Genus Diphasiastrum Holub is one of the most complicated and biosystematically very little investigated groups within Lycopodiaceae family. There are 6 species recognized in Central European region. Three of them (D. alpinum, D. complanatum, D. tristachyum) are considered basic - parental taxa, and their hybridization probably gave origin to the three adjacent species - intermediates (D. issleri, D. zeilleri, D. oellgaardii). These supposedly hybridogenous taxa often co-ocur with at least one parental species. All the taxa often meet in secondary habitats where they tend to form hybrid swarms (e.g. on ski slopes which represent an ideal biocorridor for meeting the species from alpine zone with the species from lower altitudes). In such places reciprocal crossings between all of the taxa occur, often accompanied by introgression. The degree of hybridization, including the possible introgression, was studied using absolute genome size analyses combined with classical and geometric multivariate morphometrics. Genome size was estimated for 570 plants from 83 localities, mostly from the Czech Republic. Despite the fact that each parental taxa had a specific range of absolute genome sizes, adjacent species formed more or less disconnected continuum. Multivariate statistical methods (PCA, RDA, Loess, PLS)...

Genome size or nuclear DNA content is a a trait which varies greatly among living organisms, with no apparent relashionship between genome size and organismal complexity. It is usually described using the C-value as either a number of base pairs or picograms of DNA. Unresolved questions regarding mechanisms influencing genome size and relationships between genome size and other organismal traits are together known as the C-value enigma. Genome size is known to positively correlate with cell size and negatively with developmental and cell division rate. A maximum constraint of 2 pg has also been proposed for haploid genome sizes of holometabolous insects. Despite the fact that there are about one million described insect species, genome sizes are only known in a fraction of them. This thesis thus aims to extend the dataset of known insect genome sizes by as many species of cockroaches (Blattodea) as possible. Another aim is to compare results with known phylogeny and also to compare difference in genome size between sexes. Flow cytometry was used for genome size estimations, which is a rapid, simple and effective method, also suitable for study of ploidy levels and other cellular charasteristics.

Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...

(english) Natural Killers - NK cells play an important role in immune surveilance and regulation either by direct cytotoxicity towards infected, transformed or otherwise damaged cells, or by production of cytokines and chemokines. The resulting response of NK cells is given by the sum of stimulating and inhibiting signals, tranduced by a wide array of receptors. Killer Ig-like receptors KIR2DL4 and LILRB1, which recognize self HLA-G molecules in pregnancy, as well as NKR-P1 receptors, which differ in the number of isotypes, are species-dependent and reduced during phylogenesis. NKG2D, reacting to stress-inducible proteins, and adenosine receptors (AR), which supress the inflamatory reaction, remain evolutionary conserved. The aim of this work was to study the involvement of NK cells and their receptors in several immune disorders and in various species, to provide new insights into their function and posisible immune modulation. We have shown here, that the choice of species in the study of NK cell effector functions may be crucial in some cases. The reaction to glycans, using synthetic GlcNAc-terminated glycomimetics GN8P, exerted opposing effects on NK cell function in humans and C57Bl/6 mice. In humans, the glycomimetic decreased cytotoxic activity of high NKR-P1A expressing NK cells, while in...

Primary immunodeficiencies represent a heterogeneous group of hereditary immune system malfunctions with very variable causes and symptoms. Multiparametric flow cytometry has become an important tool in primary immunodeficiency diagnostics and research because it provides detailed information on the phenotype of individual immune cells and their proportions in circulation. We have developed a complex monoclonal antibody panel composed of five eight-color tubes which is designed for immunophenotyping of basic lymphocyte subsets and further analysis of B and T cell subpopulations. We have optimized and standardized the panels so they will identify any changes originating from primary immunodeficiencies and provide comparable data on the level of cooperation between more laboratories. This was achieved by cooperation of six European research facilities which are all parts of the Euroflow consortium. The panels have been validated both on peripheral blood samples from healthy donors and patients with either gentically defined primary immunodeficiency or common variable immunodeficiency. Keywords: T lymphocyte, B lymphocyte, primary immunodeficiency, flow cytometry, immunophenotyping, Euroflow, optimization, standardization

This thesis deals with interspecific hybridization in sympatric populations of Pulsatilla patens and P. pratensis. The key aim was to assess karyological and phenotypic variation in the study group. DNA flow cytometry revealed three non-overlapping groups of genome sizes, corresponding to P. patens, P. × hackelii and P. pratensis. The frequency of interspecific hybridization was low, accounting to approx. 3% of analyzed plants. Morphometric analyses identified a set a reliable species-specific characters, including number of divisions the leaf, length of petiole the part of leaf, number of smaller parts of leaf and colour of blossom. Although the majority of hybrid individuals flowered and set fruits, their fertility was reduced considerably. The threat from interspecific hybridization to parental species is therefore rather low.

