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Production of human milk oligosaccharides in the cell factory of E. coli
Havrdová, Jana ; Bojarová, Pavla (advisor) ; Smrček, Stanislav (referee)
Human milk oligosaccharides (HMOs) are among the most abundant components of human breast milk and are essential for the development of neonatal health. It is very challenging to isolate these oligosaccharides from animal milk, especially the less abundant ones. Therefore, different approaches are to be sought after. Chemical and enzymatic syntheses of these compounds are labor-intensive, and expensive affording low yields. A newly adopted approach to HMO synthesis is through bac- terial cell factories, in which genetically engineered bacterial strains can use cheap carbohydrate substrates and convert them into specific oligosaccharides. The aim of this thesis is to examine the feasibility of using selected bacterial strain (E. coli) for the production of HMOs. With lactose as a glycan substrate, the bacterial host has to be β-galactosidase deficient, otherwise, the substrate would be degraded. In order to generate higher lactose intake in the cell, a crp gene that encodes for the positive transcriptional regulator (Catabolite Activator Protein - CAP) can be incorporated into the host organism. The cloned plasmid - pRSFDuet- 1-crp was used for the transformation of into the selected bacterial strain. Lactose from the cells was purified by gel chromatography and the influence of CAP over expression...
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Phytoremediation potential of higher plants - phytoextraction of paroxetine
Štelbacká, Kristína ; Smrček, Stanislav (advisor) ; Hýsková, Veronika (referee)
Environmental pollution has long been an important issue for humanity. There are many mechanical and chemical pollutants in water and soil that need to be removed. Xenobiotics, which are increasingly found in the environment, are also a problem. This thesis deals with the study of the phytoextraction of the drug paroxetine using the plant maize (Zea mays). The antidepressant paroxetine belongs to the group of selective serotonin reuptake inhibitors and is used for the treatment of depressive disorders. The plants were cultured in sterile medium for the time necessary for sufficient growth. Subsequently, paroxetine was added to the plant medium and samples were collected for 96 hours. For the first experiment the concentration of drug in the medium was 3 mg ∙ l−1 , for the second 2 mg ∙ l−1 and for the third 1 mg ∙ l−1 . To determine the phytoextraction activity, the collected samples were detected by HPLC system with UV detector. The phytoextraction efficiency of paroxetine was determined to be 0.0023 mg/g(fresh weight) for the first experiment and 0.0014 mg/g(fresh weight) for the second experiment. Plants used for phytoremediation have increased potential for oxidative stress and reactive oxygen species formation. After the phytoextraction experiments, the antioxidant capacities of the leaf and...
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Comparison of enantioseparation of selected drugs on commercially available and laboratory-prepared CSP based on amylose.
Sidoryk, Edvard ; Bosáková, Zuzana (advisor) ; Smrček, Stanislav (referee)
This bachelor's thesis compares the performance of two commercially available chiral columns based on derivatized amylose (amylose-tris((3,5-dimethylphenylcarbamate), bound to a silica gel as a chiral selector's linker) with two chiral columns with the same chiral selector, prepared in the laboratory at the Department of Organic Chemistry of the FoS CU, but using electrostatic binding to silica gel. The aim of the work was to evaluate the enantioselectivity and the efficacy in the separation of racemic mixtures of selected chiral drugs and verify the stability and reproducibility of the prepared columns. The results showed that commercially available chiral columns proved acceptable enantioselectivity and enantioseparation in most analyses. However, columns prepared in the laboratory - despite the use of diverse mobile phases - did not show sufficient separation capabilities in any of the analytes tested. Nevertheless, their stability and reproducibility were demonstrated, and the principal usability of electrostatic binding of modifier to silica gel was validated.
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Quantification of the stabilizing effects of cyclodextrins on volatile biocides using spectral methods
Konovalova, Olga ; Jindřich, Jindřich (advisor) ; Smrček, Stanislav (referee)
This bachelor's thesis deals with monitoring the stability of chlorine dioxide of native and methylated cyclodextrins in the presence of hydrogenating agents. It is believed that using cyclodextrins and carboxymethylcellulose can improve the stability of chlorine dioxide and slow its release from the solution. UV spectroscopy was used to monitor any changes in absorbance over time and to determine the concentration of chlorine dioxide in the solution. When the rate of chlorine dioxide loss from solution was monitored, it was found that the presence of native and permethylated cyclodextrins slowed the loss of chlorine dioxide from solution, with the slowest decomposition observed in the presence of permethylated cyclodextrin. The addition of carboxymethylcellulose to the chlorine dioxide solution slows the rate of chlorine dioxide loss from the open vial the most, by almost three times, compared to ClO2 itself. This study's results can help optimize chlorine dioxide product formulation for various applications and contribute to the development of more stable and effective chlorine dioxide- based products that can be used to control or eliminate harmful microorganisms in different environments. Key words: chlorine dioxide, CDS, cyclodextrins, carboxymethylcellulose, biocides, UV-Vis...
