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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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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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Stability of vesicular systems using fluorescence spectroscopy techniques
Máčala, Jakub ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This thesis is focused on possibility of studying stability and fusion of catanionic vesicles with Förster resonance energy transfer. The mainly used technique was Time-Correlated single photon counting. Firstly, excitation and emission spectra of chosen probes were measured and donor-acceptor pairs were suggested: 5-hexadecanoylaminofluorescein with Octadecyl Rhodamine B, Bodipy 493/503 with rhodamine or DiI, perylene with fluorescein, DiO with DiI. Then, time-resolved measurements of suggested pairs from environment of catanionic vesicles with different content of cholesterol were made in order to track the FRET associated with fusion of vesicles. It was found out, that it is not possible to use DiO as a donor because of it’s inefficient solubilisation into vesicles. It is also not possible for Bodipy to be used as a donor, because of it‘s excimer formation. In case of using fluorescein as a donor, it was found, that there is ongoing homo-fret between fluorescein molecules. Thanks to this, fusion was tracked by addition of unstained vesicles. It was also possible to track fusion in longer period of time. Also perylene-fluorescein pair was found to be capable of tracking the fusion, but with the exception of vesicles with content of cholesterol of 43 mol. %, tracking of fusion was possible only in short period of time.
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Resonance energy transfer in the study of polymer-vesicle interaction
Jízdná, Zuzana ; Kratochvíl, Matouš (referee) ; Mravec, Filip (advisor)
The presented bachelor's thesis dealt with the subject of preparation of cationic vesicular systems, formed by the CTAB and SDS surfactants, in order to study their mutual interaction with the hyaluronan polymer using Förster resonance energy transfer measurements. First, concentration series of vesicular systems with fluorophores were prepared. DPH, perylene and fluorescein were used as fluorophores. Afterwards, the fluorescence lifetime of selected fluorophores was measured by the TCSPC method. Then, the characteristics of the prepared vesicular systems were measured using the DLS and ESL methods. For the final measurement of the interaction between the vesicle and the polymer, a vesiculation solution was chosen based on all the measurements carried out, which contained DPH with a concentration of 3·10-5 mol/l and hyaluronan. Two molecular weights of hyaluronan (300 and 900 kDa) were tested at mass concentrations of 1, 5, 10, 50 and 100 mg/l. The measurement confirmed their mutual interaction, thus this system could find application in the medical sector.
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Organic luminophores with long wavelenght emission
Kolaříková, Adéla ; Kratochvíl, Matouš (referee) ; Vala, Martin (advisor)
This bachelor thesis studies the possibility of achieving intense fluorescence in the red region of the spectrum using Förster resonance energy transfer (FRET) in nanoparticles. First, the optical properties of organic molecules that could be suitable for the creation of a so-called host-guest (HG) system consisting of an acceptor-donor pair were studied. The molecules studied were based on derivatives of diphenylstilbene containing the electron-donor group diphenylamine (DPA-DPS), which is linked to a differing electron-acceptor group via a -conjugated double bond system. The acceptor groups used, i.e., indandione (-IOO), vinyl ( V) and di(methoxycarbonyl)vinyl (-V(COOMe)2), differ from each other in structure and polarity, which was reflected by a change in the position of the fluorescence spectrum. In the HG systems studied, DPA-DPS-IOO always served as the guest (G1) and DPA-DPS-V and DPA-DPS-V(COOMe)2 always served as the host (H1 or H2). Nanoparticles from these substances (G1H1 and G1H2) were prepared by the nanoprecipitation method. FRET was observed for both of these systems. Upon excitation of the matrix, the energy was transferred to the guest G1, which subsequently fluoresced in the long-wavelength region. Furthermore, an increase in the quantum yield of the guest fluorescence was also observed for the nanoparticles formed from the G1H2 system, from 7% (powder) and 3.1% (nanoparticles) to 14% compared to both the powder form and nanoparticles formed from the guest alone. No increase in the quantum yield of guest fluorescence was observed for the nanoparticles of the G1H1 system. The results indicated that FRET can be an effective tool in developing nanoparticles exhibiting intense long-wavelength fluorescence for imaging.
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Tracking membrane permeabilization on single lipid vesicles - method development.
Gücklhorn, David ; Šachl, Radek (advisor) ; Heřman, Petr (referee)
Protein complexes are challenging systems to study, especially when these complexes form on lipid membranes only for a short period of time. This is also the case of fibroblast growth factor 2 (FGF2), a protein that has many physiological and pathological functions in the human organism. It plays major role in the development of cancer as it promotes cell survival and angiogenesis. It also serves as a basis for development of novel treatments of nerve injuries. Despite being heavily studied for many years, it remains unclear how the protein is translocated into the extracellular space where it performs its function. To study complex systems such as FGF2 that self-assembles on the membrane into membrane penetrating pores we decided to develop a simple and efficient fluorescent microscopy method. This method is called double leakage single GUV assay (DLSGA). It utilizes giant unilamellar vesicles (GUVs) mimicking native cellular membranes. In a single experiment, up to 300 individual GUVs are imaged for the content of a leakage dye that reports on the presence of FGF2 pores. During three measurements and under different conditions, detailed information about pore-opening dynamics is gained for each GUV. Results of these measurements are then used to divide GUVs into six groups based on formation and...
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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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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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Analysis of Src dynamics in cellular structures
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Rozbeský, Daniel (referee)
Src kinase is a key element in many signaling pathways affecting cellular processes such as differentiation, proliferation, motility, or migration. Deregulation of its activity is associated with the promotion of cancer. Therefore, understanding its cellular function is vital. Src activity directly correlates with its structure; when Src is active, it adopts opened conformation, when inactive, it is in closed conformation stabilized by intramolecular interactions. Detection of the conformation can be used to analyze Src activity. In this thesis, conformation-sensitive FRET-based Src biosensor was improved using mNeonGreen as a new acceptor fluorophore in the existing design and the properties of the new biosensor were compared with the original Src biosensor. The new biosensor is able to detect changes in Src conformation and can be stably expressed in cells. Src activity in focal adhesion was analyzed and higher Src activity in these structures was confirmed. Although the new biosensor did not exhibit significantly better sensitivity to Src conformational changes, it still proved to be a useful tool to study Src activity, and mNeonGreens higher brightness makes it more suitable for microscopic experiments. Key words: Src, FRET, biosensor, live-cell imaging, mNeonGreen
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Construction and evaluation of a novel protein mechanosensor
Kolomazníková, Veronika ; Rösel, Daniel (advisor) ; Novotný, Ivan (referee)
The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing
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