National Repository of Grey Literature 38 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Modified nucleotides and DNA for electrochemical labelling and defined display of small molecules
Krömer, Matouš ; Hocek, Michal (advisor) ; Křen, Vladimír (referee) ; Vrábel, Milan (referee)
This thesis is focused on enzymatic construction of DNA probes for electrochemical labelling, bioconjugations and, in the final part, building on knowledge gathered in previous chapters, it describes a method useful for construction of highly functionalized base-modified DNA enabling defined multivalent display of glycosides. In first chapter, a chemical route to diol-bearing nucleotides was found. Sonogashira reaction facilitated access to alkyne-tethered diols and subsequent catalytic hydrogenation, described for the first time in the literature, provided protection-free method for obtaining nucleotide diols tethered via flexible sp3 hybridized linker. Cleavage of alkane-linked, but not alkyne-linked, nucleotide diols yielded aliphatic nucleotide aldehyde. All nucleotides were found to be good substrates for KOD XL DNA polymerase in both primer extension and polymerase chain reaction, apart from aldehyde-linked dUCHO TP nucleotide, which performed well in PEX reaction, but gave PCR products only in a mixture with natural dTTP. This could be overcome by cleavage of diol-modified DNA, which also yielded aldehyde-functionalized dsDNA. All reactive probes were examined for bioconjugations with fluorescent hydrazine, reductive amination with lysine or lysine-containing peptides or other molecules...
Multivalent carbohydrate ligands of galectins
Hovorková, Michaela ; Křen, Vladimír (advisor) ; Kavan, Daniel (referee)
Galectins are proteins, wich belong to a group of lectins that are able to bind to saccharide units and they specifically recognize glycans exposed to the surface of the cells. Galectins participate in vivo, for example, in carcinogenesis, angiogenesis or fibrosis. Their occurrence increases significantly in connection with a number of pathogenic processes, therefore they can be used as markers for some types of cancer or cardiopathology and also for the targeted binding of therapeutics and/ or imaging agents in diagnosis and therapy. Galectin-3 has a specific structure known as chimeric and it is capable of forming multivalent oligomers. The natural ligands of galectins are glycans containing terminal β-galactosides, especially N-acetyllactosamine, but the binding of monovalent glycans is very weak. Glycoconjugates with high affinity to galectin receptors are optimally multivalent, biocompatible and stable in vivo. These criteria accomplish carbohydrate ligands conjugated to soluble and structurally flexible N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In this work two types of functionalized disaccharides based on N-acetyllactosamine (Galβ4GlcNAc) and its structural analogue of N,N'-diacetyllactosamine (GalNAcβ4GlcNAc) was prepared by enzymatic synthesis. For the synthesis were used...
Mammalian Serine Racemase as a Pharmaceutical Terget
Vaníčková, Jana ; Konvalinka, Jan (advisor) ; Kotora, Martin (referee) ; Křen, Vladimír (referee)
PH.D. DISSERTATION Mammalian Serine Racemase as a Pharmaceutical Target Jana Jirásková Supervisor: Jan Konvalinka Department of Biochemistry Faculty of Science Charles University Institute of Organic Chemistry and Biochemistry Gilead Sciences & IOCB Research Centre Academy of Sciences of the Czech Republic Praha 2010 Introduction Serine racemase is a pyridoxal-5'-phosphate (PLP) -dependent enzyme that is responsible for D- serine production. D-serine is a neurotransmitter that acts, together with L-glutamate, as agonist of ionotropic N-methyl-D-aspartate (NMDA) receptors, which are important for neuronal tissue signalization. Recent serine racemase knock-out mouse studies revealed that SR produces approximately 90% of brain D-serine. SR was first isolated from a pool of rat brains about a decade ago. Its orthologs are present in mammals as well as plants and yeast. Mammalian SRs share high sequence identity, about 90%. Mouse and human SRs are similar enzymatically, suggesting that mouse SR and mouse model are suitable to shed light on human SR. SR forms homodimers in solution and has a molecular weight of approximately 37 kDa per monomer. In addition to pyridoxal-5'-phosphate, the enzyme requires divalent cations such as Ca2+ or Mg2+ , nucleotides such as ATP, and reducing agents for full activity. The...
