The Diploma Thesis deals with an implementation of the new method for an assessment of a cloning efficiency of the cancer stem cells separated by a high speed cell sorter. The cell-sowing on the microtitration plates was performed by the flow cytometry method in a combination with the high speed cell sorter. In the first part of the Diploma Thesis the new method was introduced and tested on the selected cell lines. The obtained results were compared with the results of the limiting dilution assay within four cell lines. As for the second part of my Diploma Thesis, the method was practically applied to analysis of the cloning capacity of two subpopulations of cE2 cells based on the expressions of characteristic markers of stem and cancer stem cells - CD44 and CD 133. Based on the findings, the new method can be introduced as an approved proceeding for the cloning capacity assessment of cancer stem cells in other workplaces that possess analogical device equipment.

This master's thesis is focused on the determination of 6-thioguanine (6-T) by DC voltammetry (DCV), differential pulse voltammetry (DPV) and flow injection analysis with electrochemical detection (FIA-ED) on a boron doped diamond electrode (BDDFE). The optimum conditions for determination of 6-T were found and under these conditions, concentration dependences were measured and the limits of quantification (LOQ) were calculated for each method. Medium of 60% methanol and phosphate buffer (PB) pH 2,0 was chosen as optimum for DCV and DPV determination of 6-T at BDDFE. For both DCV and DPV, the linear concentration dependences were obtained in concentration ranges of 6-T from 2 µmol·l-1 to 10 µmol·l-1 with LOQ 0,9 µmol·l-1 for DCV and 1,5 µmol·l-1 for DPV. Lower LOQ was achieved in PB pH 2,0, where LOQ was 0,6 µmol·l-1 for both DCV and DPV, but the problem was lower repeatability. In PB pH 2 6-T was determined by DCV in drinking and river water with LOQ 1,3 µmol·l-1 in both drinking and river water. The possibility of solid phase extraction was investigated as a method for preliminary separation of 6-T from urine. For FIA under optimized conditions (polarization potential 1300 mV, flow rate 5 ml·min1 and sample volume 50 µl) the linear concentrations dependences were obtained in concentration ranges...

(EN) The presented dissertation thesis summarizes the new results of electrochemical generation of volatile compounds usable in atomic spectral methods. The main aim of this work is to develop and to characterize new types of electrolytic flow-through cells and to examine their possibilities of determination of arsenic, selenium and antimony by using the electrochemical hydride generation technique coupled with atomic absorption spectrometry with a quartz tube atomizer. Individual electrolytic cells were designed and constructed to comply with two important requirements. The cathode chamber of the electrolytic cell should have a minimal volume and a high efficiency of analyte conversion to the volatile hydride. Constructed electrolytic cells are divided into the construction groups and described in the experimental part. Selenium was chosen as the first analyte. The relevant working parameters (such as type, concentration and flow rate of electrolytes, generation current and carrier gas flow rate) were optimized for each newly constructed electrolytic cell. Under the optimal working parameters, the basic characteristics of selenium determination were found out by using electrochemical hydride generation. The electrolytic cells were compared to each other and with the classical electrolytic cell...

Bladder carcinoma (BCa) is among the most common carcinomas in the Western world. Despite the availability of effective therapies, there is currently an urgent need to develop a stratification method, which would enable the accurate identification of patients responsive to therapy. In the theoretical part of my diploma project I describe the heterogeneity of BCa and the currently applied immunotherapeutic approaches. I specifically focused on the Bacillus Calmette-Guérin (BCG) vaccine instillation. For decades another use of BCG has been a prophylactic vaccination against tuberculosis (TB) infection. BCG serves as a model treatment because it is highly efficient when prescribed to the responsive patient. However, an effective stratification is yet to be developed for BCa and latent tuberculosis infection (LTBI) diagnosis and/or monitoring. In the experimental part of my project, I developed and tested a 10-parameter panel for T cell- specific activation test (TAT) applicable for a stratification of BCa patients as well as for the detection of LTBI. I tested the panel on positive controls using flow cytometry (FCM) method because it allows for detection and measurement of dozens of markers at a single cell level. It is easily applicable to available urine and blood samples obtained from BCa...

The Master´s thesis gives an overview of cord blood, its use, processing and cryopreservation. The role of cord blood as an alternative resource for transplantation is being widely discussed. In the experimental part, 50 grafts of donor cord blood were processed and the effect of cryopreservation and demographic factors of mother and child were evaluated on the cellularity of cord blood. Based on the evaluation, a recommendation about the inclusion of additional tests in routine processing of cord blood were made.

The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...

