Chitosan-glucan complex is fungal origin copolymer that finds application in medicine and cosmetics. Traditionally mycelium of Aspergillus and Penicillium is considered as industrial chitosan-glucan complex source, though utilization of Micromycetes in biotechnological productions is sometimes undesirable. The aim of the work was to study the possibility of Basidiomycete Schizophyllum commune submerged cultivation for industrial scale chitosan-glucan complex production use. Within the work there was studied effect of cultivation conditions (type and concentration of carbon sources in nutrient medium, ratio of carbon source to nitrogen source, medium initial pH and aeration intensity) on Sch. commune #127 mycelium growth, chitosan-glucan complex formation and exopolysaccharide synthesis. As the result, the method for chitosan-glucan complex production increase and exopolysaccharide synthesis suppression was suggested. Chitosan-glucan complex from Sch. commune #127 submerged mycelium was separated by successive alkali and acid treatments. Effects of alkali concentration and application technique, and type of acid on physical and chemical properties of chitosan-glucan complex were described. Analytical methods for in process control and final product characteristics were suggested.

The bacteria of genus Streptococci are among the most significant producers of hyaluronic acid in industrial scale. One of the typical representatives of that group is Streptococcus equi subsp. zooepidemicus. The production of hyaluronic acid in Streptococcus equi subsp. zooepidemicus is heavily influenced by cultivation conditions and by genetic alterations. The present work describes the deletion of genes covR and covS responsible for transcriptional regulation of stress response. According to Galeas a kol. [35] the deletion of these genes in S. pyogenes led to the hyaluronic acid capsule increase. As the S. pyogenes and S. equi subsp. zooepidemicus share approx. 80 % of genome, it was assumed, that the deletion of genes covR and covS in Streptococcus equi subsp. zooepidemicus genome would lead to the higher hyaluronic acid production. The new strain SEZ covRS was obtained by allelic replacement mutagenesis. The cultivations performed in laboratory-scale fermenters in rich Wheat E1 medium showed approx. 9% higher production over parental strain. Therefore, the covRS regulation system plays the same role in Streptococcus equi subsp. zooepidemicus and indirectly regulates the biosynthesis of hyaluronic acid.

Cell cultures are grown in special incubators ensuring appropriate culture conditions for cell survival. The introduction of the diploma thesis is dedicated to the cultivation of cells in vitro and description of sensors, especially designated for the sensing of gas concentration. The main essence of this work is a project and realization of the monoclimatic incubation chamber for the incubation of cell cultures with the opportunity to observe them under a confocal microscope. Another part of this work is dealing with the project and application of autonomous sensing system and regulation of gas concentration (O2 and CO2) in the chamber. The nal part of thesis is dedicated to the presentation of the project correctness verication of the incubation chamber.

Plants are exposed to many adverse factors during their life cycles. Abiotic stresses are significantly limiting plant growth and development. Abiotic stress response mechanisms involve compatible solute synthesis (e.g. sugars, sugar alcohols and amino acids). The aim of this study was to characterise the responses of plants from Plantago genus to different abiotic stresses (drought, salinity, cold and stress combination). The complete plants grew under in vitro conditions. I compared morphological parameters, selected metabolic parameters (carbohydrates balance and proline accumulation) under optimal conditions and stress exposure. This study was focused on plants from genus Plantago, because they differ from each other in their tolerance to the salinity. Both, the glycophyte and the halophyte species are described within this genus. Plantains produce besides widespread soluble carbohydrates (sucrose, glucose, fructose) also sugar alcohol sorbitol, which has been reported as a significant component of the stress response. In addition, the reaction of plants to different carbon and energy sources was tested. More severe growth inhibition of the glycophyte Plantago lanceolata compared to the halophyte P. maritima was observed under salt treatment. Significant accumulation of sorbitol was observed...

