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Flow injection analysis of selected glycosaminoglycans with spectrofluorimetric detection
Tichá, Renata ; Nesměrák, Karel (advisor) ; Rychlovský, Petr (referee)
The thesis is focused on a determination of heparin and chondroitin sulfate, using flow injection analysis with spectrofluorimetric detection. The determination is based on the interaction of negatively charged heparin, chondroitin sulfate resp., with a cationic dye (azure B or phenosafranine) which is manifested by the decrease in fluorescence intensity of the dye in its emission maximum. The optimal conditions for the determination in static mode were found, and calibration dependencies were measured. The conditions of FIA were optimized and following parameters were established: the volume of dispensed sample of 100 ml, the length of the reaction coil 60 cm, the flow rate 0.7 ml min-1 , the concentration of azure B 1.6×10-5 mol dm-3 , the concentration of phenosafranine 3.5×10-5 mol dm-3 . For the determination of heparin using azure B it was found: LOD = 0.023 IU ml-1 , LOQ = 0.186 IU ml-1 , and linear dynamic range 0.19-1.43 IU ml-1 . For the determination of heparin using phenosafranine it was found: LOD = 0.102 IU ml-1 , LOQ = 0.192 IU ml-1 , and linear dynamic range 0.19-1.79 IU ml-1 . For the determination of chondroitin sulfate using azure B it was found: LOD = 0.58 mg dm-3 , LOQ = 2.37 mg dm-3 , and linear dynamic range 2.37-8.32 mg dm-3 . The developed determination was applied to the...
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Preparation and characterization of cell models of lysosomal hereditary diseases - Mucopolysaccharidoses
Presová, Gabriela ; Dobrovolný, Robert (advisor) ; Dvořáková, Lenka (referee)
Mucopolysaccharidoses are a group of diseases that belong to lysosomal storage disorders. A common sign of these monogenic multisystem diseases is a gene mutation leading to a deficiency of the lysosomal enzyme participating in glycosaminoglycan degradation. It results to their accumulation in the tissues and organs, where they cause a progressive damage. There is no efficient treatment available for most mucopolysaccharidoses. Moreover, the research is complicated because of the low prevalence and type of affected tissues. Animal models of these human diseases are used for an evaluation of newly developed therapeutic approaches. However, they also have many limitations due to the different pathogenesis and catabolic pathways of the accumulated substrates between humans and animals. Therefore, animal models are replaced by human cell models. In this thesis, the development of four mucopolysaccharidoses human cell models is reported (MPS IIID, MPS IVA, MPS IVB, MPS VI). Corresponding genes (GNS, GALNS, GLB1, ARSB) were inactivated using CRISPR/Cas9 technology, where plasmids containing specific inserts are delivered to the target human induced pluripotent stem cells (iPSC), using electroporation. Isolated clones, which represent iPSC disease models, were characterized by Sanger sequencing, enzyme...
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Flow injection analysis of selected glycosaminoglycans with spectrofluorimetric detection
Tichá, Renata ; Nesměrák, Karel (advisor) ; Rychlovský, Petr (referee)
The thesis is focused on a determination of heparin and chondroitin sulfate, using flow injection analysis with spectrofluorimetric detection. The determination is based on the interaction of negatively charged heparin, chondroitin sulfate resp., with a cationic dye (azure B or phenosafranine) which is manifested by the decrease in fluorescence intensity of the dye in its emission maximum. The optimal conditions for the determination in static mode were found, and calibration dependencies were measured. The conditions of FIA were optimized and following parameters were established: the volume of dispensed sample of 100 ml, the length of the reaction coil 60 cm, the flow rate 0.7 ml min-1 , the concentration of azure B 1.6×10-5 mol dm-3 , the concentration of phenosafranine 3.5×10-5 mol dm-3 . For the determination of heparin using azure B it was found: LOD = 0.023 IU ml-1 , LOQ = 0.186 IU ml-1 , and linear dynamic range 0.19-1.43 IU ml-1 . For the determination of heparin using phenosafranine it was found: LOD = 0.102 IU ml-1 , LOQ = 0.192 IU ml-1 , and linear dynamic range 0.19-1.79 IU ml-1 . For the determination of chondroitin sulfate using azure B it was found: LOD = 0.58 mg dm-3 , LOQ = 2.37 mg dm-3 , and linear dynamic range 2.37-8.32 mg dm-3 . The developed determination was applied to the...
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