The aim of this Thesis is development of electroanalytical methods for determination of cholesterol and 7-dehydrocholesterol on boron doped diamond electrodes based on electrochemical oxidation of products of acid-induced dehydration reaction. Two approches have been applied, relying on their detection in 0,1 mol.l-1 perchloric acid in acetonitril containing variable water content 0,43 % - 40 %. Cyclic voltammetry revealed substantial difference in oxidation peak pontecials for 7-dehydrocholesterol (+850 mV) and cholesterol (+1500 mV) in 0,1 mol.l-1 perchloric acid in acetonitrile containing 5 % of water. The limit of detection using differential pulse voltammetry were 6,19 µmol.l-1 a 19,2 µmol.l-1 for a mixture of 7-dehydrocholesterol and cholesterol, respectively. For flow injection analysis with amperometric detection (FIA-AD) the water content in run buffer, detection potencial and the distance between the capillary outlet and electrode surface in wall-jet arrangement of amerometric flow-through detection cell were optimized. Achieved detection limits are in range of 0,61 - 3,43 µmol.l-1 depending on the detection potencial and water concent in the run buffer. The developed FIA-AD method was used for determination of a model mixture solution of 7- dehydrocholesterol and cholesterol. Key words...

Melampyrum nemorosum agg. is very complicated group of hemiparasitic plants. According to the traditional concept, 15 species is distinguished. Recent molecular analyses show a need of critical taxonomic revision of group and a potential importance of ancient hybridization. Analysis of 3 regions of cpDNA (trnTUGU-trnLUAA, psbA-trnHGUG, rpl32-trnLUAG) and 2 regions of nuclear DNA (Agt1 and At103) reveals well supported lineage with limited geographical distribution. Morphology and genome size of genetically supported lineages were compared.

Melampyrum nemorosum agg. is very complicated group of hemiparasitic plants. According to the traditional concept, 15 species is distinguished. Recent molecular analyses show a need of critical taxonomic revision of group and a potential importance of ancient hybridization. Analysis of 3 regions of cpDNA (trnTUGU-trnLUAA, psbA-trnHGUG, rpl32-trnLUAG) and 2 regions of nuclear DNA (Agt1 and At103) reveals well supported lineage with limited geographical distribution. Morphology and genome size of genetically supported lineages were compared.

The data from public herbal collections were used to determine the frequency of the occurence of fertile bracken populations. Samples of bracken were collected to assess the degree of fertility and also to reveal the frequency of both diploid and triploid cytotypes using flow cytometry. Collected samples were also used for SSR analysis to characterize the population structure of both cytotypes and the propagation of triploid plants of bracken.

The aim of this thesis was to find optimal therapy based on combination of membrane-anchored phagocytic ligands (mannose-(G)5-(K)10-STE, mannan-BAM, mannan-SMCC) with LPS (ligand of signal receptor)for treatment of murine melanoma B16-F10. Mixture of mannan-BAM with LPS applied in pulse regime proved to be the most effective, resulting in heigh reduction of tumor growth and significant prolongation of survival.

The thesis aims at clarifying the taxonomic difficulties in the Tortula muralis complex. For the first time, the group was studied by means of morphometric analysis, ploidy level assessment by flow cytometry, and molecular analysis based on sequencing of ITS region of nuclear ribosomal DNA. Morphometric and cytometric studies found only two well distinguished groups within the complex. These groups corresponded to the markedly variable species T. muralis and to the rather uniform species T. lingulata. Variability in ITS sequences suggested extensive gene flow among some of traditional morphologically defined taxa of the complex. Multiple polytopic autopolyploid origin of polyploids was revealed in some taxa. Changes in taxonomic conception of T. muralis complex were proposed in order to reflect structure of morphological, karyological, and DNA variability in the group. Natural hybridization is probably an important evolutionary mechanism that generated morphological diversity and taxonomic complexity in the mosses of T. muralis complex.

