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Directed evolution of enzymes on microfluidic chip
Kohúteková, Táňa ; Chmelíková, Larisa (oponent) ; Prokop,, Zbyněk (vedoucí práce)
This thesis focuses on the directed evolution of enzymes using Fluorescence–Activated Droplet Sorting (FADS) on a microfluidic chip. A new FADS system was constructed, validated for high-throughput optical detection of microfluidic droplets, and coupled with real-time dielectrophoretic sorting. The system was benchmarked using two different populations (DhaA31, ancHLD-RLuc) of Haloalkane dehalogenases (HLD) expressed in Escherichia coli cells and one population of empty pIDR9 vector in a ratio of 5:45:50, mimicking the protein library. The collected and analyzed sorting data show that the droplet intensities were recorded in the same proportion as the theoretical ratio. The library of HLD mutants with insertions and deletions was then sorted using the system to obtain 1 % of the variants with the highest activity and the results are reported. The FADS data analyzing system was programmed in LabVIEW bundled with Python, with functions for initial analysis, prediction of threshold voltage, validation and graphical interpretation of data, and final reporting of processes.
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The Process Of Ct Image Classifier Optimization For Pedatric Spine Segmentation
Kohúteková, Táňa
Computed tomography (CT) is a method commonly used when generating images ofa spine diagnosed with scoliosis. The main reason for this common employment is found in itspracticality: the spine in the image is clearly visible. However, while portraying, a child is exposedto a dose of radiation. To eliminate a risk of children being negatively affected, this article is tryingto find the best technique of segmentation, that could be applied also while using magneticresonance imaging (MRI). It implements a 3D Slicer equipped with Support Vector Machines(SVM) Classifier plug-in to search for the best feature combination. Afterwards, segmentationresults are compared to ground truth image, where the area of hard tissue is determined. The aim isto get Dice coefficients as close as possible to 1.
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