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Genetically encoded biosensors of cellular tension and their use in cellular biology
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Lánský, Zdeněk (referee)
1 Abstract and key words Mechanical forces have great impact on the life of cells. They influence cell proliferation, migration or differentiation and defects in cellular mechanosensing were reported to be the cause of various diseases, such as deafness, atherosclerosis or cancer. However, mechanisms of mechanical sensing are not thoroughly examined and not many tools for doing such research are available. Genetically encoded FRET-based biosensors are one of the existing methods for studying transfer of mechanical signal in cells. It is a non-invasive method allowing to observe changes in mechanical tension across proteins in living cells. In this thesis, different types of existing genetically encoded FRET-based tension biosensors are introduced together with the process of their development and knowledge gained by their use in research. Key words: mechanical force, mechanosensing, FRET, tension sensor, biosensor development
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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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Influence of lipid composition and model peptides on lateral organization of lipid layers
Veľas, Lukáš ; Heřman, Petr (advisor) ; Malínský, Jan (referee)
Oxidized phospholipids (OxPLs) are known to be present in living organisms due to oxidative stress. However, the physiological function of OxPLs is still not fully understood. They have been shown to be present in many inflammatory diseases such as atherosclerosis and neurodegenerative diseases like Parkinson's and Alzheimer's disease. In this work we present the influence of two truncated OxPLs on the lateral heterogeneity of a model lipid membrane. Specifically, we studied the effect of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) on the formation of nanodomains present in giant unilamellar vesicles containing 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol and sphingomyelin. Only few techniques are capable of detecting nanometer-sized domains in the membrane with high resolution. Time resolved Förster resonance energy transfer (TR-FRET) combined with Monte Carlo (MC) simulations provide a strong tool not only to detect lateral heterogeneities but also characterize them with the resolution of 2 nm. Profound effects on the nanodomain size were observed in the presence of both studied OxPLs and differences were detected, as PGPC with a carboxylic group drives formation of larger nanodomains than POVPC...
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Využití simulace jako komplementární metody pro interpretaci experimentálních dat ve výzkumu fluorescence jednotlivých molekul
CARDA, Zdeněk
Fluorescence single-molecule methods represent mighty tools for researchers in the field of structural and molecular biology. These methods are bringing in many advantages when compared to the statistical data processing of multi-molecular species. We can directly compare true statistical distributions and their kinetics. Here belongs fluorescence correlation spectroscopy, FRET and burst variance analysis. As the research advances, new methods are being developed which at the very beginning do not have proper analytical relations for data interpretation and which experimental limits we can't tell. That is the moment when the computer simulations can be used to our advantage. They can help us to specify the right direction of the future research, which is a great money-saver, while opening new perspectives and insights into the explored matter. Correct interpretation of the simulations results is key for the consequent defining of new theoretical models.
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Spliceosome assembly
Hausnerová, Viola ; Staněk, David (advisor) ; Chalupníková, Kateřina (referee)
Pre-mRNA splicing is a process in which introns are removed from eukaryotic transcripts and exons are ligated together. Splicing is catalyzed by spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and more than 100 additional proteins, which recognizes 5' splice site, branch point site and 3' splice site and performs two transesterification reactions to produce mRNA molecules. 5' splice site is recognized by U1 snRNP and U2 auxiliary factor (U2AF) is involved in branch point and 3' splice site recognition in the early splicing complex. There is some evidence of splice sites cooperation during intron recognition in vitro but little is known about the situation in vivo. Using Fluorescence resonance energy transfer (FRET) and RNA immunoprecipitation (RIP) methods, we have investigated the early stages of spliceosome assembly. We have employed splicing reporters based on -globin gene and MS2 stem loops to detect interactions of proteins on RNA molecule directly in the cell nucleus. Results of FRET indicate that intact 5' splice site is required for U2AF35 interaction with 3' splice site and that U1C recruitment to 5' splice site is partially limited upon 3' splice site mutation. We have also confirmed by RIP that U2 snRNP association with pre-mRNA molecule requires presence of 5'...
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Methods of measurement of electrical voltage on the cell membrane
DIVOKÝ, Karel
The aim of this work was to compile a comprehensive list of methods that deal with the research of voltage on the cell membrane. The work should help in orientation in these methods, as well as understanding of their physical and biological principle. In the first part is analysis of physical properties of membrane potential; in the second part are the most important methods for measuring membrane potential. Very important is the comparison of these methods in terms of their focus, localization, physical demands or in terms degree of damage of object under examination.
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Single-molecule fluorescence detection in molecular biology
FESSL, Tomáš
SMFD techniques offer genuine detection possibilities which are often inaccessible using ensemble methods. This was demonstrated in three projects investigating translocation activity of CHD4 protein, analysis of MS2 phage capsid assembly and in-cell characterization of DNA structure. In other projects, binding interactions between two fluorescent probes and a short oligonucleotide were characterized and all optical depth of focus extended microscope configuration for imaging of individual molecules inside bacterial cells was developed and tested.
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