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Biochemical analyses of the DNA interaction partners
Valchová, Michaela ; Coufal,, Jan (referee) ; Brázda, Václav (advisor)
This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.
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Ordered and disordered arrays of colloidal nanoparticles for biomolecule detection
Ligmajer, Filip ; Rezek, Bohuslav (referee) ; Kolíbal, Miroslav (advisor)
This thesis deals with guided self-assembly of gold nanoparticles from their colloidal solutions onto silicon substrates and possible employment of nanoparticles for detection of biomolecules. It was found that by adjustment of solution pH and surface chemistry modification by means of electron beam irradiation it is possible to facilitate nanoparticle deposition to patterns with almost single particle precision. Spectroscopic ellipsometry was then employed in analysis of self-assembled layers of nanoparticles and its combination with a theory of effective medium approximation has proven the ability to assess nanoparticle dimensions and volume fractions. By experiments with thiolated oligonucleotides it has been shown that using ellipsometry one can detect even with very subtle changes in nanoparticle environment caused by biomolecules, thus promising its possible use in the field of biodetection.
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The development of an HPLC method for monitoring the preparation of oligodeoxynucleotide probes
Šemlej, Tomáš ; Kučera, Radim (advisor) ; Pilařová, Pavla (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Mgr. Tomáš Šemlej Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title of rigorosum thesis: The development of an HPLC method for monitoring the preparation of oligodeoxynucleotide probes An artificially prepared oligodeoxynucleotide sequence with the attached label is referred to as a molecular probe. Molecular probes are mainly used to quantify multiplication in RT-PCR and identify mutations in PCR analyses. There are typically two different types of probes - mono-labeled molecular probes and double-labeled molecular probes. The mono-labeled molecular probes are constructed from fluorophore- labeled oligodeoxynucleotide sequence or quencher-labeled oligodeoxynucleotide sequence. Whereas double-labeled molecular probes consist of an oligodeoxynucleotide sequence with a fluorophore attached at one end together with a quencher attached at the other end of the chain. In this work, we first paid attention to the separation of double- labeled oligodeoxynucleotides from mono-labeled oligodeoxynucleotides. Several types of stationary phases were tested and based on that the column Clarity® Oligo-RP™ was chosen to separate a mixture of labeled oligodeoxynucleotides. This...
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Synthesis, Biological Profiling and Photophysical Properties of Polycyclic Hetero-Fused 7-Deazapurine Nucleosides
Yang, Chao ; Hocek, Michal (advisor) ; Soural, Miroslav (referee) ; Nencka, Radim (referee)
This thesis describes the synthesis, photophysical properties and biological profiling of several series of polycyclic hetero-fused 7-deazapurine nucleosides. Modified 7-deazapurine ribonucleosides display a variety of biological effects. Previously, small (hetero)aromatic rings-fused 7-deazapurine nucleosides show submicromolar cytostatic effects or antiviral activities. Thus, the two isomeric series of new benzothieno-fused deazapurine nucleosides were designed as the extended analogues to the cytotoxic thieno-fused nucleosides and hetero-analogues of antiviral naphtho-fused nucleosides. The goal of the first part of my work was to synthesize these target compounds. Key steps include Negishi coupling of zincated pyrimidine with iodobenzothiophene, thermal or photochemical cyclization, glycosylation and final diversification. The furyl and benzofuryl derivatives exerted moderate anticancer and anti-HCV activities. Most of the free nucleosides showed moderate to strong fluorescence, and the corresponding 2′-deoxyribonucleoside triphosphate was incorporated into modified DNA and their fluorescence properties were studied The tri- and tetracyclic fused nucleobases can be synthesized either by multistep heterocyclization approach or through cross-coupling of zincated pyrimidine with hetaryl halides,...
