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Synthesis of quantum dots for proteins detection
Šibíková, Anna ; Fohlerová, Zdenka (referee) ; Pekárková, Jana (advisor)
This thesis is focused on synthesis of quantum dots (QDs) for protein detection. It comprises three parts. The first part summaries the theory of QDs, their synthesis, functionalization, interactions and applications in medicine. In the second part synthesis of CdTe/ZnS core/shell QDs modified by glutathione (GSH) is described, followed by the conjugation with biomolecules BSA and IgG. Several coupling agents such as EDC with NHS and CDI were used. In the last part, the final products were characterized by fluorescence spectroscopy and capillary electrophoresis. The results show the dependence of the fluorescence intensity of the QDs on pH range, concentration of BSA and IgG concentrations using different crosslinkers.
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Effect of elution solution on the enrichment and purification of glycopeptides using SPE-HILIC
Chobotová, Michaela ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
Changes in protein glycosylation are related to various diseases such as cancer or Alzheimer's disease. Unfortunately, the analysis of glycoproteins remains a difficult task due to the low proportion of glycopeptides compared to peptides obtained after glycoprotein cleavage. As a result, it is almost impossible to analyze glycopeptides without specific enrichment steps. A suitable method for the enrichment of glycopeptides is solid phase extraction (SPE) using the principle of hydrophilic interaction liquid chromatography (HILIC). Since the choice of solvent significantly affects the enrichment of glycopeptides, the aim of this thesis was to compare the effect of different solvents on the efficiency of glycopeptide extraction using SPE-HILIC. The extraction was carried out on a silica column modified with aminopropyl groups. Model analytes were glycopeptides obtained by tryptic digestion of human IgG. During the comparsion of acetonitrile, methanol and isopropanol, acetonitrile was chosen as a suitable elution solvent. During the conditioning and washing step, the content of acetonitrile was changed (65%, 75% and 85%), whereby the elution of glycopeptides is accelerated as the amount of acetonitrile decreases. In the elution step, the effectiveness of different acetonitrile contents (5%, 10% and...
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Study of Cellular Toxicity of Representative Nanoparticles in Tissue Cultures.
Filipová, Marcela ; Holada, Karel (advisor) ; Benson, Veronika (referee) ; Hubálek Kalbáčová, Marie (referee)
Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...
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The role of B cells in transplantation reactions
Brožová, Jitka ; Slavčev, Antonij (advisor) ; Stříž, Ilja (referee)
Kidney transplantation is the best treatment for patients with end-stage renal failure. The main problem of kidney transplantation is however the development of a cellular and antibody-mediated (humoral) rejection. During the last decade, thanks to the advanced immunosuppression, prognosis of survival and function of transplanted organs has significantly improved. Nevertheless, humoral rejection remains very serious obstacle in high-risk patients, because it can permanently damage the graft. Therefore, before transplantation it is necessary to stratify patients into high and low risk groups for development of antibody-mediated rejection. Current immunogenetic tests performed before transplantation include, in addition to HLA typing, detection of panel-reactive antibodies. However, this test does not provide information about B cells which participate in the humoral response of the kidney recipient. Therefore, in the presented thesis we studied B cell reactivity and its regulation in transplanted patients. In this retrospective analysis we measured levels of the B cell activating factor, a cytokine regulating the function of B lymphocytes (BAFF). Current reports suggest that BAFF could serve as a marker of humoral rejection. Furthermore, we focused on B lymphocytes and their capacity to produce...
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Synthesis of quantum dots for proteins detection
Šibíková, Anna ; Fohlerová, Zdenka (referee) ; Pekárková, Jana (advisor)
This thesis is focused on synthesis of quantum dots (QDs) for protein detection. It comprises three parts. The first part summaries the theory of QDs, their synthesis, functionalization, interactions and applications in medicine. In the second part synthesis of CdTe/ZnS core/shell QDs modified by glutathione (GSH) is described, followed by the conjugation with biomolecules BSA and IgG. Several coupling agents such as EDC with NHS and CDI were used. In the last part, the final products were characterized by fluorescence spectroscopy and capillary electrophoresis. The results show the dependence of the fluorescence intensity of the QDs on pH range, concentration of BSA and IgG concentrations using different crosslinkers.
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