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Construction of modified DNAs with selected reactive or protective groups
Vaníková, Zuzana ; Hocek, Michal (advisor) ; Křen, Vladimír (referee) ; Zimčík, Petr (referee)
This PhD thesis is focused on the synthesis of DNA modified with photocleavable 2- nitrobenzyl protecting groups in major groove and its applications in the regulation of gene expression in the level of transcription. In the first part of my thesis, the synthesis of photocaged 2'-deoxyribonucleosides triphosphates and their photolysis to unprotected 5-hydroxymethylated nucleotides is described. All prepared nucleoside triphosphates were good substrates for their enzymatic incorporation into DNA. Synthesized 5-(2-nitrobenzyloxy)methyl-2'-deoxyuridine-5'- monophosphate (dUNBMP) and DNA with one 5-(2-nitrobenzyloxy)methyl- modification in the sequence were used for the detailed kinetic studies of photocleavage reactions. In the second part of the thesis, the series of modified DNAs with specific sequences were prepared by primer extension (PEX) and/or polymerase chain reaction (PCR). A cleavage of prepared modified DNAs was studied by selected restriction endonucleases (REs). In all cases, the nitrobenzylated DNA fully resist the cleavage by REs. The deprotection/ photocleavage conditions for nitrobenzylated DNA were studied in the case of DNAs with positive restriction endonuclease digestion of hydroxymethylated DNA. The resulting photocleaved DNA was fully digested by REs, therefore 2-nitrobenzyl...
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Corporate social responsibility of SAP
Vaníková, Zuzana ; Müllerová, Františka (advisor) ; Sitter, Barbora (referee)
The thesis deals with corporate social responsibility. The subject of investigation is the CSR strategy of the chosen company. The theoretical part describes the corporate social responsibility from a historical and a current perspective. Above all, the thesis puts emphasis on the social pillar, namely the activities of the company in relation to its stakeholders, particularly employees and volunteering. Volunteering is an important part of corporate social responsibility. The questionnaire and subsequently its statistical evaluation answers the question whether the students currently studying in two economic universities consider SAP as a responsible company and how they relate to volunteerism.
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Polymerase synthesis of new photocaged DNA
Vaníková, Zuzana ; Hocek, Michal
5-[(2-nitrobenzyl)oxymethyl]-2’-deoxyuridine 5’-O-triphosphate (dUNBTP) and 5-hydroxymethyl-2’- deoxyuridine 5’-O-triphosphate (dUHMTP) were incorporated to diverse DNA sequences by polymerase synthesis (PEX or PCR). UHM modified DNA was synthesized also through the photolysis of photocaged, UNB-linked DNA. The presence of bulky NB-modification in the recognition sequence of DNA resulted in blocking of cleavage by all restriction endonucleases (REs), however sequences with small HM-modification (formed after irradiation by UV) were perfectly cleaved by all enzymes. Using of photoremovable protecting group in the DNA sequence presents bioorthogonal chemical masking and it has a potential in gene manipulation and cloning.
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Polymerase synthesis of base-modified DNA: New methods and new applications
Balintová, Jana ; Daďová, Jitka ; Kielkowski, Pavel ; Ménová, Petra ; Vaníková, Zuzana ; Riedl, Jan ; Raindlová, Veronika ; Fojta, Miroslav ; Hocek, Michal
Diverse base-modified oligonucleotides and double-stranded DNA molecules were prepared by polymerase incorporation of modified nucleoside triphosphates. The methods include primer extension, PCR, nicking enzyme amplification reaction and mixed incorporations. The modified nucleic acids were used in redox and fluorescence labelling and coding, as well as regulation of binding and cross-linking with proteins.
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