The thesis is focused on a determination of heparin and chondroitin sulfate, using flow injection analysis with spectrofluorimetric detection. The determination is based on the interaction of negatively charged heparin, chondroitin sulfate resp., with a cationic dye (azure B or phenosafranine) which is manifested by the decrease in fluorescence intensity of the dye in its emission maximum. The optimal conditions for the determination in static mode were found, and calibration dependencies were measured. The conditions of FIA were optimized and following parameters were established: the volume of dispensed sample of 100 ml, the length of the reaction coil 60 cm, the flow rate 0.7 ml min-1 , the concentration of azure B 1.6×10-5 mol dm-3 , the concentration of phenosafranine 3.5×10-5 mol dm-3 . For the determination of heparin using azure B it was found: LOD = 0.023 IU ml-1 , LOQ = 0.186 IU ml-1 , and linear dynamic range 0.19-1.43 IU ml-1 . For the determination of heparin using phenosafranine it was found: LOD = 0.102 IU ml-1 , LOQ = 0.192 IU ml-1 , and linear dynamic range 0.19-1.79 IU ml-1 . For the determination of chondroitin sulfate using azure B it was found: LOD = 0.58 mg dm-3 , LOQ = 2.37 mg dm-3 , and linear dynamic range 2.37-8.32 mg dm-3 . The developed determination was applied to the...

The increasing incidence of children suffering from allergic diseases could be caused by sensitization of immature immune system during the intrauterine development. Several important scientific papers have demonstrated the ability of cord blood cells to respond by elevated proliferation activity after stimulation by common allergens. Following these findings, present study follows the production of cytokines which play a role in the pro- and anti-allergenic tuning of the immune system. Umbilical cord blood cells were stimulated with polyclonal activators (phytohaemagglutinin) and common allergens (ovalbumin, timothy grass, birch, mite). Subsequently, cytokine production was monitored using selected methods that reflect different stages of cell activation - at the level of mRNA by quantitative real time PCR (qRT-PCR), by flow cytometry detection of the presence of intracellular cytokines in different cell subpopulations and by ELISA measurement of cytokines in CBMC culture supernatants. The results obtained point to a very weak ability of these common allergens (timothy grass, birch, mite, ovalbumin) to stimulate CBMC to produce cytokines observed by all of these methodological procedures. Although we did not observe significant differences in CBMC cytokine production (IL-2, IL-4, IL-10, IL-12,...

The aim of this thesis has been design, construction and application of a flow-through nephelometric detection cell based on photodiode as a sensor and a laser source. The developed device has been tested and its function has been proven on the determination of sulphate by flow injection analysis with nephelometric detection after precipitation by barium chloride. The results achieved were comparable with those obtained by spectrometric detection. Furthermore, the device has been successfully applied to the determination of promethazine based on formation of its insoluble ion associate with bromophenol blue. Powered by TCPDF (www.tcpdf.org)

Polyploidisation plays a major role in plant evolution. It can also cause taxonomic complexities as morphological differences are often blurred in higher ploidy levels. One of the taxonomically-intrigued groups of Central Europe is C. rotundifolia agg. Three ploidy- heterogeneous species are traditionally recognized in the Czech Republic: C. gentilis (2x, 4x), C. rotundifolia (2x, 4x) and C. moravica (4x, 6x). The thesis examines karyological and phenotypic variation of C. moravica, in the centre of its distribution (the Czech Republic and Slovakia). With the aid of modern biosystematics tools (DNA flow cytometry, multivariate morphometrics), marked discrepancies were revealed between published data and actual cytotype distribution pattern and morphological variation. In contrast to literature records, cytotype distribution in C. moravica is largely parapatric, with hexaploids occurring in Pannonian basin and tetraploids in Central and Eastern Slovakia. The type population of presumed tetraploids turned out to be hexaploid. The coexistence of one majority (4x) and six minority cytotypes (2x, 3x, 5x, 6x, 8x, 9x) was found in one population in central Slovakia and represent the most salient case of ploidy coexistence ever reported. Multivariate morphometrics showed that species identification on the...