Nové fluorescenční nukleotidy pro metabolické značení a konstrukci DNA sond

The aim of the thesis was to synthesize new nucleosides, nucleotides and the corresponding DNA probes bearing various fluorescent labels, which can be used for bioanalytical applications. In the first part of the thesis, 2'-deoxycytidine and the corresponding nucleoside triphosphate bearing tryptophan-based imidazolinone fluorophore were synthesized by Sonogashira cross-coupling reaction. The fluorophore showed sensitivity to pH and viscosity. Nucleotide was used for the construction of modified oligonucleotides (ON) and DNA by primer extension (PEX) or polymerase chain reaction (PCR). Labelled ON probe was used for sensing interaction with single-strand binding protein, which resulted in increased fluorescence intensity of modified ON. Next, thymidine and thymidine triphosphate labelled by bezylidene- tetrahydroxanthylium fluorophore were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Fluorescence of the fluorophore is dependent on the polarity and viscosity of the environment. Incorporation of the modified nucleotide into DNA, by PEX or PCR, led to dramatic increase of the fluorescence presumably due to the interactions of the fluorophore in the major groove. Unfortunately, the modified nucleotide was not suitable for in cellulo imaging due to its cytotoxicity. The modified...Cílem této dizertační práce byla syntéza nových nukleosidů, nukleotidů a příslušných DNA sond nesoucích různé fluorescenční značky pro bioanalytické aplikace. V první části práce byl pomocí Sonogashiry cross-kapling reakce syntetizován 2'-deoxycytidin a jeho trifosfát nesoucí imidazolinový fluorofor na bázi tryptofanu. Daný fluorofor vykazoval citlivost na pH a viskozitu. Nukleotid byl použit pro konstrukci modifikovaných oligonukleotidů (ON) a DNA prodlužováním primeru (PEX) nebo polymerázovou řetězovou reakcí (PCR). Značená ON sonda byla použita pro snímání interakce se single-strand vazebným proteinem, což vedlo ke zvýšení intenzity fluorescence modifikovaného ON. Dále byl připraven thymidin a thymidin-5'-O-trifosfát, nesoucí benzyliden- tetrahydroxanthyliový fluorofor, pomocí mědí katalyzované azid-alkynové cykloadice (CuAAC). Fluorescence fluoroforu je závislá na polaritě a viskozitě prostředí. Inkorporace modifikovaného nukleotidu do DNA pomocí PEX nebo PCR vedlo k dramatickému zvýšení fluorescence, pravděpodobně díky interakcím fluoroforu ve velkém žlábku. Bohužel, modifikovaný nukleotid nebyl vhodný pro vizualizace v buňkách kvůli své cytotoxicitě. Modifikovaná dsDNA byla použita jako fluorescenční sonda pro snímání interakcí s malými molekulami a proteiny změnou fluorescence. Nakonec byl...Katedra organické chemieDepartment of Organic ChemistryFaculty of SciencePřírodovědecká fakult

Spared cognitive processing of visual oddballs despite delayed visual evoked potentials in patient with partial recovery of vision after 53years of blindness

AbstractWe examined the visual and cognitive functions of a 72-year-old subject, KP, who recovered his sight after 53years of visual deprivation. We used visual evoked potentials (VEPs) to pattern-reversal and motion-onset stimuli and cognitive responses (ERPs) during the oddball paradigm to assess the effect of long-term deprivation on a mature visual system. KP lost his sight at the age of 17years, and light projection onto his right retina was restored at 71years by a corneal implant. Nine months after sight recovery we recorded reproducible responses to all examined stimuli. The response to pattern reversal contained two P100-like peaks with the later peak being dominant and significantly delayed (260ms) when compared to the P100s of two control subjects, to whom the stimuli were adjusted in size and contrast to mimic KP’s vision. KP’s motion-onset VEPs to full-field and peripheral stimuli had a characteristic shape with a well-defined N2 peak; however, both peaks were significantly delayed (262 and 272ms) compared to control responses. Unlike the P100 and N2 peaks, which represent sensory detection, the P3b/P300 component of the ERP to a target event in the oddball paradigm was not further delayed. In spite of degraded vision and sensory deprivation lasting 53years, KP displayed reproducible responses to all reported stimuli. Long-term visual deprivation and retinal detachment degraded KP’s visual sensory processing, assessed by pattern-reversal and motion-onset VEPs, whereas the cognitive processing of appropriate visual stimuli was not compromised

Novel fluorescent nucleotides for metabolic labelling and for the construction of DNA probes

The aim of the thesis was to synthesize new nucleosides, nucleotides and the corresponding DNA probes bearing various fluorescent labels, which can be used for bioanalytical applications. In the first part of the thesis, 2'-deoxycytidine and the corresponding nucleoside triphosphate bearing tryptophan-based imidazolinone fluorophore were synthesized by Sonogashira cross-coupling reaction. The fluorophore showed sensitivity to pH and viscosity. Nucleotide was used for the construction of modified oligonucleotides (ON) and DNA by primer extension (PEX) or polymerase chain reaction (PCR). Labelled ON probe was used for sensing interaction with single-strand binding protein, which resulted in increased fluorescence intensity of modified ON. Next, thymidine and thymidine triphosphate labelled by bezylidene- tetrahydroxanthylium fluorophore were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Fluorescence of the fluorophore is dependent on the polarity and viscosity of the environment. Incorporation of the modified nucleotide into DNA, by PEX or PCR, led to dramatic increase of the fluorescence presumably due to the interactions of the fluorophore in the major groove. Unfortunately, the modified nucleotide was not suitable for in cellulo imaging due to its cytotoxicity. The modified..