Presence and range of phenotypic plasticity in the Agrostis stolonifera polyploid complex (Poaceae) was studied in the territory of the Czech Republic. Plants were cultivated under different experimental conditions. Stomatal size of different Agrostis stolonifera cytotypes was measured. Flow cytometry was applied for genome size estimation. Ploidy levels were determined for more than 150 samples of Agrostis stolonifera as well as several specimens of Agrostis canina, A. capillaris, A. gigantea, A. rupestris and A. vinealis. Absolute DNA content was estimated in all studied species. Isozyme analysis was used to test the possibility of the hybrid origin of pentaploid individuals of Agrostis stolonifera species.

B-lymphocytes are lymphoid cells, which are a part of the adaptive/innate immune system and generate antibodies. Recently, many studies have supported hypothesis that different rather minor B-lymphocyte subpopulations may play a direct and indirect role in immunopathogenesis in human pathologies such as Crohn's disease (CD). The aim of current study was therefore to investigate distribution of frequencies of B lymphocyte subpopulations (from transient to mature effector B cell stages) in peripheral blood of healthy subjects (CO), patients with Crohn's disease (CD) and ulcerative colitis (UC). Thus, using 11-colour flow cytometry we have analysed 30 blood samples of individuals, including 14 healthy controls, 11 patients with Crohn's disease and 5 with UC. In 6 patients with CD we have had an opportunity to analyze blood samples collected 2 hours after an administration of anti-TNF therapy. Higher frequencies of memory B-lymphocytes (CD19+ CD27+ , CD19+ CD20+ CD27+ and CD19+ CD20+ CD27+ IgM+) were found in patients with CD as compared to COs. (20.06±13.58%; 17.61±13.48%; 88.60±20.56% vs. 11.75±26.47%; 11.25±26.50%; and 66.82±22.60%), in case of CD19+CD20-CD27-IgM+ B-lymphocytes the difference was statistically significant (57.15±17.21% in CD vs. 19.59±31.79% in CO; p=0.0341), which is in accordance...

Myriophyllum L. (watermilfoil) belongs to one of the most invasive genus of the Northern Hemisphere. The three most agressive species, one of which is native to Europe (M. spicatum), are widespread throughout the majority of United states and two of them (M. aquaticum and M. heterophyllum) are becoming invasive in several Europaean countries as well. Therefore, Europaean populations of both native and invasive species, represents ideal place where to study processes which account for watermilfoils' invasivness. In this study, flow cytometry, morphometric analyses and germination experiments were employed to evaluate cytotype and morphological variability of Myriophyllum along with its potential to hybridize and propagate generatively. Five ploidy levels were detected in Europe, however no ploidy variability has been found within any species except for M. sibiricum (hexaploids and nonaploids) and one population of M. aquaticum (hexaploids and oktoploids). Flow cytometry proved to be useful tool for purposes of distinguishing species of different ploidy level and their potential hybrids. Nevertheless, the key species - M. sibiricum and M. spicatum - among which probably even more aggressive hybrids were recently identified, have similar hexaploid level. Thus flow cytometry cannot facilitate...

This thesis was focused on a determination of nadroparin using sequential injection analysis and flow injection analysis with fluorimetric and spectrophotometric detection. The principle of determination was based on the interaction of nadroparin with phenothiazine dye. Methylene blue was used as phenothiazine dye. The determination was performed on a laboratory made FIA instrument and on the commercial SIA instrument. Optimal conditions for SIA were concentration of dye 6 ∙ 10-5 mol dm-3 , injected volume 100 µl, flowrate 1,5 ml min-1 , for FIA: concentration of dye 3 ∙10-5 mol dm-3 flowrate 2 ml ∙ min-1 , injected volume= 100 µl, for fluorimetric detection was sensivity of the detector 1000 V, Emission was measured by 2 nm and excitation wavelenght 621 nm. For spectrophotometric detection, absorbance was detected at the absorption maximum wavelength. Repeatability ranged from 2.01 to 2.85%. The limit of detection for FIA was 0.05-0.09 IU ml-1 , for SIA were limits of detection in range 0,25 - 1,63 IU ml-1 , limits of quantification in range 0,83 - 5,44 and linear range was from 0,5 - 20 IU ml-1 . The limits of detection, limits of determination and the linear range could be corrected for the SIA by the injected volume of phenothiazine dye.