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Study of isotopically labeled substances in living cells by means of Raman microscopy
Bura, Radek ; Mojzeš, Peter (advisor) ; Pilát, Zdeněk (referee)
Unicellular algae (microalgae) are able to produce a number of substances such as starches, oils, proteins, carotenoids, polyphosphates, or crystalline purines directly from inorganic sources by photosynthesis. Different species of microalgae can be used for the economic production of various biomolecules. Due to their autotrophic nature, microalgae are also unique as they can synthesize complex isotopically labeled biomolecules from simple isotopically labeled inorganic substances. Analysis of the chemical composition of microalgae by means of chemical-analytical methods is relatively complex, time-consuming, and laborious. Confocal Raman microscopy represents one of the optical methods by which the chemical composition of microalgae can be determined in situ, i.e. directly within intact cells. This technique combining confocal optical microscopy with Raman spectroscopy enables fast and non- destructive analysis of the chemical composition of substances in the investigated objects, including the effect of isotopic labeling. The chemical composition of the investigated objects is reflected by their Raman spectra, in the case of Raman mapping of microscopic objects by their chemical maps. In this work, a specific case of isotopic labeling was studied, namely the effect of heavy water (D2O) on the deuteration...
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Photogeneration of singlet oxygen by Verteporfin
Laubrová, Veronika ; Dědic, Roman (advisor) ; Mojzeš, Peter (referee)
Photodynamic therapy is a method that can be used to treat a number of diseases, such as oncological diseases. The basis of this method is the elimination of diseased tissue by singlet oxygen obtained by energy transfer from photosensitizers, including verteporphin. The experimental part of this work examines the absorption and fluorescence spectra of verteporphin for different concentrations. Furthermore, from the kinetics of singlet oxygen luminescence for different concentrations, lifetimes of singlet oxygen, verteporphin triplets and their dependence on the sample concentration were obtained. The quenching constant was determined from the dependence of singlet oxygen lifetimes on verteporphin concentration. From the comparison of the measurements in the sample in equilibrium with air and with pure oxygen, the quenching constant of the verteporphin triplets with dissolved oxygen was determined.
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Conformational transitions and stability of quadruplex nucleic acids
Johanovská, Zuzana ; Mojzeš, Peter (advisor) ; Profant, Václav (referee)
The work deals with guanine quadruplexes, i.e., non-canonical four-stranded structures of nucleic acids rich in repetitive guanine sequences with potential biological and nanotechnological significance. The first part summarizes basic information about the structure and topology of quadruplexes, in the second part, the present knowledge about the quadruplexes in vivo are overviewed. The third part is devoted to experimental methods used in the present study, namely Raman spectroscopy, circular dichroism, and absorption spectroscopy. The next sections are devoted to the acquisition and treatment of your own experimental results. Five sequentially related oligonucleotides differing in the bases in a single-member loop were investigated. The effect of concentration of oligonucleotide and potassium ions K+ on the stability of these quadruplexes was investigated by Raman spectroscopy, and an unusually high thermal stability was observed. Other experiments included measurements of CDs and absorption spectra at lower oligonucleotide concentrations, two different potassium ion concentrations, and a comparison of the stability of individual modifications with the previously published results. Furthermore, the possible interaction with the cationic porphyrin CuTMPyP4, similar to the interaction with the antiparallel...
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Thermodynamics of complex formation by miRNA
Vacková, Anežka ; Štěpánek, Josef (advisor) ; Mojzeš, Peter (referee)
The bachelor thesis concerns microRNA (miRNA), short RNA molecules that mediate post-transcriptional regulation of gene expression by binding to a target mRNA. They usually form extended duplexes consisted of short tracks of Watson-Crick basepairs separated by internal loops, mismatched pairs, or bulges. The thesis represents a study of the effect of structural anomalies, the internal loop and the bulge, on the structural stability miRNA Let7 complex with its target LCS1 sequence at mRNA lin-41 from Caenorhabditis Elegans. The task was solved by recording temperature dependent UV absorption spectra of solutions containing mixtures of the mentioned above RNA sequences and their mutated analogs, analysis of the spectral changes by using a singular decomposition method followed by fitting to a thermodynamic model. The determined thermodynamic characteristics of the complexes and spectral changes caused by a temperature increase or the complex dissociation are the outcome of the work.
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Raman microspectroscopy of living cells and biological tissues
Moudříková, Šárka ; Mojzeš, Peter (advisor) ; Matthäus, Christian (referee) ; Samek, Ota (referee)
Title: Raman microspectroscopy of living cells and biological tissues Author: Šárka Moudříková Department / Institute: Institute of Physics of Charles University Supervisor of the doctoral thesis: doc. RNDr. Peter Mojzeš, CSc., Institute of Physics of Charles University Abstract: Raman microscopy combines Raman spectroscopy with optical confocal microscopy and thus provides information on chemical composition of a sample with a µm3 resolution. In this thesis, Raman microscopy has been used to study microalgae-unicellular photosynthetic organisms that are greatly relevant for the Earth's environment as well as for biotechnological applications. Raman microscopy of photosynthetic organisms struggles with a highly intensive background of the spectra, which is formed by fluorescence of cellular photosynthetic apparatus. In this thesis, we have developed a fast and reliable photobleaching method that suppresses the unwanted background; this method has enabled us to study intracellular distribution of algal biomolecules such as proteins, starch, lipids and polyphosphate. We have investigated an evolution of these structures during a cell cycle of a model microalga Desmodesmus quadricauda. Next, we have developed a method for quantitative analysis of polyphosphate in a cellular culture of a microalga Chlorella...
