National Repository of Grey Literature 7 records found  Search took 0.00 seconds. 
Enzymatic synthesis of DNA modified in the minor groove
Matyašovský, Ján ; Hocek, Michal (advisor) ; Hlaváč, Jan (referee) ; Urban, Milan (referee)
In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA....
Synthesis and studies of modified DNA: (i) development of DNA targeting molecular scissors and (ii) competitive enzymatic incorporation of base-modified nucleotides
Panattoni, Alessandro ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Fojta, Miroslav (referee)
In the first part of this work, a series of site-specific artificial metallonucleases (AMNs) was developed conjugating clamped-phenanthroline (Clip-Phen) copper complexes to triplex- forming oligonucleotides (TFOs). Several synthetic routes were explored for the synthesis of the TFO-AMNs hybrids, all sharing a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction as the key step. As a consequence, building blocks for enzymatic or chemical synthesis of oligonucleotides (ONs) containing clickable groups, or already conjugated to the Clip-Phen ligand via CuAAC, were prepared. Two new alkynyl-linked nucleoside-5'-O-triphosphates (dNTPs) were designed and developed in order to obtain an efficient polymerase incorporation of clickable alkynyl-tethers into ONs and, at the same time, enhance the efficiency of CuAAC reactions on modified DNA. The relative 3'-O- phosphormaidites were also prepared in order to insert the same alkynyl-linkers into ONs via solid-phase synthesis. The AMN was linked at the 5'- or 3'-ends or in the middle of the TFO stretch, using diverse likers. The hybridization of all the synthesized TFOs with a target DNA duplex was studied. Finally, an extensive study of cleavage efficiency and specificity of the TFO-AMN conjugates towards the target DNA was performed, exploring the...
New redox labels for DNA
Simonova, Anna ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Vyskočil, Vlastimil (referee)
The aim of my thesis was the synthesis of the modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing electrochemically oxidizable labels and their incorporation into DNA for the application in bioanalysis. In the first part of my thesis, I developed the synthesis of modified dNTPs bearing 2,3- dihydrobenzofuran (DHB) or 2-methoxyphenol (MOP) labels at 5-position of 2'- deoxycytidine 5'-O-triphosphate and at the 7-position of 7-deaza-2'-deoxyadenosine 5'-O- triphosphate by Suzuki-Miyaura cross-coupling reactions. Then modified dNTPs were used as substrates for DNA polymerases in enzymatic synthesis of modified DNA by PCR and primer extension. Electrochemical properties of the DHB and MOP-labeled nucleosides, dNTPs and DNA were studied by using of a square-wave voltammetry (SWV) at the pyrolytic graphite electrode (PGE) giving signals of MOP oxidation around 0.5 V and DHB oxidation around 0.85 V. The use of DHB group in combination with other electrochemical active labels was limited by close position of its oxidation peak to the signals of oxidation of natural nucleobases, whereas MOP moiety was successfully used for redox coding of nucleobases in combination with aminophenyl or benzofurazane label giving two independently readable redox signals in each case. In the second part of this...
The physiological and proteomic characterisation of winter oilseed rape upon abiotic stress
Urban, Milan ; Vítámvás, Pavel (advisor) ; Hnilička, František (referee) ; Valentová, Olga (referee)
- Ph.D. thesis - Milan Urban, 2017 In some years, the agricultural production of oilseed rape, an important crop in the Czech Republic, is - besides biotic stress - facing the problem of damage caused by frost or drought. Together with special attention paid to proteins revealing responses between crop genotypes with differential abiotic stress tolerance levels we reviewed possible applications of proteomic results in crop breeding programs aimed at an improvement of crop stress tolerance (paper 1). For first original result, cold temperature was imposed upon non-vernalized plants in the stage of leaf rosette. The article (paper 2) shows a significant correlation between frost tolerance (FT), dehydrin (DHN) accumulation, and photosynthetic acclimation in five cultivars (cvs). Newly, the specific DHN D97 was shown to accumulate and other DHNs were shown to have qualitative differences in accumulation. These results imply that proper FT assessment is based on rapid photosynthetic acclimation together with higher accumulation of protective compounds. Drought stress (paper 3) was imposed in the water- demanding stem prolongation phase before flowering, because late-spring drought before and during flowering decreases the yield and seed quality significantly. This paper newly describes two water-uptake...
Role of brassinosteroids in plant response to drought stress
Trubanová, Nina ; Rothová, Olga (advisor) ; Urban, Milan (referee)
Brassinosteroids are steroid phytohormones with a wide range of effects. They improve survivance of plants exposed to drought stress, increase their resistence and yield therethrough. They regulate the response of plants directly (via synthesis of metabolits) and/or indirectly (via the induction of antioxidant compounds and enzymes), often in interaction with other phytohormones. Experiments studying influence of brassinosteroids on response of plants stressed with water deficit differ in several parameters. Their correct interpretation is complicated conseguently.
Development of a general and modular approach to C-nucleosides
Kubelka, Tomáš ; Štefko, Martin ; Bárta, Jan ; Joubert, Nicolas ; Urban, Milan ; Chapuis, Hubert Jean ; Hocek, Michal
Highly efficient and modular approach was developed for the synthesis of various types of new (het)aryl C-nucleosides. This protocol consists of the synthesis of haloaryl-C-nucleoside intermediates, followed by a functional group transformation to introduce various substituents. Using this approach protected 2′-deoxy-C-nucleosides bearing halogenated benzene, pyridine, thiophene, furane and pyrimidine were prepared. These intermediates were then submitted to a wide range of palladium-catalyzed reactions. The same approach was also used for preparation of C-nucleosides bearing ribofuranose moiety. Functional ribofuranosides bearing diverse substituted pyridine and benzene nucleobases were prepared in this way.
Modulární metodiky pro přípravu různých aryl C-2'-deoxyribonukleosidů
Joubert, Nicolas ; Bárta, Jan ; Urban, Milan ; Hocek, Michal
A modular methodology for the syntheses of various substituted aryl C-2´-deoxyribonucleosides has been developed. In each series, a larger scale synthesis of a versatile halogenated C-nucleoside intermediate has been accomplished, followed by its use for a generation of diverse derivatives by displacement of the halogen for alkyl, aryl or amino substituents by cross-coupling or amination reactions. Subsequent deprotection gave the final desired C-2´-deoxyribonucleosides. This methodology has been applied for the development of modular syntheses of 4- or 3-substituted benzene C-nucleosides, 6-substituted pyridin-2-yl C-nucleosides, 6-substituted pyridin-3-yl C-nucleosides and 5-substituted thiophen-2-yl C-nucleosides.

See also: similar author names
15 URBAN, Martin
20 URBAN, Michal
4 URBAN, Miroslav
4 Urban, Marek
3 Urban, Marián
15 Urban, Martin
1 Urban, Matěj
11 Urban, Michael
20 Urban, Michal
4 Urban, Miroslav
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