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Synthesis and delivery of novel fluorescently-labelled nucleotides and their nucleic acids for bio-analytical applications
Güixens Gallardo, Pedro ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Klán, Petr (referee)
1 Abstract The goals of the thesis were to synthesise novel fluorescently labelled nucleotides and the corresponding nucleic acids for bio-analytical applications as well as their delivery into cells. The thesis also aimed at the development of an effective method to inhibit non-templated incorporation of nucleotides. The problematic non-templated enzymatic incorporation of nucleotides is addressed by using several commercially available 5'-modified-oligonucleotides. The oligonucleotides (ONs) that we tested bore ortho twisted intercalating nucleic acid (oTINA), a trityl group, or biotin at the 5'-end. The modified ONs were used as templates in the enzymatic primer extension (PEX) experiments in the presence of either modified nucleotides or only natural deoxynucleoside triphosphates (dNTPs). The oTINA templates underwent PEX reaction using natural dNTPs and different DNA polymerases of the A or B family. In parallel, two types of fluorescent nucleoside derivatives were independently designed and synthesised. Firstly, we envisaged new fluorescent nucleotide tags containing the hexamethylated BODIPY moiety as a bright fluorescent label. Conversely, we focused on the improvement of fluorescent nucleotide probes sensitive to the viscosity or polarity. The fluorescently labelled methylated BODIPY nucleotides...
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Photodissociation of small polyatomic molecules investigated via molecular beam and velocity map imaging techniques
Vinklárek, Ivo ; Fárník, Michal (advisor) ; Wester, Roland (referee) ; Klán, Petr (referee)
Velocity map imaging (VMI) combined with supersonically expanded molecules pro- vides molecular-level insight into many fundamental natural phenomena. This compre- hensive work delivers an introduction to the photodissociation theory and applied VMI and mass spectrometric experimental techniques, followed by an overview of our achieve- ments in the investigation of photodissociation and clustering on the selected molecular systems. Also, new implementations of the centroiding mode and the current status of the apparatus for imaging are commented on for future measurements. The first part of the experimental results focuses on fundamental research of photodissociation of isolated molecules. We demonstrate an efficient coupling between the stretching vibration and ejection of hydrogen radical in pyrrole and, furthermore, describe the impact of singlet- triplet dynamics and molecular frame on energy redistribution during the fragmentation of selected chloroalkanes. The second part revolves around chloroalkane clusters in the perspective of atmospheric chemistry, which is represented by the observations of differ- ent dynamics for CH3 and Cl fragments in (CH3Cl)n cluster photodissociation. Finally, the clustering research is extended by our recent pickup experiments with clusters of polyaromatic hydrocarbons...
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Investigation of monomolecular organic layers and their interaction with atmospheric oxidants and pollutants
Habartová, Alena ; Jungwirth, Pavel (advisor) ; Jedlovszky, Pal (referee) ; Klán, Petr (referee)
Title: Investigation of monomolecular organic layers and their interaction with atmospheric oxidants and polutants Author: Mgr. Alena Habartová Institute: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic Advisor: prof. Mgr. Pavel Jungwirth, DSc. Abstract: Using classical molecular dynamics simulations, we studied interac- tions between Langmuir monolayers of palmitic acid (PA) and haloalkanes as serious atmospheric pollutants, to model naturally occurring and atmospherical- ly relevant complex surfaces of extended water bodies and aqueous aerosols. We investigated partitioning, orientation, solvation, as well as structure and morphol- ogy of the individual components and their mixture at water/vapor interfaces at different temperatures in order to validate our computational model and com- plement experimental data with molecular-level details. First, we have shown that halocarbon molecules adsorbed on amorphous ice nanoparticles at 100 K remain mostly isolated and do not form aggregates, in contrast to their cluste- ring on cryogenic argon nanoparticles. Next, we report that chloro-, bromo-, and iodoalkanes with short alkyl chains (up to C5) exhibit high preference for the outermost region of the water/vapor interface. Their average residence times of several...