New Microbial Glycosidases
Charvátová, Andrea ; Křen, Vladimír (advisor) ; Králová, Blanka (referee) ; Walterová, Daniela (referee)
Sunrvranv Glycosidases from fungi are useful in the preparation of various glycosides mainly by transglycosylation or reversed glycosylation. The lack ofany requirement for protection- deprotection sequences, mild conditions and easier synthesis of thermodynamically not preferred glycosidic bonds are the main advantages of glycosidase-catalysed synthesis of glycosides. In ttris thesis we concentratedon exoglycosidases,mainly B.N.aceýlhexosaminidases, cr-galactosidases and cr-L-rhamnosidases. For the enzyme preparation taxonomically characterised fungal strains from public collections were used. A library comprising more than 200 various glycosidases was developed by modification ofcultivation conditions and by the use ofspecific inducers. The enrymes were used for screening ofsubstrate specificity and stability in organic solvents and subsequently for synthesis and modification of various substrates. Both B-N-acetylgalactosaminidase and B-N-acetylglucosaminidase activities in the series of B-N-acetylhexosaminidases were determined. Saccharides with strong immunomodulation activity $-o-GalpNAc-(i-+4)-o-GlcpNAc and B-o-GalpNAc-(1-+6)-o-GlcpNAc were synthesisedby transglycosylation using B-N-acetylhexosaminidase from Penicillium oxalicum CCF 2430' enryme having the highest B.N-acetylgalactosaminidase activiý....
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...
Multivalent carbohydrate ligands of galectins
Hovorková, Michaela ; Křen, Vladimír (advisor) ; Kavan, Daniel (referee)
Galectins are proteins, wich belong to a group of lectins that are able to bind to saccharide units and they specifically recognize glycans exposed to the surface of the cells. Galectins participate in vivo, for example, in carcinogenesis, angiogenesis or fibrosis. Their occurrence increases significantly in connection with a number of pathogenic processes, therefore they can be used as markers for some types of cancer or cardiopathology and also for the targeted binding of therapeutics and/ or imaging agents in diagnosis and therapy. Galectin-3 has a specific structure known as chimeric and it is capable of forming multivalent oligomers. The natural ligands of galectins are glycans containing terminal β-galactosides, especially N-acetyllactosamine, but the binding of monovalent glycans is very weak. Glycoconjugates with high affinity to galectin receptors are optimally multivalent, biocompatible and stable in vivo. These criteria accomplish carbohydrate ligands conjugated to soluble and structurally flexible N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In this work two types of functionalized disaccharides based on N-acetyllactosamine (Galβ4GlcNAc) and its structural analogue of N,N'-diacetyllactosamine (GalNAcβ4GlcNAc) was prepared by enzymatic synthesis. For the synthesis were used...
Differences in Oxidation Mechanism of Selected Bioflavonoids, UV-Vis and IR Spectroelectrochemical Study
Sokolová, Romana ; Fiedler, Jan ; Ramešová, Šárka ; Kocábová, Jana ; Degano, I. ; Quinto, A. ; Křen, Vladimír
The oxidation of selected bioflavonoids (quercetin, rhamnazin, fisetin, rhamnetin, taxifolin, luteolin, silybin and 2,3-dehydrosilybin) was studied by electrochemical and spectroscopic methods. The oxidation mechanism and formation of oxidation products depends on the chemical structure. Only small differences in the chemical structure of compounds with flavone, flavanone and flavonol motif result in extremely variable oxidation pathways and products. Products of oxidation and further decomposition were determined by HPLC-ESIMS/MS and HPLC-DAD.

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