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Detection of Pt derivatives using ICP mass spectrometry
Zelinová, Karin ; Hložková, Michaela (referee) ; Vašinová Galiová, Michaela (advisor)
This Bachelor´s thesis deals with the monitoring of chemotherapeutic penetration into tumor cells. Due to the toxicity of drugs, targeting them is absolutely essential from the point of view of minimizing the interference with healthy tissue of the patient. In order to achieve the best possible targeting, it is necessary to monitor the penetration of chemotherapeutics into cells. The subject of study was platinum-based drugs therefore the ICP-MS method was chosen to analyse the drug content in cells, because it is suitable for fast and reliable detection of trace amounts of elements. The theoretical part of the Bachelor´s thesis focuses on the description of ICP-MS, as a method, which was chosen for the detection of platinum derivates. It also summarizes the use of platinum-based drugs in cancer therapy. The practical part of the thesis deals with the analysis of cells exposed to platinum-based chemotherapeutics. Detection and quantification of platinum in the cells were determined by both SN-ICP-MS and LA-ICP-MS. To verify the results, the analysis of the solution was also performed by the AAS method. The results show, that the drug was most readily taken up by A2780 cells. It was also shown that cisplatin was the most accumulated drug.
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The study of metallocene anticancer therapeutics-cells interaction
Bilavčíková, Kristýna ; Mravcová, Ludmila (referee) ; Vašinová Galiová, Michaela (advisor)
There were almost 600 thousand patients in Czech Republic until 2017 and the number keeps rising. Chemotherapeutics used today have lots of undesirable side effects, so scientists around the world are trying hard to find more specific and less cytotoxic drugs. In this bachelor’s thesis, potential drugs based on metallocene with a central atom of Ru, Hf or Zr were studied, always in three different drugs for each element. The permeability and distribution of these drugs into lung cancer cell lines were investigated. These drugs were detected by analysis of solutions and laser ablation with an ICP mass spectrometer. Based on the experiment, the greatest potential was found for the ruthenium-based drug 133, which had the highest permeability and was able to get into the cells in the highest volume.
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Utilisation of fluorescence techniques for analysis of industrially relevant microorganisms
Müllerová, Lucie ; Kráčmar, Stanislav (referee) ; Krčma, František (referee) ; Obruča, Stanislav (advisor)
Techniky analýzy jednotlivých buněk neboli single-cell analýzy, jsou velmi důležité v kontextu studia důležitých biotechnologických mikroorganismů. V biotechnologických procesech je optimální mít rychlé, efektivní a real-time analýzy, které lze provést s minimální přípravou vzorku. Proto jsme v rámci předložené disertační práce využili autofluorescenci jako marker fyziologického stavu mikrobiální kultury, který je možné analyzovat pomocí fluorescenční mikroskopie a/nebo průtokové cytometrie. Nejdříve bylo potvrzeno, na základně emisních spekter, že zelená autofluorescence emitovaná bakteriemi Cupriavidus necator H16 a jejím polyhydroxyalkanoáty (PHA) neprodukujícím mutantem Cupriavidus necator PHB-4, pochází skutečně z flavinů. Emisní maximum bylo naměřeno v rozmezí 510–550 nm pro FAD, FMN a riboflavin – bohužel jednotlivá spektra od sebe nebylo možné rozeznat, a 515 nm pro oba bakteriální kmeny Cupriavidus necator. Byly nalezeny dvě maxima excitačních spekter, jedno v rozmezí 360–370 nm a druhé okolo 440 nm. Z těchto poznatků byl sestaven nový protokol pro fluorescenční mikroskopii – laser byl nastaven na hodnotu 467 nm a emisní filtr pro detekci byl vybrán 520/35 nm. Flaviny a jejich autofluorescence může být take použita v kombinaci s jinými fluorofory, když jsou použity multiparametrické analýzy, ale je důrazně doporučeno, aby vybrané fluorescenční sondy buď emitovaly v jiném rozmezí, než je 500–550 nm anebo byla jejich doba života excitovaného stavu jiná, než je interval 3,2–3,7 a 4,2 – 4,9 ns. Tyto