of Saccharomyces cerevisiae were diluted in fluid medium YPD and the proliferation of the culture was...
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Electromagnetic-based nano-resolution microscopies for biological research
Kučera, Ondřej ; Cifra, Michal
Research of cell biology is principally related to progress in imaging under diffraction limit of visible light and to the functional imaging. This paper brings short review of experimental techniques including Photonic force microscopy, Scanning microwave microscopy and other relevant techniques. The possibility of employing these techniques for elucidation of endogenous biological electromagnetic activity is discussed
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Electric field generated by higher vibration modes of microtubule
Cifra, Michal ; Havelka, D. ; Kučera, Ondřej ; Pokorný, Jiří
Certain structures in a living cell may generate electric oscillations. Microtubules, which form a part of a cellular skeleton, belong to this class of structures and ful-fill all conditions for generation of electric oscillations in kHz÷GHz band. We present selected results from calculations of the oscillatory electric field generated by higher vibration modes of microtubules. We propose that the electric field of certain modes may play specific function in cellular organization
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Calculation of the Electromagnetic Field Around Microtubule
Havelka, D. ; Cifra, Michal
Microtubules are important structures in cytoskeleton which organizes the cell. Single microtubule is composed of electrically polar structures, tubulin heterodimers, which have strong electric dipole moment. Vibrations are expected to be generated in microtubules, thus tubulin heterodimers as electric dipoles are oscillating. This gives rise to electromagnetic field, which is detected around the cells. We calculate here the electromagnetic field of microtubules if they are excited at 1 GHz. This paper includes the work done in bachelor thesis of the first author.
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Measurement and Computer Simulation of Electromagnetic Oscillations of Living Cells
Cifra, Michal ; Pokorný, Jiří ; Vrba, J.
Frohlich postulated coherent polar oscillations as a fundamental biophysical property of biological systems. Recently, Pelling et al. (2004, 2005) detected mechanical vibrations of yeast cell membrane with atomic force microscope (AFM) and analyzed by Fourier analysis in the frequency range 0.5-2 kHz with amplitudes of the order of 1 nm. This article describes the measurement of electric activity of yeast cells in the acoustic frequency range and of mechanical vibrations of cell membrane. Spectrum analyzer and electrically and > electromagnetically screened box with point sensor and amplifiers fed by batteries were used for measurement of synchronized and non synchronized tubulin mutants of yeast cells. We show that the electric activity of synchronized cells in the M phase is greater that of non synchronized cells. That corresponds to the findings of Pohl et al. (1981). Obtained results of measurement of cell electric activity are in good agreement with AFM findings.
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