|
|
Novel fluorescent nucleotides for metabolic labelling and for the construction of DNA probes
Kuba, Miroslav
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...
|
|
|
Novel fluorescent nucleotides for metabolic labelling and for the construction of DNA probes
Kuba, Miroslav ; Hocek, Michal (advisor) ; Slanina, Tomáš (referee) ; Míšek, Jiří (referee)
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...
|
|
| |
|
| |
|
|
Selected QEEG variables in early prediction of response to antidepressant treatment
Vlček, Přemysl ; Brunovský, Martin (advisor) ; Kuba, Miroslav (referee) ; Praško Pavlov, Ján (referee)
Electroencephalography is a sensitive method that is able to register neurophysiological changes (operations) in the cerebral cortex, which can be interpreted on the basis of the outputs of various complex computational transformations and in accordance with current neuroscience knowledge. The dynamic course of electrocortical activity is conditioned by functional and structural processes, which can have their origin both in the cortex itself and in various subcortical areas that are associated with it. The development of mathematical methods for the analysis of biological signals (respectively the development of the theory and practice of signal processing in general) has enabled a significant increase in EEG information yield, which is now far from limited to visual inspection of EEG recordings (although it still remains very important for the clinic). Current computer-controlled analysis of EEG signals from individual electrodes allows to obtain a valid reflection of neuronal activity derived from the values of various variables of quantitative electroencephalography (QEEG; spectral power, asymmetry, cordance, frequency peak, current density distribution, etc.), which can specifically correlate with psychopathological processes. Important QEEG methods in electrophysiology include those that are...
|
|
|
Functional imaging of cortical evoked potentials following painful and nonpainful stimulation in healthy volunteers and chronic pain patients
Poláček, Hubert ; Stančák, Andrej (advisor) ; Šonka, Karel (referee) ; Kuba, Miroslav (referee)
A close relationship between painful and non-painful (somatosensory) percepction was noticed already in the past centuries and led into development of many analgesic methods. Only basic neuroanatomical and neurophysiological research using animal models of nociception was able to shed more light on their proper mechanisms until the era of modern non- invasive imaging methods. The main aim of this thesis was to analyze non-invasively, in human volunteers and patients with chronic pain, spatiotemporal relations between brain evoked responses to painful (or aversive) and non-painful stimuli. In next step, to discuss the roles of different engaged mechanisms in found interactions and suggest recommendations for further research of pain. 4 experimental studies (3 in healthy volunteers and 1 in patients with failed back surgery syndrome) are presented. Using high-resolution EEG, phasic electrical stimulation of median, tibial or sural nerve(s), and source analysis of recorded data, modulations of all repersentative components of somatosensory evoked potentials (SEPs) by several interfering conditions were analyzed. In healthy volunteers, effects of heterotopic repetitive heat pain administered to the right side of the body (ipsilateral to electrically stimulated nerve) were tested in Experiment 1 (compared to the...
|
|
|
Functional imaging of cortical evoked potentials following painful and nonpainful stimulation in healthy volunteers and chronic pain patients
Poláček, Hubert ; Stančák, Andrej (advisor) ; Šonka, Karel (referee) ; Kuba, Miroslav (referee)
A close relationship between painful and non-painful (somatosensory) percepction was noticed already in the past centuries and led into development of many analgesic methods. Only basic neuroanatomical and neurophysiological research using animal models of nociception was able to shed more light on their proper mechanisms until the era of modern non- invasive imaging methods. The main aim of this thesis was to analyze non-invasively, in human volunteers and patients with chronic pain, spatiotemporal relations between brain evoked responses to painful (or aversive) and non-painful stimuli. In next step, to discuss the roles of different engaged mechanisms in found interactions and suggest recommendations for further research of pain. 4 experimental studies (3 in healthy volunteers and 1 in patients with failed back surgery syndrome) are presented. Using high-resolution EEG, phasic electrical stimulation of median, tibial or sural nerve(s), and source analysis of recorded data, modulations of all repersentative components of somatosensory evoked potentials (SEPs) by several interfering conditions were analyzed. In healthy volunteers, effects of heterotopic repetitive heat pain administered to the right side of the body (ipsilateral to electrically stimulated nerve) were tested in Experiment 1 (compared to the...
|
|
| |
|
| |
|
| |
guest ::
RSS feed.