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Properties and interactions of nucleoproteins studied by advanced lifetime-based imaging methods
Vacková, Anežka ; Heřman, Petr (advisor) ; Krůšek, Jan (referee)
The main aim of this thesis was implementation and testing of an additional imaging method for investigation of protein-protein interactions in our laboratory and to extend already established heteroFRET (Förster resonance energy transfer) method used with the fluorescence lifetime imaging (FLIM). We performed homoFRET experiments with both the steady-state and time-resolved anisotropy-imaging methods. The presence of homoFRET between fluorophores was done by partial fluorophore bleaching and by the "red edge" analysis. We compared the methods, especially their sensitivity, experimental requirements, complexity, and informational value. We effectively applied steady-state and time-resolved anisotropy imaging to six proteins of interest; both proved comparably effective and reliable as heteroFRET-FLIM for detection of the protein interactions. The red edge method appeared problematic due to background contribution, which could not be suppressed with the current setup and instrumentation. We validated steady-state and time-resolved anisotropy imaging by studying well- described oligomerizing nucleophosmin variants (NPMwt and NPMmut) and noninter- acting mutant NPMcut, expressed in living HEK-293T cells. Then, we also probed nu- cleolin (NCL) and free fluorescent tags NowGFP and mVenus, aiming to find...
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Thermodynamics of complex formation by miRNA
Vacková, Anežka ; Štěpánek, Josef (advisor) ; Mojzeš, Peter (referee)
The bachelor thesis concerns microRNA (miRNA), short RNA molecules that mediate post-transcriptional regulation of gene expression by binding to a target mRNA. They usually form extended duplexes consisted of short tracks of Watson-Crick basepairs separated by internal loops, mismatched pairs, or bulges. The thesis represents a study of the effect of structural anomalies, the internal loop and the bulge, on the structural stability miRNA Let7 complex with its target LCS1 sequence at mRNA lin-41 from Caenorhabditis Elegans. The task was solved by recording temperature dependent UV absorption spectra of solutions containing mixtures of the mentioned above RNA sequences and their mutated analogs, analysis of the spectral changes by using a singular decomposition method followed by fitting to a thermodynamic model. The determined thermodynamic characteristics of the complexes and spectral changes caused by a temperature increase or the complex dissociation are the outcome of the work.
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