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Investigation of Non-Heme Iron-Oxo Intermediates in the Gas Phase
Andris, Erik ; Roithová, Jana (advisor) ; Nachtigall, Petr (referee) ; Klán, Petr (referee)
I used helium-tagging infrared photodissociation spectroscopy of ions in the IR (IRPD) and vis (visPD) range in conjunction with gas-phase reactivity studies to study biomimetic terminal iron-oxo complexes. In first part of the work, I measured IRPD spectra of iron(IV)- oxo complexes of TMC, N4Py and PyTACN amine ligands in different charge states. Results show that the gas-phase Fe=O stretching frequencies are, on average, blue-shifted by 9 cm-1 with respect to the condensed phase. I prepared stereoisomers of [(PyTACN)FeIV (O)(X)]+ complexes (X = CF3COO, NO3) that were in quintet and triplet state using nitrate cleavage method and showed that the Fe=O frequency of these iron(IV)-oxo complexes is not affected by the spin state. Gas-phase reactivity shows that dicationic complexes react with 1,4- cyclohexadiene by hydride transfer and monocationic complexes engage in hydrogen atom transfer (HAT) from the methylene group and oxygen atom transfer to the C=C bond. Variation of the ligand trans to the Fe=O unit has the greatest influence on reactivity, whereas the spin state has a modest effect. In the second part of the work, I prepared and studied gas- phase iron(III)-oxo complexes with N4Py, TPA, TQA and TMC ligands. IRPD and visPD spectra in conjunction with DFT calculations allowed me to assign the...
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Nanofiber materials simultaneously photogenerating NO and 1O2 species; Reversible NO binding on boron-containing clusters
Dolanský, Jiří ; Mosinger, Jiří (advisor) ; Klán, Petr (referee)
This project is concerned with the preparation of electrospun polystyrene (PS) nanofiber materials with covalently bonded NO-photodonor and electrostatically attached tetracationic porphyrinoid photosensitizers. These photofunctional nanofiber materials exhibit effective simultaneous photogeneration of small antibacterial NO and O2(1 ∆g) species under irradiation with daylight creating an antibacterial surface and near surrounding. NO species can be also generated just by gentle heating. Nanofiber materials were analyzed with SEM, FTIR, emission and UV/vis spectroscopy and time-resolved emission and absorption spetroscopy. The antibacterial effect was tested on Escherichia coli. The dual antibacterial action, in combination with the nanoporous character of the material that detains pathogens like bacteria on its surface, is ideal for any application where a sterile environment is neces- sary. The known bimetallic cluster system [(PMe2Ph)4Pt2B10H10] that possesses the propen- sity to reversibly bind small gaseous molecules (O2, SO2, CO) was synthesized in good yields for NO reversible binding investigation. Seven new monometallic precursors (Pt, Pd and Ni) to new bimetallic species were succesfully synthesized with the aim of future study of NO reversible binding. All new compounds were purified by...
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Implementation of ion imaging technique in experiments with free molecules, clusters and nanoparticles
Kočišek, Jaroslav ; Fárník, Michal (advisor) ; Klán, Petr (referee) ; Glosík, Juraj (referee)
Title: Implementation of ion imaging technique in experiments with free molecules, clusters and nanoparticles Author: Jaroslav Kočišek Department: Department of Surface and Plasma Science Supervisor: Mgr. Michal Fárník, PhD., DSc., Ústav fyzikální chemie J. Hey- rovského, AV ČR, v.v.i. Abstract: The experimental work is focused on implementation of novel tech- niques to study clusters and nanoparticles in molecular beams. A new exper- imental system was tested, which combines the technique of velocity map ion imaging with pulsed molecular beam source. The same method and new mass spectrometer were implemented on the apparatus with molecular beam of size selected clusters. The new methods were used to study environmental effects on photo and electron induced chemistry. The most important results concerns on influence of expansion conditions on the structure of formed neutral clusters of HBr and C2H2. Results of experiments with HNO3 and CF2Cl2 molecules are then crucial for understanding heterogeneous processes in the Stratosphere. Keywords: ion imaging, mass spectrometry, molecular beams, photochemistry, nanoparticles
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Synthesis and delivery of novel fluorescently-labelled nucleotides and their nucleic acids for bio-analytical applications
Güixens Gallardo, Pedro ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Klán, Petr (referee)
