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Analysis of motility in leukemia cells using incoherent holographic quantitative phase imaging
Smrčková, Zuzana ; Škarková, Aneta (referee) ; Zicha, Daniel (advisor)
This diploma thesis deals with the issue of motility analysis in leukemia cells. An accurate description of the cell movement and the detection of differences in motility under experimental conditions can be obtained by quantitative analysis of cell motility using time-lapse recording. The first part of this work describes various types of tumor cell migraton. The second part focuses on methods of analysis of cell motility in tissue culture using time-lapse recording, which include image acquisition and processing. Part of this chapter describes a coherence-controlled holographic microscope, which was used in the practical part and for which an insert was designed to ensure the exact and stable position of the individual chambers. The last part is focused on the research of leukemic cell motility, which is concluded by a discussion of the obtained results. The appendix contains a published study included acknowledgement to the author of this diploma thesis for participation in the project.
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Automated bioreactor for the cultivation of living cells
Ukropcová, Iveta ; Tolde, Ondřej (referee) ; Dostál, Zbyněk (advisor)
Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.
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Analysis of Src dynamics in cellular structures
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Rozbeský, Daniel (referee)
Src kinase is a key element in many signaling pathways affecting cellular processes such as differentiation, proliferation, motility, or migration. Deregulation of its activity is associated with the promotion of cancer. Therefore, understanding its cellular function is vital. Src activity directly correlates with its structure; when Src is active, it adopts opened conformation, when inactive, it is in closed conformation stabilized by intramolecular interactions. Detection of the conformation can be used to analyze Src activity. In this thesis, conformation-sensitive FRET-based Src biosensor was improved using mNeonGreen as a new acceptor fluorophore in the existing design and the properties of the new biosensor were compared with the original Src biosensor. The new biosensor is able to detect changes in Src conformation and can be stably expressed in cells. Src activity in focal adhesion was analyzed and higher Src activity in these structures was confirmed. Although the new biosensor did not exhibit significantly better sensitivity to Src conformational changes, it still proved to be a useful tool to study Src activity, and mNeonGreens higher brightness makes it more suitable for microscopic experiments. Key words: Src, FRET, biosensor, live-cell imaging, mNeonGreen
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Analysis of motility in leukemia cells using incoherent holographic quantitative phase imaging
Smrčková, Zuzana ; Škarková, Aneta (referee) ; Zicha, Daniel (advisor)
This diploma thesis deals with the issue of motility analysis in leukemia cells. An accurate description of the cell movement and the detection of differences in motility under experimental conditions can be obtained by quantitative analysis of cell motility using time-lapse recording. The first part of this work describes various types of tumor cell migraton. The second part focuses on methods of analysis of cell motility in tissue culture using time-lapse recording, which include image acquisition and processing. Part of this chapter describes a coherence-controlled holographic microscope, which was used in the practical part and for which an insert was designed to ensure the exact and stable position of the individual chambers. The last part is focused on the research of leukemic cell motility, which is concluded by a discussion of the obtained results. The appendix contains a published study included acknowledgement to the author of this diploma thesis for participation in the project.
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Automated bioreactor for the cultivation of living cells
Ukropcová, Iveta ; Tolde, Ondřej (referee) ; Dostál, Zbyněk (advisor)
Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.
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