Nanocrystalline diamond (NCD) films possess great mechanical properties (low friction coefficient, high hardness etc.), chemical properties (e.g. low corrosivity or chemical inertness) and good biocompatibility. This makes them perspective materials for protective coatings of medical implants and devices. As bacteria biofilms are often very resistant to antibacterial treatment, materials with anti-bacterial or at least anti-adhesive properties are needed. The interaction of NCD films with bacteria has not been properly examined yet. The aim of this thesis was to introduce and optimize the methods for routine bacterial biofilm cultivation and analysis, use them to investigate the ability of NCD films to inhibit the attachment and biofilm formation of Escherichia coli and correlate it with the NCD surface hydrophobicity. The materials used for the study were hydrogenated NCD (hydrophobic), oxidized NCD (hydrophilic) and uncoated glass. For bacterial biofilm growth, cultivation in six-well plates and continuous cultivation in CDC Bioreactor was used. Several methods were tested for quantitative biofilm detachment and analysis. The putative anti-bacterial properties of NCD material were not confirmed in this work. Higher bacterial attachment to NCD films in comparison to the uncoated glass was...

Plants are exposed to the stress of the environment throughout their entire live. They therefore had to develop sophisticated mechanisms to avoid or tolerate stress. In some plants, repeated exposure to stress increases tolerance, which is known as stress memory. The aim of this study is to assess stress memory in terms of oxidative stress level. Content of malondialdehyde (MDA), reactive oxygen species (ROS) and the electrolyte leakage were chosen as markers of oxidative stress. Fresh weight, plant height and water content were also evaluated. The level of oxidative stress was assessed in several genotypes of Solanum tuberosum subspecies, Andigena and Tuberosum. In vitro culture did not work well for the evaluation of stress memory, ex vitro partially did. The content of MDA in 66B was significantly lower than in cv. Lada (Tuberosum) in root (in vitro) and shoot (ex vitro) in control groups as well as during water shortage. Treatment of acclimation-inducing cold before the stress-inducing cold had a variable effect on growth and electrolyte leakage according to the culture conditions. In vitro, the effect on electrolyte leakage in roots of cv. Lada was positive, in leaves of 66B (Andigena) it was negative. In ex vitro, a positive effect was found on electrolyte leakage in 66B leaves, but at the...

Cultivation of cells in bioreactors with mechanical load simulates the physiological conditions to which cells in the body are exposed. This technology has been used to induce the differentiation of stem cells from adipose tissue towards the phenotype of vascular smooth muscle cells, which can further serve to form vascular replacements. At present, there is no established strategy for cultivating stem cells while being exposed to mechanical stress. The main aim of this work was therefore to optimize the cultivation strategy and determine the ideal load parameters. Differentiation was analyzed by immunofluorescence of specific smooth muscle cell markers, α-actin and h1-calponin, which were quantified by Western blot. Extracellular matrix production was also detected by immunofluorescence staining. The outcome of this work is the establishment of ideal conditions of cell culture in a bioreactor with mechanical load, during which they differentiate into smooth muscle cells. Three types of scaffolds were used for cultivation; plasma treated glass, fibrin-coated glass and decelularized pericardium. Preliminary results show that smooth muscle differentiation was succesfully induced in human and porcine adipose tissue stem cells. Cells were analyzed after 3 and 7 days of culture. Developing a stem cell...

Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...

4 Abstract The main goal of my thesis was to create our own proposals for laboratory work in biology of unicellular organisms (Protista). I designed the laboratory tasks, so that they can be mastered by the pupils of sixth year of primary school. Creating the proposals for laboratory exercises was preceded by the work in the laboratory at Charles University in Prague, Faculty of Education, the Department of Biology and Environmental Studies. In the laboratory, I verified the methods of cultivation of selected representatives of ciliates (Paramecium caudatum), amoebae (Amoeba proteus) and euglenids (Euglena gracilis) and selected methods of dyeing their different organels. Key words: protists, Paramecium caudatum, Amoeba proteus, Euglena gracilis, cultivation, dying

7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....