1 Abstract The goals of the thesis were to synthesise novel fluorescently labelled nucleotides and the corresponding nucleic acids for bio-analytical applications as well as their delivery into cells. The thesis also aimed at the development of an effective method to inhibit non-templated incorporation of nucleotides. The problematic non-templated enzymatic incorporation of nucleotides is addressed by using several commercially available 5'-modified-oligonucleotides. The oligonucleotides (ONs) that we tested bore ortho twisted intercalating nucleic acid (oTINA), a trityl group, or biotin at the 5'-end. The modified ONs were used as templates in the enzymatic primer extension (PEX) experiments in the presence of either modified nucleotides or only natural deoxynucleoside triphosphates (dNTPs). The oTINA templates underwent PEX reaction using natural dNTPs and different DNA polymerases of the A or B family. In parallel, two types of fluorescent nucleoside derivatives were independently designed and synthesised. Firstly, we envisaged new fluorescent nucleotide tags containing the hexamethylated BODIPY moiety as a bright fluorescent label. Conversely, we focused on the improvement of fluorescent nucleotide probes sensitive to the viscosity or polarity. The fluorescently labelled methylated BODIPY nucleotides...
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Investigation of Non-Heme Iron-Oxo Intermediates in the Gas Phase
Andris, Erik ; Roithová, Jana (advisor) ; Nachtigall, Petr (referee) ; Klán, Petr (referee)
I used helium-tagging infrared photodissociation spectroscopy of ions in the IR (IRPD) and vis (visPD) range in conjunction with gas-phase reactivity studies to study biomimetic terminal iron-oxo complexes. In first part of the work, I measured IRPD spectra of iron(IV)- oxo complexes of TMC, N4Py and PyTACN amine ligands in different charge states. Results show that the gas-phase Fe=O stretching frequencies are, on average, blue-shifted by 9 cm-1 with respect to the condensed phase. I prepared stereoisomers of [(PyTACN)FeIV (O)(X)]+ complexes (X = CF3COO, NO3) that were in quintet and triplet state using nitrate cleavage method and showed that the Fe=O frequency of these iron(IV)-oxo complexes is not affected by the spin state. Gas-phase reactivity shows that dicationic complexes react with 1,4- cyclohexadiene by hydride transfer and monocationic complexes engage in hydrogen atom transfer (HAT) from the methylene group and oxygen atom transfer to the C=C bond. Variation of the ligand trans to the Fe=O unit has the greatest influence on reactivity, whereas the spin state has a modest effect. In the second part of the work, I prepared and studied gas- phase iron(III)-oxo complexes with N4Py, TPA, TQA and TMC ligands. IRPD and visPD spectra in conjunction with DFT calculations allowed me to assign the...
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Designing and Implementation of a web based clothes delivery service
Feldstein, David ; Klán, Petr (advisor) ; Šretr, Jiří (referee)
The objective of the thesis is to create a web based service designed for sending packages of clothes from the initial designing to final implementation using authors content management system FeldCode CMS. It is planned to put this service into actual operation in summer 2017. This is why another goal of this thesis is to lay out key success identifiers of the service assuming that my masters final thesis would build on these identifiers later in my studies aiming to evaluate success of the whole project. The content of the thesis will be divided traditionally to theoretical and practical part. The theoretical part would describe the process of the whole service and its participants. It will outline technologies used while developing, some design principles followed and of course it will specify key success identifiers as mentioned above. The practical part consists of the designing and the output application itself which is indivisible electronical attachment to this thesis containing all the source codes and SQL commands to create its database structure.
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Website development with web analytics implementation
Kolařík, Miroslav ; Klán, Petr (advisor) ; Galba, Alexander (referee)
The aim of this bachelor thesis is to create a company website according to the latest trends and standards including inherit web analytics implementation. The website should inform new and current customers, raise public awareness about the company and provide data to analyze its usage. The theoretical part briefly describes key terms, such as HTML, CSS and web analytics methods. The practical part focuses on creating website optimized for mobile devices with a focus on basic search engine optimization and successful Google Analytics implementation.
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