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Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (oponent) ; Sedláček, Petr (vedoucí práce)
The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.
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Polyphenols in nutritions and their effect on DNA
Osorio, Juan ; Černayová, Diana (oponent) ; Brázda, Václav (vedoucí práce)
Epidemiological studies have shown the effect of plant-derived food consumption in the prevention of a wide range of diseases. The natural antioxidants present in these foods, among which polyphenols are very relevant, may be responsible for this health-promoting activity. The aim of this bachelor thesis is to submit evidence in the interaction of certain polyphenols with the genetic material through different signaling mechanisms, especially regarding the stabilization of the non-canonical DNA structure G-quadruplex and therefore point out the most selective substance to inhibit biochemical processes. Furthermore, the thesis includes detailed information that might help understand how polyphenol compounds can be attached to the DNA through epigenetic mechanisms and G4 structures, and which factors may affect the efficiency of these. Different experiments, by biological and experimental repetition, were used to corroborate the interaction between the compounds and DNA.
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Metody stanovení obsahu polyhydroxyalkanoátů v bakteriálních buňkách
Černayová, Diana ; Enev, Vojtěch (oponent) ; Sedláček, Petr (vedoucí práce)
Táto bakalárska práca sa zaoberá metódami na stanovenie polyhydroxyalkanoátov (PHA) v bunkách baktérie Cupriavidus necator H16. Medzi použité metódy patrí infračervnená spektroskopia s Fourierovou transformáciou (FTIR), Ramanova spektroskopia, turbidimetria a termické analýzy (termogravimetrická analýza a diferenčná skenovacia kalorimetria). Výsledky z metód boli porovnávané s obsahom PHA stanoveného plynovou chromatografiou. Pri infračervenej spektroskopií s Fourierovou transformáciou a Ramanovej spektroskopií sa zistila pozitívna korelácia pomeru charakteristických pásov k obsahu PHA. S použitím FTIR bolo možné okrem kvantitatívneho stanovenia PHA zistiť aj kvalitu vzorky (kryštalickosť /amorfnosť). Ďalej sa použila turbidimetrická analýza, pri ktorej sa sledovala závislosť rozptylu UV-VIS žiarenia na obsahu PHA v bakteriálnych bunkách. Najefektívnejšou technikou sa ukázala byť termogravimetrická analýza. Závislosť úbytku hmotnosti v oblasti spaľovania PHA, na jeho obsahu (zisteného plynovou chromatografiou) bola stanovená s najvyššou koreláciou k plynovej chromatografií. Metóda DSC sa neukázala byť vhodnou alternatívnou náhradou na stanovenie obsahu PHA, ale umožnila stanoviť či sa polymér nachádza v stave kryštalickom, alebo amorfnom.
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Produkce kopolymerů na bázi polyhydroxyalkanoátů pomocí bakterie Schlegelella thermodepolymerans
Černý, Vojtěch ; Černayová, Diana (oponent) ; Pernicová, Iva (vedoucí práce)
Cílem této práce bylo prostudovat schopnost různých kmenů termofilní bakterie Schlegelella thermodepolymerans inkorporovat do struktury polyhydroxyalkanoátů (PHA) různé monomerní jednotky a tvořit tak kopolymery. Byly testovány prekurzory pro produkci monomerů mcl-PHA, 4-hydroxybutyrátu (4HB) a 3-hydroxyvylerátu (3HV). Teoretická část práce pojednává o charakteristice studovaných polymerů, jejich vlastnostech a využití, a také o termofilních organismech a jejich možnosti uplatnění v průmyslu. V experimentální části práce byly prováděny kultivace s různými prekurzory, u kterých se měnily časy přidání a koncentrace. Během těchto kultivací byly použity čtyři bakteriální kmeny S. thermodepolymerans pocházející z různých veřejných sbírek mikroorganismů. Po sérii prvotních screeningů byl vyhodnocen jako nejlepší prekurzor kyselina levulová přidaná v čase zaočkování produkčního média inokulem. Následně byla provedena kultivační série koncentrační řady, kde bylo zjišťováno, zda úprava koncentrace prekurzoru ovlivní složení kopolymeru. Při zvyšování množství prekurzoru klesalo množství biomasy a PHA, avšak vznikly velmi zajímavé materiály s vysokým obsahem 3HV. Při přidání 8 g/l prekurzoru to bylo až 52 %. Nakonec byla u vzniklých materiálů zjištěna molekulová váha polymeru pomocí gelové permeační chromatografie (SEC) s detektorem statického rozptylu světla ve více úhlech (MALS).
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Polyphenols in nutritions and their effect on DNA
Osorio, Juan ; Černayová, Diana (oponent) ; Brázda, Václav (vedoucí práce)
Epidemiological studies have shown the effect of plant-derived food consumption in the prevention of a wide range of diseases. The natural antioxidants present in these foods, among which polyphenols are very relevant, may be responsible for this health-promoting activity. The aim of this bachelor thesis is to submit evidence in the interaction of certain polyphenols with the genetic material through different signaling mechanisms, especially regarding the stabilization of the non-canonical DNA structure G-quadruplex and therefore point out the most selective substance to inhibit biochemical processes. Furthermore, the thesis includes detailed information that might help understand how polyphenol compounds can be attached to the DNA through epigenetic mechanisms and G4 structures, and which factors may affect the efficiency of these. Different experiments, by biological and experimental repetition, were used to corroborate the interaction between the compounds and DNA.
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Analytical methods for qualitative and quantitative determination of PHA in cyanobacteria
Černayová, Diana ; Samek, Ota (oponent) ; Sedláček, Petr (vedoucí práce)
The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.
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Metody stanovení obsahu polyhydroxyalkanoátů v bakteriálních buňkách
Černayová, Diana ; Enev, Vojtěch (oponent) ; Sedláček, Petr (vedoucí práce)
Táto bakalárska práca sa zaoberá metódami na stanovenie polyhydroxyalkanoátov (PHA) v bunkách baktérie Cupriavidus necator H16. Medzi použité metódy patrí infračervnená spektroskopia s Fourierovou transformáciou (FTIR), Ramanova spektroskopia, turbidimetria a termické analýzy (termogravimetrická analýza a diferenčná skenovacia kalorimetria). Výsledky z metód boli porovnávané s obsahom PHA stanoveného plynovou chromatografiou. Pri infračervenej spektroskopií s Fourierovou transformáciou a Ramanovej spektroskopií sa zistila pozitívna korelácia pomeru charakteristických pásov k obsahu PHA. S použitím FTIR bolo možné okrem kvantitatívneho stanovenia PHA zistiť aj kvalitu vzorky (kryštalickosť /amorfnosť). Ďalej sa použila turbidimetrická analýza, pri ktorej sa sledovala závislosť rozptylu UV-VIS žiarenia na obsahu PHA v bakteriálnych bunkách. Najefektívnejšou technikou sa ukázala byť termogravimetrická analýza. Závislosť úbytku hmotnosti v oblasti spaľovania PHA, na jeho obsahu (zisteného plynovou chromatografiou) bola stanovená s najvyššou koreláciou k plynovej chromatografií. Metóda DSC sa neukázala byť vhodnou alternatívnou náhradou na stanovenie obsahu PHA, ale umožnila stanoviť či sa polymér nachádza v stave kryštalickom, alebo amorfnom.
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