The neuropathogenic trematode Trichobilharzia regenti (Schistosomatidae) infects the central nervous system of birds and mammals. During its migration through the spinal cord, the parasite causes tissue damage and triggers inflammation which is likely responsible for the elimination of the parasite. In this thesis, the proinflammatory cytokines IL-1β and IL-17 were detected by immunohistochemistry in the affected spinal cord of C57BL/6J mice during the infection. Additionally, IL-4 and IL-6, participating in the regulation of the inflammation and tissue repair, respectively, were noticed. Astrocytes, microglia and other, yet unidentified cells, produced these cytokines. Furthermore, splenic T-lymphocytes were phenotyped by flow cytometry to characterize peripheral immune response. At the peak of nervous tissue inflammation, mixed (Th1/Th2) cellular immune response was observed. Taken together, this thesis extended the knowledge of cytokine immune response of mice infected with T. regenti and also confirmed that inflammation in the nervous tissue influences the polarization of peripheral immune response. Key words: cytokines, spleen, CNS, microglia, astrocytes, Trichobilharzia regenti, immunohistochemistry, flow cytometry

The aim of this study was to induce the triploidy in pikeperch (Sander lucioperca) with use of a cold shock. To induce the triploidy, fertilised egg were (spawning temperature 14,5 °C) submerged in a cold bath at the temperature of 2 °C. Time of initiation was 1; 3; 5; 7 and 10 minutes post activation. The exposure time was 20 and 40 minutes. Ploidy level of freshly hatched larvae was assessed with use of the flow cytometry. In both exposure times, the hatching rate was getting lower with later time of initiation (20 minutes exposure, hatching rate: 58,4-13,4 %; 40 minutes exposure, hatching rate: 28- 9,6 %). Number of malformed larvae increased with later time of initiation and longer exposure time (20 minutes exposure, malformed larvae 0-47,2 %; 40 minutes exposure, malformed larvae 0-58,8 %). None of the tested combination of exposure time and time of initiation led to a population containing 100 % triploid larvae. However, percentage of triploid larvae grew up with longer exposure time and later time of initiation. The best cold shock combination with highest yield of triploids were after 20 minutes long treatment initiated 10 minutes post activation (57,1 +- 14,2 %) and after 40 minutes long treatment initiated 10 minutes post activation (61,9 +- 8,2 %). The most important finding of this study is that cold shock treatment leads to triploidy in pikeperch. To obtain 100% triploid larvae, shorter exposure time and different shock temperature might be applied. It may also eliminate low hatching rate and high appearance of malformed larvae.

In the present study, I dealt with morphological, cytological, and ecological research on a rare Central-European species Spergularia echinosperma and its comparison with a similar weedy species S. rubra. Existence of two cytotypes of S. echinosperma significantly differing in their morphology was revealed, as well as distinct morphological differences between the two species were found. Moreover, the analyses revealed one possibly hybridogenous population. In addition, both the species and the cytotypes were also proven to display different germination behavior, which I correlate with their individual ecological adaptations.

B-cells of peripheral blood in humans represent a heterogeneous cellular environment displaying many important functions in the immune system. Recently, there is an increasing amount of evidence that B cell subpopulations are involved in the pathogenesis of many different diseases. However, there is little or no knowledge on how the individual differentiation stages of B lymphocytes are involved in pathological processes, and how they are distributed and represented under the physiological state and under pathological conditions. There is a reasonable assumption that, as with dendritic cells, NK / NKT cells and T lymphocytes, also B cell populations will contain minor and/or rare subpopulations reaching relative frequencies in the range of 0.01% to 0, 1 ‰. The primary aim of this thesis was to investigate the extent of phylogenetic and ontogenetic heterogeneity of the peripheral B cell population and lymphopoietic tissues on the basis of a comparative study across different vertebrate species. Another goal of the work was to use polychromatic flow cytometry with 183 individuals, out of them 50 controls and 133 patients with different (immune) pathologies or tumors in order to identify an optimal combination of surface features, and to use it to detect and demonstrate the existence of minor/rare...

The immune system will maintain the integrity of the organism from harmful non-malicious recognizes and protects the body against exo- and endogenous toxic substances and together with the nervous and endocrine system are among regulatory systems of the organism. Recently the evidence has supported the emerging concept of different B cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B cell homeostasis. This work is focused on the study of peripheral CD27+ B lymphocytes (one of the least explored human B lymphocytes) in healthy controls and patients with various immunopathologies, in this case we present representative results for patients with inflammatory bowel disease. Using polychromatic flow cytometry we examined 31 of peripheral blood samples, including 14 controls, 7 patients with Crohn's disease (CD) and 5 with ulcerative colitis (UC). In 6 patients with CD, we were able to perform immunophenotyping also 2 hours after intravenous administration of infliximab, and in one patient 14 days after drug...