Nové fluorescenční nukleotidy pro metabolické značení a konstrukci DNA sond

The aim of the thesis was to synthesize new nucleosides, nucleotides and the corresponding DNA probes bearing various fluorescent labels, which can be used for bioanalytical applications. In the first part of the thesis, 2'-deoxycytidine and the corresponding nucleoside triphosphate bearing tryptophan-based imidazolinone fluorophore were synthesized by Sonogashira cross-coupling reaction. The fluorophore showed sensitivity to pH and viscosity. Nucleotide was used for the construction of modified oligonucleotides (ON) and DNA by primer extension (PEX) or polymerase chain reaction (PCR). Labelled ON probe was used for sensing interaction with single-strand binding protein, which resulted in increased fluorescence intensity of modified ON. Next, thymidine and thymidine triphosphate labelled by bezylidene- tetrahydroxanthylium fluorophore were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Fluorescence of the fluorophore is dependent on the polarity and viscosity of the environment. Incorporation of the modified nucleotide into DNA, by PEX or PCR, led to dramatic increase of the fluorescence presumably due to the interactions of the fluorophore in the major groove. Unfortunately, the modified nucleotide was not suitable for in cellulo imaging due to its cytotoxicity. The modified...Cílem této dizertační práce byla syntéza nových nukleosidů, nukleotidů a příslušných DNA sond nesoucích různé fluorescenční značky pro bioanalytické aplikace. V první části práce byl pomocí Sonogashiry cross-kapling reakce syntetizován 2'-deoxycytidin a jeho trifosfát nesoucí imidazolinový fluorofor na bázi tryptofanu. Daný fluorofor vykazoval citlivost na pH a viskozitu. Nukleotid byl použit pro konstrukci modifikovaných oligonukleotidů (ON) a DNA prodlužováním primeru (PEX) nebo polymerázovou řetězovou reakcí (PCR). Značená ON sonda byla použita pro snímání interakce se single-strand vazebným proteinem, což vedlo ke zvýšení intenzity fluorescence modifikovaného ON. Dále byl připraven thymidin a thymidin-5'-O-trifosfát, nesoucí benzyliden- tetrahydroxanthyliový fluorofor, pomocí mědí katalyzované azid-alkynové cykloadice (CuAAC). Fluorescence fluoroforu je závislá na polaritě a viskozitě prostředí. Inkorporace modifikovaného nukleotidu do DNA pomocí PEX nebo PCR vedlo k dramatickému zvýšení fluorescence, pravděpodobně díky interakcím fluoroforu ve velkém žlábku. Bohužel, modifikovaný nukleotid nebyl vhodný pro vizualizace v buňkách kvůli své cytotoxicitě. Modifikovaná dsDNA byla použita jako fluorescenční sonda pro snímání interakcí s malými molekulami a proteiny změnou fluorescence. Nakonec byl...Department of Organic ChemistryKatedra organické chemiePřírodovědecká fakultaFaculty of Scienc

The kinetics of poly(2,6-dimethylphenyleneoxide) dissolution and swelling

Práce se zabývá studiem botnání a rozpouštění filmů na bázi poly(2,6-dimetylfenylenoxidu). Dominantní role při rozpouštění se přikládá vnitřní difuzi molekul rozpouštědla do studovaného makromolekulárního vzorku a expanzi příslušného polymerního řetězce.A solid thin polymeric film dissolution in a liquid solvent, at the temperature below the Tg, is characteized by a few kinetic parameters depeAnding on the static or dynamic conditions of the experimental arrangement. The dominant role in the dissolution has geen given to the internal diffusion of solvent molecules into the studied macromolecular sample and a proper polymer chains expansion

Grid Infrastructure Monitoring as Reliable

Abstract. A short overview of Grid infrastructure status monitoring is given followed by a discussion of key concepts for advanced status monitoring systems: passive information gathering based on direct application instrumentation, indirect one based on service and middleware instrumentation, multidimensional matrix testing, and on-demand active testing using non-dedicated user identities. We also propose an idea of augmenting information provided traditionally using Grid information services by information from the infrastructure status monitoring which gives verified and thus valid information only. The approach is demonstrated using a Testbed Status Monitoring Tool prototype developed for a GridLab project.