doby života navíc zůstaly konstantní, i když byly buňky vystaveny kultivačním a stresovým podmínkám. To z nich dělá velmi stabilní marker v porovnání s dynamickým charakterem intenzit fluorescence. Dále byl optimalizován protokol pro značení bakterie C. necator s využitím PHA specifické sondy BODIPY 493/503. Optimální koncentrace této lipofilní sondy byla stanovena na 2,5 µg ml-1 a sondu je možné dle našich dat aplikovat společně s viabilitní sondou propidium iodidem. Tento protokol byl také využit pro studium morfologie PHA-syntetizující halofilní bakterie Halomonas halophila. Pro stanovení UV-protektivních vlastností PHA obsaženého v bakterii Cupriavidus necator, byl optimalizován další protokol, tentokrát pro ROS citlivou sondu CM-H2DCFDA, která byla stanovená jako citlivější v porovnání s její nemethylovanou formou. V této studii byly navíc potvrzeny UV-protektivní vlastnosti PHA na bakterii Cupriavidus necator H16 v porovnání s PHA nesyntetizujícím kmenem Cupriavidus necator PHB-4.
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Detection of Pt derivatives using ICP mass spectrometry
Zelinová, Karin ; Hložková, Michaela (referee) ; Vašinová Galiová, Michaela (advisor)
This Bachelor´s thesis deals with the monitoring of chemotherapeutic penetration into tumor cells. Due to the toxicity of drugs, targeting them is absolutely essential from the point of view of minimizing the interference with healthy tissue of the patient. In order to achieve the best possible targeting, it is necessary to monitor the penetration of chemotherapeutics into cells. The subject of study was platinum-based drugs therefore the ICP-MS method was chosen to analyse the drug content in cells, because it is suitable for fast and reliable detection of trace amounts of elements. The theoretical part of the Bachelor´s thesis focuses on the description of ICP-MS, as a method, which was chosen for the detection of platinum derivates. It also summarizes the use of platinum-based drugs in cancer therapy. The practical part of the thesis deals with the analysis of cells exposed to platinum-based chemotherapeutics. Detection and quantification of platinum in the cells were determined by both SN-ICP-MS and LA-ICP-MS. To verify the results, the analysis of the solution was also performed by the AAS method. The results show, that the drug was most readily taken up by A2780 cells. It was also shown that cisplatin was the most accumulated drug.
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The study of metallocene anticancer therapeutics-cells interaction
Bilavčíková, Kristýna ; Mravcová, Ludmila (referee) ; Vašinová Galiová, Michaela (advisor)
There were almost 600 thousand patients in Czech Republic until 2017 and the number keeps rising. Chemotherapeutics used today have lots of undesirable side effects, so scientists around the world are trying hard to find more specific and less cytotoxic drugs. In this bachelor’s thesis, potential drugs based on metallocene with a central atom of Ru, Hf or Zr were studied, always in three different drugs for each element. The permeability and distribution of these drugs into lung cancer cell lines were investigated. These drugs were detected by analysis of solutions and laser ablation with an ICP mass spectrometer. Based on the experiment, the greatest potential was found for the ruthenium-based drug 133, which had the highest permeability and was able to get into the cells in the highest volume.
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Sample preparation for single cell analysis
Adamová, Eva ; Basova, E. Y. ; Potáčová, Anna ; Foret, František ; Matalová, Eva ; Klepárník, Karel
Development of sensitive, miniaturized and fast methods aims to reliable analyze even small amounts of samples. Micromanipulation systems such as laser capture microdissection enable to collect just single cells from cell suspensions or tissue samples. One option for further analysis is the bioluminescence reaction based on luciferin/luciferase conversion. This approach was used for detection of active caspases, caspase-3/7. Droplets based microfluidic systems provide benefits in biological, pharmaceutical and chemical research.
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