Polyploidization belong to principal evolutionary mechanisms in plants. Although it is rather rare in the family Apiaceae, there are several interesting polyploid complexes here. The present study deals with Libanotis pyrenaica, species three previously reported ploidy levels, namely diploid (2n = 2x = 22), tetraploid (2n = 4 x = 44) and most likely very rare triploid (2n = 3x = 33). The present study aims at geographical pattern of ploidal diversity in central Europe, genetic variation and selected biological features of this species. Flow cytometru, analysis of two chloroplast DNA markers (trnF-trnL and cp018-cp020), morphometrics and in vitro germination experiments were used. The tetraploid cytotype occurs throughout the studied area; in the Czech Republic it was found in the České středohoří Mts., southern Bohemia and Moravia, in Slovakia it especially in the Carpathian mountains in western and central parts of the country and in the Pieniny Mts. in the northeast. Tetraploid cytotype occurs mainly in central-east Europe; in the Czech Republic it is common in the lowlands along the river of Labe and locally in Moravia; in Slovakia it was found along the river of Váh, in the Slovenské rudohorie Mts. and in the Belanské Tatry Mts. A mixed-ploidy population (2x, 4x) was discovered in southern...

The diploma thesis is focused on the study of the mechanism of plasticization of polyhydroxyalkanoates (PHA) in vivo, focusing mainly on methods of isolation native PHA granules and physical and chemical methods of initiation their phase transitions. The literature search prepared in this work deals with this microbial polyester and further focuses on individual methods of isolating PHA from bacterial cells. The main task of the experimental work was to optimize the isolation process of native PHA granules, as well as to design and test processes that initiate the PHA crystallization in isolated granules, as well as analytical procedures to correctly detect this phase transition. Isolation of PHA granules from Cupriavidus necator was performed by enzymes, centrifugation several times and sonication of the cells. Fourier transform infrared spectroscopy (FTIR) and an enzymatic method in which amorphous PHA is selectively degraded by PHA-depolymerase with turbidimetric detection of its loss in dispersion was used to detect PHA crystallization. Techniques based on fluorescence staining of granules (fluorescence spectroscopy and flow cytometry) were used in a more detailed study of the mechanism of PHA plasticization in granules. The results of the analyzes confirmed, among other things, that the optimized procedure makes it possible to isolate granules in the amorphous state, which remain amorphous even after drying and are comparable to native granules in their basic physicochemical properties.

This Diploma thesis deals with preparation of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium breve enriched with prebiotics meant for application in baby food products. Natural extracts from matcha, moringa, young beat, young barley, chlorella and spirulina were selected as prebiotics. The theoretical part is focused on probiotic bacteria, their biological effects and their effects on the child´s body. The experimental part deals with the cultivation of probiotic bacteria with plant extracts, monitoring their viability and stabilization in an encapsulated form. Mixtures of probiotic cells with prebiotics were encapsulated into alginate particles to increase stability. Some of the alginate particles were processed by freeze drying. Mixtures of probiotic cultures with plant extracts were subjected to model human digestion by the action of model digestive juices in unencapsulated, encapsulated and lyophilized form. Selected extracts of plant materials were characterized in terms of amount of total and reducing sugars, total phenolic substances, individual phenolic substances and antioxidant activity. Further, two baby commercial dietary supplements containing probiotics were selected, which were characterized in terms of cell number and viability. Probiotic products were also subjected to model digestion.

The aim of the diploma thesis is to characterize the adsorption properties of probiotic bacteria. Characterization was performed using of designed and optimized spectrophotometric method of determination of adhesion of probiotic bacteria to mucin as the main viscoelastic component of mucus. In order to provide a more detailed description of the adhesion interactions involved in the adhesion of probiotic bacteria to mucin, the viability of selected probiotic bacterial strains Lactobacillus rhamnosus CCM 1825, Lactobacillus plantarum CCM 7039, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190, Lactobacillus acidophilus CCM 4833, Lactobacillus casei CCM 4798, Bifidobacterium breve CCM 7825, Bacillus coagulans CCM 2658 and a potential probiotic strain of Lactobacillus zeae CCM 7069. Moreover, bacterial adhesion to carbohydrates was also determined for all the bacteria tested. The last proposed and optimized technique was dynamic and electrophoretic light scattering characterizing surface properties such as -potential, average size distribution and isoelectric point. From this method, information was obtained on aggregation of bacterial cells and on electrostatic interactions. The combination of these methods was used as a complex tool to characterize adhesion of the tested bacterial cultures as a very specific, sensitive and key parameter of a successful probiotic microorganism that is influence by the multiple effects.