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Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
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Program-controlled freeze drying for the preparation of the delivery system of native hyaluronan and hydrophobic molecules
Waczulíková, Kristína ; Sedláček, Petr (referee) ; Enev, Vojtěch (advisor)
The amphiphilic nature of the hyaluronan biopolymer facilitates interactions between its hydrophobic portions of the chain and the hydrophobic ligands, making it one of the promising compounds that can be used as a suitable carrier for drug delivery. Therefore, the aim of this diploma work was by means of a program-controlled freeze-drying to prepare a system based on native hyaluronan, which would be capable of transporting hydrophobic drugs. Perylene, pyrene, prodan and 1,6-diphenyl-1,3,5-hexatriene fluorescence probes were used as model compounds to simulate hydrophobic drug-hyaluronan interactions. Freeze-drying efficiency was studied using infrared spectrometry and scanning electron microscopy. ATR-FTIR spectrometry confirmed for all probes but pyrene the hydrophobic interactions between the hyaluronan and probe molecules based on the presence of the C=C bond valence vibration absorption band in the aromatic probe rings. From the IR spectra, it was not possible to confirm with certainty the presence of residual tert-butyl alcohol in the samples. SEM photographs revealed that increasing the pressure during drying process had a positive effect on the quality of the lyophilized sample in the second series of samples. Interactions of individual fluorescent probes with hyaluronan were studied using steady-state and time-resolved fluorescence spectrometry. Positive results have been obtained, indicating the presence of fluorescent probes interacting with the hydrophobic regions of the hyaluronan chains. The results of this work can contribute to the development of carrier systems in the field of targeted drug distribution.
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Aggregation Behaviour of Polysaccharides in Aqueous Solutions
Mravec, Filip ; Valko, Marián (referee) ; Hrdlovič, Pavol (referee) ; Valko, Marián (referee) ; Pekař, Miloslav (advisor)
Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.
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Effect of freeze-drying conditions on the hydrophobic interaction between native hyaluronan and fluorescent probe
Říha, Karel ; Venerová, Tereza (referee) ; Enev, Vojtěch (advisor)
The biopolymer native hyaluronan possesses the amphiphilic character of its molecule. Which means that it contains both a hydrophilic and a hydrophobic part. This character of the molecule favors interactions between hydrophobic ligands and hydrophobic parts of the chain, making hyaluronan one of the compounds potentially suitable as drug carrier systems. The aim of this bachelor thesis was to prepare a carrier system based on hyaluronan and model hydrophobic compounds (fluorescent probes). The fluorescent probe we used was pyrene. Furthermore, the work was focused on the study of lyophilization conditions, which would lead to the actual influence of hydrophobic interactions between the substances we used. The main techniques that served us to verify the effectiveness of lyophilization (in the sense of supporting the creation of a hydrophobic effect between individual hyaluronan chains) were: thermogravimetric analysis, fluorescence spectrometry, and last but not least, infrared spectroscopy with Fourier transformation.
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Program-controlled freeze drying for the preparation of the delivery system of native hyaluronan and hydrophobic molecules
Waczulíková, Kristína ; Sedláček, Petr (referee) ; Enev, Vojtěch (advisor)
The amphiphilic nature of the hyaluronan biopolymer facilitates interactions between its hydrophobic portions of the chain and the hydrophobic ligands, making it one of the promising compounds that can be used as a suitable carrier for drug delivery. Therefore, the aim of this diploma work was by means of a program-controlled freeze-drying to prepare a system based on native hyaluronan, which would be capable of transporting hydrophobic drugs. Perylene, pyrene, prodan and 1,6-diphenyl-1,3,5-hexatriene fluorescence probes were used as model compounds to simulate hydrophobic drug-hyaluronan interactions. Freeze-drying efficiency was studied using infrared spectrometry and scanning electron microscopy. ATR-FTIR spectrometry confirmed for all probes but pyrene the hydrophobic interactions between the hyaluronan and probe molecules based on the presence of the C=C bond valence vibration absorption band in the aromatic probe rings. From the IR spectra, it was not possible to confirm with certainty the presence of residual tert-butyl alcohol in the samples. SEM photographs revealed that increasing the pressure during drying process had a positive effect on the quality of the lyophilized sample in the second series of samples. Interactions of individual fluorescent probes with hyaluronan were studied using steady-state and time-resolved fluorescence spectrometry. Positive results have been obtained, indicating the presence of fluorescent probes interacting with the hydrophobic regions of the hyaluronan chains. The results of this work can contribute to the development of carrier systems in the field of targeted drug distribution.
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Characterization of newly developed fluorescence probes in cellular systems
Kadlecová, Julie ; Hubálek Kalbáčová, Marie (advisor) ; Hendrych, Tomáš (referee)
Nanoparticles (NP) are currently a progressive area of scientific research. The possibility of synthesizing them according to the required parameters opens up possibilities for their wide use also in biomedicine. One example is a nanoparticle that can detect cellular processes, such as pH. We already know that the pH of healthy and cancer cells differs by the opposite gradient on the intracellular and extracellular side of the membrane. In this context, this work deals with the study of fluorescent silicon nanoparticles (SiNP) tested on a human keratinocyte cell line from a healthy donor (HaCaT) and from skin cancer donor (A431). Once found that even the highest concentrations of SiNP used are not cytotoxic, they can be further studied by fluorescence, confocal and super-resolution microscopy. In order to assess the pH detection properties of these SiNPs, a method for measuring intracellular pH with a fluorescent raciometric probe SNARF-1 using fluorescence spectroscopy and flow cytometry was introduced. Since the pH values of the intracellular environment are closely related to cellular metabolism, the metabolism of A431 and HaCaT cells was characterized and compared. To do this, methods for measuring analog glucose consumption (2-NBDG) and another new method for measuring real-time metabolism...
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Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
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Aggregation Behaviour of Polysaccharides in Aqueous Solutions
Mravec, Filip ; Valko, Marián (referee) ; Hrdlovič, Pavol (referee) ; Valko, Marián (referee) ; Pekař, Miloslav (advisor)
Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.
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Photoluminiscent diamond nanoparticles - new diagnostic and visualization probes
Cígler, Petr
Selected problems connected with surface modification of nanosized diamond crystals, attachment of chemical sensing architectures to them and their colloidal stability were addressed. Fluorescent nanodiamonds modified by proper functional structures were shown as unique unbleachable nanoprobes for construction of future sensors useful in biological and medicinal applications.
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