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Microcrystalline inclusions in microalgae studied via Raman microscopy
Suja, Matyáš ; Mojzeš, Peter (advisor) ; Šloufová, Ivana (referee)
Many freshwater, terrestrial or marine microalgae contain various microcrystalline inclusions that they use in their life cycle. However, the identification of the molecular composition of these inclusions via many physical or chemical methods is often very difficult and susceptible to many measurement errors. Therefore, the chemical composition of these microcrystals in many microalgae has not been determined at all or may be incorrect. One of the high precision methods capable of determining the composition of microcrystalline bodies within microalgae is Raman confocal microscopy. This very promising method of optical vibrational spectroscopy allows rapid and non-destructive molecular analysis of objects. Raman microscopy does not require chemical extraction, modification or other color marking or staining of the sample. Therefore, it can directly measure living cells at various stages of their natural development. The chemical composition of the sample is then characterized by its corresponding Raman vibrational spectrum. The aim of this diploma thesis is to determine the presence of microcrystals in different species of microalgae, study the conditions of their occurrence and identify their chemical composition via Raman microscopy.
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Polyphosphates in microalgae: monitoring of their accumulation and intracellular localization by means of Raman microscopy
Suja, Matyáš ; Mojzeš, Peter (advisor) ; Kočišová, Eva (referee)
Phosphorus is widely used in agriculture, where it promotes the growth of crops and increases the profitability of soil. Together with a growing world population, there is a dramatic increase in consumption of this important element. However, the global phosphate deposits are finite and are expected to be depleted in the next few decades. It is therefore important to find an effective way of recycling, which could be represented by single-celled microscopic algae. Microscopic algae are able to accumulate large quantities of phosphorus from the surrounding environment and to store this phosphorus, among others, in the form of polyphosphates. Polyphosphates are high-energetic biomolecules which are contained in the cells of plant as well as animal kingdom. They play a key role in a vast number of vital processes. Raman microscopy can be applied to study metabolism and distribution of biomolecules at a cellular level without the need of a special preparation of the specimen before the measurement. The subject of this bachelor thesis consists of development of methodologies that are needed for studying the accumulation and intracellular localization of polyphosphates in microalgae by means of Raman microscopy.
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Non-resonant Raman Spectroscopic Study of Guanine Quadruplex Structures
Golan, Martin ; Mojzeš, Peter (advisor) ; Mašek, Vlastimil (referee)
Parts of human telomere sequences containing at least 4 guanine subsequences show the ability to form intrastrand quadruplexes of remarkable conformational diversity. Former studies using conventional Raman spectroscopy have revealed that the sequence G3(TTAG3)3 at milimolar concentrations in phosphate buffer solution doped with Na+ ions (ionic strength 150 mM) adopts antiparallel conformation regardless of the length of standing at room temperature or annealing, whereas K+ ions cause gradual transition to "3+1" or even parallel conformation. On the other hand, measurements carried out upon sequence AG3(TTAG3)3 at similar concentrations using Photonic Crystal Fibre-enhanced Raman Spectroscopy (PCFRS) suggest that in the respective presence of both Na+ and K+ (ionic strength 100 mM), a parallel structure is adopted. The hereby presented work employs conventional Raman spectroscopy and Drop Coating Deposition Raman spectroscopy to examine the sequence AG3(TTAG3)3 at concentrations ranging from units to hundreds of milimoles in strands. It concludes that the structure adopted in the presence of Na+, resp. K+ ions is antiparallel, resp. "3+1", and doesn't change over time despite both long standing and annealing. Two hypotheses about the cause of the differences between the results obtained by PCFRS and...
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Raman Microspectroscopy and Mapping of Single Cells
Gregorová, Šárka ; Mojzeš, Peter (advisor) ; Plášek, Jaromír (referee)
Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.
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Biologically important non-canonical structures of nucleic acids in complexes with cationic porphyrins
Palacký, Jan ; Mojzeš, Peter (advisor) ; Setnička, Vladimír (referee) ; Víglaský, Viktor (referee)
Guanine quadruplexes are a class of unusual nucleic acids conformations based on stacked planar guanine tetrads stabilized via Hoogsteen pairing and cation coordination. They are implicated in numerous cellular processes including replication, recombination or transcription. Guanine quadruplexes are widespread within the human genome but their occurrence is highest in the single stranded guanine-rich regions at telomeres. Telomeric guanine quadruplexes are gaining growing interest due to their ability to inhibit the activity of the telomerase enzyme, which is responsible for the proliferation of tumor cells. Specifically, we investigated the conformational polymorphism of the human core telomeric sequence G3(TTAG3)3 conditioned by the concentration of DNA, metal cations (K+ , Na+ ) and/or annealing. Raman spectroscopy was employed as the primary method for this study because, unlike common spectroscopic methods, it allowed us to monitor the quadruplex structure at very high DNA concentrations mimicking molecular crowding conditions in the cell. We demonstrate that the G3(TTAG3)3 quadruplex switch between the antiparallel and parallel strand alignment as a function of nucleoside and potassium concentration. In addition, we demonstrate that cationic porphyrins can be used as sensitive probes of the quadruplex...
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