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Study of biomolecular interactions by the method of surface plasmon resonance
Bocková, Markéta ; Homola, Jiří (advisor)
Title: Study of biomolecular interactions by the method of surface plasmon resonance Author: Markéta Bocková Department: Department of Biochemistry, Faculty of Science Supervisor: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics of the Czech Academy of Sciences Abstract: In this diploma work, a surface plasmon resonance (SPR) biosensor technology was exploited to study two different classes of molecular interactions: hybridization of partially complementary oligonucleotides (ONs) and interactions between enzymes and peptides involved in pathogenesis of Alzheimer disease (AD). In particular, the study of interactions between ONs was focused on determining kinetic parameters of the hybridization proces. It was found that the association equilibrium constant decreases with the increasing surface probe density. An SPR biosensor-based method for rapid and sensitive detection of the 17-HSD10 enzyme and its complex with amyloid β (Aβ) peptides was designed and developed. It was demonstrated that the developed method is capable of detecting 17β-HSD10/Aβs complex in real cerebrospinal fluid (CSF) samples and that CSF samples from AD patients exhibited elavated levels of 17β- HSD10/Aβs complex compared to the control groups. The research reported in this thesis resulted in two publications in...
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Study on the Electrophoretic Behaviour of Short Oligodeoxyribonucleotides in Fused Silica Capillaries. A Preliminary Communication
Vítová, Lada ; Fojta, Miroslav ; Vespalec, Radim
Effects of background electrolyte pH, composition and concentration on the electrophoretic transport of short oligodeoxyribonucleotides (ODNs) in fused silica capillaries have been investigated in the presented study. Mobilities and separation efficiencies of ODNs peaks varied with all three background electrolyte characteristics. Just changes in the background electrolyte characteristics and acid-base properties of ODNs are not sufficient for a systematic explanation of the observed variations. Despite the indicated complexity of ODNs electrophoretic behaviour, we show that very high separation efficiencies of ODNs peaks can be achieved in background electrolytes of physiological pH.
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Electrochemical Reduction and Oxidation of Nucleic Acids Bases and their Analogues: a Brief Overview
Fojta, Miroslav ; Špaček, Jan ; Dudová, Zdenka ; Pivoňková, Hana ; Daňhel, Aleš ; Fojt, Lukáš ; Havran, Luděk
Nucleic acids are known as electroactive biomacromolecules containing electrochemically reducible or oxidizable constituents. Nucleobases cytosine, 5-methylcytosine, adenine and guanine can be reduced in aqueous media on mercury or silver amalgam electrodes. Oxidation of all natural nucleic acids bases (in addition to the above mentioned ones, also uracil and thymine) was demonstrated using various types of carbon electrodes. Some of synthetic nucleobases or nucleotide analogues (e g., 7-deazapurines, cytidine analogues used as epigenetic modulators, etc.) exhibit specific electrochemical properties that differ from those of the parent bases and can be utilized to determine the given substance in the presence of natural nucleic acids or their components.
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Biochemical analyses of the DNA interaction partners
Valchová, Michaela ; Coufal,, Jan (referee) ; Brázda, Václav (advisor)
This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.
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Study of biomolecular interactions by the method of surface plasmon resonance
Bocková, Markéta ; Homola, Jiří (advisor)
Title: Study of biomolecular interactions by the method of surface plasmon resonance Author: Markéta Bocková Department: Department of Biochemistry, Faculty of Science Supervisor: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics of the Czech Academy of Sciences Abstract: In this diploma work, a surface plasmon resonance (SPR) biosensor technology was exploited to study two different classes of molecular interactions: hybridization of partially complementary oligonucleotides (ONs) and interactions between enzymes and peptides involved in pathogenesis of Alzheimer disease (AD). In particular, the study of interactions between ONs was focused on determining kinetic parameters of the hybridization proces. It was found that the association equilibrium constant decreases with the increasing surface probe density. An SPR biosensor-based method for rapid and sensitive detection of the 17-HSD10 enzyme and its complex with amyloid β (Aβ) peptides was designed and developed. It was demonstrated that the developed method is capable of detecting 17β-HSD10/Aβs complex in real cerebrospinal fluid (CSF) samples and that CSF samples from AD patients exhibited elavated levels of 17β- HSD10/Aβs complex compared to the control groups. The research reported in this thesis resulted in two publications in...
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