Acute lymphoblastic leukaemia (ALL) is the most frequent childhood malignancy. One of the recent improvements in ALL treatment was the introduction of minimal residual disease (MRD) monitoring that enables risk stratification based treatment adaptation. The same MRD monitoring helps to choose relapse treatment, to guide indication for stem cell transplantation (SCT) and allows for a more personalized management of patients undergoing SCT. One of the main routes of MRD levels detection is characterisation of leukemic blasts using flow cytometry. However, flow cytometry is limited by its mainly manual expertise-based analysis. Such analysis is subjective and clearly insufficient for current complex data. While new computational tools are available for multidimensional flow cytometry data, there is an urgent need to test and adapt them for the use in clinical environment. The goal of this thesis is to detect immunophenotypes associated with leukaemia and their development by leveraging machine-assisted analysis of a set of diagnostic files selected based on information about more than three hundred thousand of multiparameter flow cytometry datasets. Advanced bioinformatic tools will help to detect blast and healthy haematopoietic populations, to derive their immunophenotypes and to identify individual...

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.

B-lymphocytes are a subset of immune cells that can be distinguished mainly by carrying clonally diversified membrane-bound immunoglobulin specialized to specific antigen recognition. Together with other immunocytes B-lymphocytes play a central role in adaptive immune system which takes part in defense of the host against wide variety of pathogens. 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. That is the reason for the thesis to be focused on an analysis of B-cell population profile changes in disease, identification of any association present among specific B-cell subpopulations, as well as association between these subpopulations and clinical parameters. Using polychromatic flow cytometry we analyzed frequencies of 11 B-cell subpopulations including stages of transient B-lymphocytes through effector antibody-producing plasma cells. We examined 81 individuals including 22 healthy controls and 59 patients with...

Abstract This MSc. thesis deals with karyological, genetic and phenotypic variation of selected taxa from the genus Dianthus L. in the Czech Republic and adjacent countries. The evolutionary history of the genus has been shaped by several microevolutionary processes, including interspecific hybridization, genome duplication, and edaphic speciation. These processes led to the origin of a number of phenotypically similar taxa, which are often restricted to a narrow geographic area. One subendemic and three endemic taxa occur in the Czech Republic. These (sub)species were used as model groups to gain insight into microevolutionary processes in small populations and the postglacial development of the genus in Central Europe. The thesis consists of three parts, each addressing different evolutionary phenomenon: Dianthus arenarius subsp. bohemicus is a critically endangered endemic psammophyte currently known from a single population in Central Bohemia. The site is also inhabited by widespread D. carthusianorum. Interspecific hybridization has been suspected on the basis of morphological characters, but this has never been confirmed by any other technique. I exploited differences in the number of chromosomes between both species and, with the aid of DAPI flow cytometry, estimated relative DNA contents of...

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.