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Cell transplantation methods in cardiology
Kukhta, Dziyana ; Hežová,, Reneta (referee) ; Skopalík, Josef (advisor)
Tato diplomová práce se zabývá tkáňovým inženýrstvím, zejména tvorbou homogenní, izotropní a planární vrstvy buněk srdečního svalu pomocí dvou technologii:”scaffold-based” a ”scaffold-free”. Nejprve popsaný histologie srdeční stěny, buňky srdečního svalu a buněčné kultury. Následuje popis tkáňového inženýrství, který zahrnuje technologii “cell sheet” a tkáňové inženýrství na bázi scaffoldů. Na konci teoretické části je popsána aplikace tkáňového inženýrství a buněčná vizualizace. Praktická část věnovaná tvorbě planární buněčné vrstvy z kardiomyocytů a fibroblastů s využitím informací z teoretické části.
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Mathematic Modelling of Reverse Osmosis System Design for Detection of Estrogens in Water
Siegel, Jan ; Skopalík, Josef (referee) ; Bittner, Michal (advisor)
Estrogen compounds are a subgroup of the environmental pollutants named Endocrine Disrupting Chemicals. It is a large group of chemicals which are capable of causing hormonal imbalance of an organism and affecting its reproductive, developmental or behavioral functions. In the environment, they are found in very low concentrations (below ng/l). This makes their detection and elimination highly problematic. The Research Centre for Toxic Compounds in the Environment (RECETOX) has been developing an experimental device which employes reverse osmosis (RO) to concentrate the trace amounts of micropollutants (e.g. estrogens). The aim is to lower the detection limit of subsequent analytical methods. RO seems to be a promising alternative to commonly used methods which are more time consuming and costly. The aim of this thesis is to create a mathematical model of RO, which would describe the process of estrogen concentration and therefore could contribute to the optimization of experimental device. The mathematical model was created in MATLAB-simulink and verified by comparison to thirteen experimental results. The solutions used to test the model were NaCl at 0.002–2 g/l and drinking water with 17-ethinylestradiol of 25 ng/l. In order to concentrate the retentate on a flat-sheet RO membrane the model achieves a mean relative error in the range of 0.9–4.9 % for NaCl and drinking water. For the concentration of estrogens the deflection is 3.6 %. Comparing the model and experimental concentration of NaCl on a spiral wound RO-membrane, the deviation is in the range of 5.9–8.7 %. So far, ratio of retentate volume from the beginning to the end of the process has been used to determine the process recovery. However, due to the results obtained in the thesis, this approach appears inaccurate. A more accurate recovery would likely be achieved by using the theoretical concentration value obtained by the proposed mathematical model. However, to increase reliability, the further measurements with estrogens are needed, and also to determine the causes of deflection of model and experimental retentate volume values at the end of the concentration
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Study of mesenchymal stem cell migration based on principles of chemotaxis
Pošustová, Veronika ; Skopalík, Josef (referee) ; Chmelíková, Larisa (advisor)
The purpose of this Master thesis is to verify migration of mesenchymal stem cells on the principle known as chemotaxis. First part of this study is focused on cell migration in order to explain the whole migration process. Next part describes various chemotaxis methods and selected studies dealing with clinical applications of mesenchymal stem cells in different medical and biomedical fields. The following step describes confocal microscopy, which is used for acquiring images of the cells. The experimental part is focused on cultivation of mesenchymal stem cells in a laboratory, which is necessary for cell vitality. Furthermore, there are designed two main experiments. Firstly there is a 2D experiment with adherent cells for chemotaxis using -Slide Chemotaxis. Secondly Transwell migration test is designed and executed. Finally, the acquired images from confocal microscope are used for image processing, which was done in Matlab R2020a programming environment. The result of this processing is evaluation of cell confluence and migration. In the end, experimental part of this study was optimized according to recommended studies. The results are summarized in the conclusion with proposal for improvements of those methods.
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The cell migration monitoring in a microfluidic system by the "Scratch Wound Healing Assay" method
Morgaenko, Katsiarina ; Skopalík, Josef (referee) ; Chmelíková, Larisa (advisor)
Tato diplomová práce se zabývá popisem principů kultivace embryonálních fibroblastových buněk myší (3T3), lidských endoteliálních buněk odebraných z pupečníkové žily (HUVEC) a epiteliálních buněk vaječníku čínského křečka (CHO) v mikrofluidních systémech simulujících kapiláry. Byly provedeny literární rešerše v oblasti realizací experimentu “Scratch Wound Healing Assay” v mikrofluidních systémech s použitím fibroblastů a endotheliálních buněk. V práci jsou dále popsány principy konfokální a fluorescenční mikroskopie a metody zpracování obrazů pro sledování buněčné migrace. Experimentální nastavení pro mikrofluidní realizaci “Scratch Wound Healing Assay” s použitím trypsinu – EDTA pro vytvoření rýhy, a konfokálního mikroskopu Leica TCS SP8 X pro následující snímání pořízených dat bylo navrženo a otestováno s dostatečným počtem opakování. Vhodný algoritmus pro analýzu buněčné migrace byl napsán v programovacím prostředí Matlab. Závěrem této práce je diskuze získaných výsledků.
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Classification of marked cells migration in tissue
Solař, Jan ; Skopalík, Josef (referee) ; Čmiel, Vratislav (advisor)
This diploma thesis deals with analysing of modern methods for cell detection, visualization and quantification in 3D space. The first section deals with optical methods for cells detection. There is detailed discussion about cell labeling and detection on confocal microscopy. There is also description about developed algorithm for whole cell volume quantification from microscopy images. This could made a comparsion of fluorescence signal according to time of cell labeling and according to cell shapes. There was also optimalization of handmade tissue phantoms visualization. It could be compared a possibilities of cell detections in these phantoms by confocal microscopy and OCT. It was also implemented algorithm for quantification of cells from OCT images. Besides confocal microscopy and OCT cells are also analyzed by other methods. The last part is the Conclusion of results and comparison of used methods.
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Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
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Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
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Development of parallel visualisation of subdermal regeneration by tomographic and hystological methods
Ruszová, Kristína ; Macíček, Ondřej (referee) ; Skopalík, Josef (advisor)
The aim of the thesis is to review suitable methods for quantifying the regeneration of subcutaneous layers and collagen structures in tissue defect, especially MRI methods and histological techniques. The theoretical chapters contains basic overview of skin anatomy and healing process, brief overview of histological methods and description of MRI as well. The practical part contains a description of data acquisition and optimization of methods for obtaining these MRI images with the best possible visualization of the condition of the skin layers. It also describes the designed software for comparing the acquired MRI images and the classical histological image from the tissue post-mortem. Finally, the evaluation of suitable modifications of gels and coatings that would affect the contrast characteristic of these planar medication layers is summarized.
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