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Production of polyesters by extremophilic bacteria
Bednárová, Kristína ; Mravec, Filip (oponent) ; Obruča, Stanislav (vedoucí práce)
This bachelor thesis deals with the study of the production of polyhydroxyalkanoates using thermophilic bacterial strains Schlegelella thermodepolymerans, Schlegelella aquatica and Thermomonas hydrothermalis. The aim of the thesis is to optimize the conditions of polymer production. In bacterial strains of the genus Schlegelella, phaC gene at the genotype level was confirmed. The ability to produce polyhydroxyalkanoates in the bacterial strain Thermomonas hydrothermalis at the phenotype level was demonstrated. The utilization of selected carbon sources was studied experimentaly. The production of PHA was optimized by selecting the appropriate substrate and cultivation temperature. The effect of precursors on the production of copolymers P(3HB-co-4HB) and P(3HB-co-3HV) was observed. The most promising carbon substrate for bacterial strain Schlegelella aquatica was glycerol while the best metabolic activity was at 45 °C. Strain Thermomonas hydrothermalis was able to produce the copolymer P(3HB-co-4HB) with extreme high molar fraction of 4HB. Optimal cultivation temperature was 55 °C. The highest yield of P(3HB) among selected bacteria, synthesized Schlegelella thermodepolymerans cultivated on xylose at 55 °C. For this strain, the effect of aeration on biomass growth and PHA production was observed. In an effort to reduce production costs, Schlegelella thermodepolymerans was cultivated on model hydrolysates of lignocellulose-rich materials. Finally, batch cultivation in a larger production volume in laboratory fermentor was performed.
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Use of Hsp90 as a target of biological therapy of tumors
Bednárová, Kristína ; Bouchalová,, Pavla (oponent) ; Müller, Petr (vedoucí práce)
Antibody-drug conjugates (ADCs) represent a relatively new class of highly potent anti-tumor drugs. Thanks to highly specific monoclonal antibodies, ADCs are able to deliver a cytotoxic payload directly to tumor cells and thus minimize damage to healthy cells. Therapeutic efficacy depends on the selection of an appropriate antigen that undergoes internalization upon conjugate binding. For this project, pro-oncogenic Hsp90 and c-Met were selected as potential targets. Hsp90 is a molecular chaperone that is overexpressed in tumor cells and, in addition, can be translocated to the membrane of these cells. Overexpressed Hsp90 contributes to angiogenesis, tumor cell motility or metastasis. C-Met is a receptor tyrosine kinase that plays a central role in epithelial morphogenesis and malignant transformation. Its increased activity induces pathways responsible for the proliferation, invasion and migration of malignant cells. The aim of the diploma thesis was to study the potential use of antibodies with anti-Hsp90 and anti-c-Met activities in anti-tumor therapy. The experimental part involved the purification of the EEV1-2.1 antibody with anti-Hsp90 activity and its subsequent characterization. Furthermore, it included the characterization and selection of anti-c-Met antibody clones. It was also focused on selection and optimization of the right conjugation strategy. The activity of the antibodies and their conjugates was examined by fluorescence microscopy and flow cytometry. In vivo experiments were further aimed at verifying the efficacy of ADC by monitoring the rate of inhibition of proliferation of selected tumor cell lines. The results revealed that the EEV1 antibody does not enter the cells specifically by antigen-mediated way, and is therefore not suitable for use in conjugation with a cytostatic drug. On the other hand, anti-c-Met antibody ADC conjugates exhibit high affinity for native antigen, internalization through antigen binding, and additionally inhibited proliferation of c-Met overexpressing OE33 cells.
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Use of Hsp90 as a target of biological therapy of tumors
Bednárová, Kristína ; Bouchalová,, Pavla (oponent) ; Müller, Petr (vedoucí práce)
Antibody-drug conjugates (ADCs) represent a relatively new class of highly potent anti-tumor drugs. Thanks to highly specific monoclonal antibodies, ADCs are able to deliver a cytotoxic payload directly to tumor cells and thus minimize damage to healthy cells. Therapeutic efficacy depends on the selection of an appropriate antigen that undergoes internalization upon conjugate binding. For this project, pro-oncogenic Hsp90 and c-Met were selected as potential targets. Hsp90 is a molecular chaperone that is overexpressed in tumor cells and, in addition, can be translocated to the membrane of these cells. Overexpressed Hsp90 contributes to angiogenesis, tumor cell motility or metastasis. C-Met is a receptor tyrosine kinase that plays a central role in epithelial morphogenesis and malignant transformation. Its increased activity induces pathways responsible for the proliferation, invasion and migration of malignant cells. The aim of the diploma thesis was to study the potential use of antibodies with anti-Hsp90 and anti-c-Met activities in anti-tumor therapy. The experimental part involved the purification of the EEV1-2.1 antibody with anti-Hsp90 activity and its subsequent characterization. Furthermore, it included the characterization and selection of anti-c-Met antibody clones. It was also focused on selection and optimization of the right conjugation strategy. The activity of the antibodies and their conjugates was examined by fluorescence microscopy and flow cytometry. In vivo experiments were further aimed at verifying the efficacy of ADC by monitoring the rate of inhibition of proliferation of selected tumor cell lines. The results revealed that the EEV1 antibody does not enter the cells specifically by antigen-mediated way, and is therefore not suitable for use in conjugation with a cytostatic drug. On the other hand, anti-c-Met antibody ADC conjugates exhibit high affinity for native antigen, internalization through antigen binding, and additionally inhibited proliferation of c-Met overexpressing OE33 cells.
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Production of polyesters by extremophilic bacteria
Bednárová, Kristína ; Mravec, Filip (oponent) ; Obruča, Stanislav (vedoucí práce)
This bachelor thesis deals with the study of the production of polyhydroxyalkanoates using thermophilic bacterial strains Schlegelella thermodepolymerans, Schlegelella aquatica and Thermomonas hydrothermalis. The aim of the thesis is to optimize the conditions of polymer production. In bacterial strains of the genus Schlegelella, phaC gene at the genotype level was confirmed. The ability to produce polyhydroxyalkanoates in the bacterial strain Thermomonas hydrothermalis at the phenotype level was demonstrated. The utilization of selected carbon sources was studied experimentaly. The production of PHA was optimized by selecting the appropriate substrate and cultivation temperature. The effect of precursors on the production of copolymers P(3HB-co-4HB) and P(3HB-co-3HV) was observed. The most promising carbon substrate for bacterial strain Schlegelella aquatica was glycerol while the best metabolic activity was at 45 °C. Strain Thermomonas hydrothermalis was able to produce the copolymer P(3HB-co-4HB) with extreme high molar fraction of 4HB. Optimal cultivation temperature was 55 °C. The highest yield of P(3HB) among selected bacteria, synthesized Schlegelella thermodepolymerans cultivated on xylose at 55 °C. For this strain, the effect of aeration on biomass growth and PHA production was observed. In an effort to reduce production costs, Schlegelella thermodepolymerans was cultivated on model hydrolysates of lignocellulose-rich materials. Finally, batch cultivation in a larger production volume in laboratory fermentor was performed.
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