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Small Molecules as Immune Modulators in Anticancer Therapy
Pavlovová, Anna ; Míšek, Jiří (advisor) ; Smrček, Stanislav (referee)
The aim of this bachelor thesis was to summarize knowledge about several selected therapeutic targets used for cancer immunotherapy and small molecules that can have an immunomodulatory effect on these targets. This is a relatively new and attractive topic in the field of biomedical sciences, which is constantly evolving. Some small molecules have already been approved for treatment of specific cancer diseases, and many more are currently undergoing various stages of clinical trials. This work should provide the reader with an overview of possible approaches to modulate the immune system using small molecules. Key words: small molecules, immunotherapy, cancer, checkpoint inhibitors, tumor microenvironment
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Effect of selected perfumes on aromatase activity
Drejslarová, Iva ; Hodek, Petr (advisor) ; Smrček, Stanislav (referee)
Cytochromes P450 are enzymes involved in many physiological processes in the body, besides the metabolism of xenobiotics also in the biosynthesis and catabolism of endogenous substances such as hormones and steroids. Cytochromes P450 that are responsible for the transformation of endogenous compounds can be affected by xenobiotics, which interfere with the secretion, metabolism, transport or elimination of endogenous hormones by various mechanisms. These substances are also referred to as endocrine disruptors and include, for example, pesticides, insecticides, certain pharmaceuticals, or compounds contained in cosmetic products. One of their mechanisms of action is to interfere with steroid hormone biosynthesis through interactions with steroidogenic enzymes. One such enzyme is cytochrome P450 19, aromatase, which catalyses the conversion of testosterone to ß-estradiol in the final step of steroid hormone biosynthesis. Modulation of the activity of this enzyme by endocrine disruptors results in an imbalance of estrogen levels in the body, which can lead to impaired reproduction, osteoporosis, atherosclerosis, dementia, and the development of certain types of cancer. In this context, the effect of commercially available perfumes on the conversion of testosterone to ß-estradiol, which is catalyzed by...
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Uptake and translocation of buspirone in higher plant cultures
Schmidová, Anna ; Smrček, Stanislav (advisor) ; Petrová, Šárka (referee)
This bachelor thesis is devoted to the phytoextraction of the anxiolytic buspirone from a cultivation medium and to the study of the effect of plant stress on the plant metabolism. Two experiments were carried out with maize (Zea mays) grown hydroponically in a sterile medium for 10 and 14 days. After the addition of a solution of medium containing buspirone at concentrations of 10 and 5 mg · L-1 , samples of medium were collected at 1-day intervals from all experimental plants. The decreasing concentration of buspirone in the medium was monitored by reversed phase HPLC/UV at a wavelength of 240 nm. In the first experiment with 10 mg · L-1 as the initial concentration of buspirone in the medium, a 65 % decrease was observed over the 96 hours during which the phytoextraction was carried out. The overall decrease in buspirone concentration was 53 % in 144 hours of phytoextraction in the second experiment with an initial concentration of 5 mg · L-1 in the medium. After the completion of the phytoextraction experiments, the activities of total peroxidases, membrane-bound peroxidases, guaiacol peroxidase and ABTS-peroxidase were determined in the leaves and roots of the plants. Furthermore, the content of phenolic substances, which belong to the group of so-called secondary metabolites and their effects...
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Effect of triazole fungicides on soil properties
Račko, Ján ; Ryšlavá, Helena (advisor) ; Smrček, Stanislav (referee)
Plant protection still depends on the use of pesticides, often in mixtures that pose a potential threat to non-target organisms. Triazole fungicides (tebuconazole, penconazole and others) are widely used in agriculture against fungal pathogens, but they can affect both the metabolism of the plants themselves and the composition of soil organisms. Biochemical indicators of soil health and quality include the content of microorganisms and the activity of glycosidase, protease and dehydrogenase enzymes. In this work, it was investigated how the application of penconazole and tebuconazole and their combination by watering the soil and spraying on the leaves of edible tomato plants (Solanum lycopersicum L., cv. Cherrola) affects the activities of enzymes involved in carbon and phosphorus metabolism. Endoglycosidases involved in the degradation of plant and fungal cell walls (cellulases, chitinases) were found to have reduced activities in the soil contaminated with triazoles, while the activity of endo-β-1,3-glycanase was increased in the sPT group. The activity of exoglycosidases (β-glucosidases, β-hexosaminidases, β-galactosidases and α-mannosidases) in the soil was increased. The activity of these enzymes was also determined in the roots of tomato plants, from which enzymes can be secreted into the soil....
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Phytoextraction of mixed drug samples from aqueous solutions
Hájková, Eliška ; Smrček, Stanislav (advisor) ; Soudek, Petr (referee)
A number of contaminants have been detected in the environment, including pharmaceuticals. Their presence in soil, water sources can have a toxic effect on organisms due to their constantly increasing concentration. The method of phytoremediation uses the ability of plants to absorb these contaminants and detoxify them by various mechanisms. The aim of this work was the phytoextraction of ibuprofen using maize (Zea mays) from aqueous solutions. Phytoextraction of ibuprofen after cultivation with added dextromethorphan was also carried out. The quantitative amount of extracted ibuprofen was detected by HPLC with UV detection. Ibuprofen has been very well extracted by plants. In the presence of dextromethorphan, the efficiency of phytoextraction was decreased. The phytoextraction efficiency of ibuprofen was 0.10 mg per gram of leaf fresh weight, while in the presence of dextromethorphan, the phytoextraction efficiency was 0.09 mg per gram of leaf fresh weight. After phytoextraction of ibuprofen, the antioxidant capacity was determined by FRAP method of leaf and root extracts of the plants, of which the highest values were observed in the roots of plant grown with ibuprofen in combination with dextromethorphan. The phenolic compounds were also detected in leaves and roots, where the highest percentage...
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