The presented diploma thesis is focused on the preparation and the monitoring of the biological stability of beer enriched with probiotics. Probiotic bacterial strains of Lactobacillus acidophilus, Bifidobacterium breve and Bifidobacterium bifidum were used in this study. The theoretical part is divided into two sections. In the first section, probiotics are generally characterized, and their role within a gut microbiota is described. Next, the microbiology of particular probiotic microorganisms including the genera of Lactobacillus and Bifidobacterium is described. Then, some factors influencing the viability and the growth of probiotics are stated. In this section, biological effects of probiotics on the human organism and their potential clinical applications are described. The second section of the theoretical part deals with the technology of brewing, the chemical composition of beer and particular beer styles. In the experimental part, the methods of probiotic bacterial cell concentration and viability determination were optimized. Several techniques for determination of these parameters were selected, particularly the cultivation method, flow cytometry and the spectrophotometric measurement of turbidity. Then, the growth curves of the probiotic strains were measured in MRS medium. Probiotic bacteria were cultivated in model beer samples, i.e. in MRS media with several different concentrations of ethanol. It is possible to say that ethanol did not have significant effect on probiotics growth. Next, experimental cultivations of individual probiotic bacteria and their mixtures in nine real beer samples were conducted. No increase of viable cells concentrations was detected in the samples. On the contrary, a decrease of the concentrations was observed, mainly in the samples with individual bacterial strains. However, certain values of viable cells concentrations were determined at the end of the cultivations in all cases. A pale, top-fermented beer was brewed and supplemented with probiotics, and the concentrations of viable probiotic cells were monitored during 37 days of fermentation. A decrease of concentrations by two orders of magnitude of CFU/ml was observed in almost all samples. Yet, viable cells of probiotic bacteria were detected in all samples of beer at the end of the fermentation. Maximal concentration of viable probiotic cells in the brewed beer was determined with the cultivation method at (3,80 ± 0,14)10^5 CFU/ml. Chosen samples were analyzed with HPLC-RI method that quantified the common beer concentrations of ethanol in all chosen samples, lactic acid was not detected. Sensory analysis was conducted as well. Based on the results of the experimental part and the bibliography, an optimal technology of the preparation of beer enriched with probiotics is discussed in this study.

Humic acids are part of humus material abounded in nature. Humic acids are generally aromatic stuffs with sidechains and there can be linked various functional groups as carboxylic and fenolic mainly. This functional groups are able to form transitions elements ions and complexes of different stability. This property can be used in the environment, because certain polutants should be imobilized in nature. Diploma thesis studies adsorption of metal ions Cu2+, Cd2+, Zn2+ and Pb2+ on humic acids by electroanalytical method called flow – through coulometry.

The perfusion chamber for long term observing of live cells by the means the Coherence-Controlled Holographic Microscope (CCHM) was designed. CCHM was built and designed at the Laboratory of the optical microscopy at the Institute of Physical Engineering, Brno University of Technology. CCHM can quantitatively evaluate dynamical changes inside live cells thanks to the quantitative information about phase shift in each pixel of the image. In order to demonstrate advantages of CCHM experimentally, it is important to keep the live cells in the good conditions. This is made by adding the fresh cultivation medium for studied cells directly in the microscope. In contrast to the stationary chamber the perfusion chamber allows both the cultivation medium exchange and the application of biological reagents without the necessity of removing the chamber from the microscope. Therefore we can study the vital signs of cells before and after the application of reagents. An original perfusion system with accessories compatible with CCHM was designed. The design is based on the previously published perfusion system solutions that are referred to in this thesis. The flow characteristics and medium exchange process was discussed and a modification of the internal geometry, based on numerical simulations, was introduced. The applicability of this perfusion chamber has been proven for the CCHM and even for different types of microscopes. The reactions of tumor and epithelial cells during the change of the environment from the cultivation medium to the physiologically solution were studied.

This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.

This diploma thesis is focused on encapsulation probiotics and co-encapsulation with some types of prebiotics. In theoretical part is aimed to probiotics, their general characteristics and application of probiotics in food industry. There are described prebiotics and their classification, there is described principles of encapsulation and encapsulation techniques. Methods, which are used for analysis of particles and encapsulation components were introduced too. The experimental part describes methods of prebiotics characterization by high performance liquid chromatography, thin layer chromatography and spectrophotometric methods. Cultivation of probiotics with prebiotics - hydrolyzed and non-hydrolyzed wad tested. Using flow cytometry cell viability was measured too. Finally probiotics and prebiotics were encapsulated, mainly by encapsulator machine. Long-term stability of particles during 6 week storage was observed. The particles were exposed to effect of artificial intestinal, gastric and bile juices.