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.

Tumor malignancies are the second leading cause of death worldwide. One of the reasons for the failure of oncological treatment are the uniformly set clinical guidelines, which neglect the effect of high intertumoral heterogeneity. The in vitro chemosensitivity and resistance (CSRA) assays allow for the stratification of patients prior to therapy. Therefore, the CSRA are a long-considered method for personalization of components of chemotherapy regime. Nevertheless, none of them is being routinely used in clinical practice. Certain chemotherapeutics used for their cytotoxic and cytostatic effect are also able to induce so-called immunogenic cell death (ICD) of tumor cells and activate an anti-tumor immune response. Monitoring of changes in the expression of molecules associated with the regulation of the innate immune system on the surface of dying tumor cells would enable to predict the patient's ability to respond to treatment involving modern immunotherapeutics. The feasibility of CSRA using flow cytometry and microscopy is critically evaluated in this thesis on a model of bladder cancer. Simultaneously, the correlation of the immunogenic phenotype of tumor cells and their sensitivity to selected chemotherapeutics is discussed.

In last decades, with expansion of immunological and biological methods are developed new diagnostical and treatment processes, which enable stratification of patients into sanative groups and trend to individual therapy. Absolutely transparent are effects relevant to leukemia. Present treatment procedures enable not only longer survivance of patients, but often their stable sanation. In present time is in progress intesive research imunotherapy NK cells, which could be able to finish minimal residual disease after chemotherapeutical treatment, which is evoke by persistant malignant cells. Next advantage of this treatment procedure is elimination of system disease in cosequence of exactly pointed cure. In this work he attended in vitro testing to possibility of utilization imunotherapeutic treatment by NK cells acute and chronic myeloid leukemia and chronic lymfoblastic leukemia. Using flow cytometry methods we detected activation and inhibitory ligands which are recognized by NK cells on the cell surface of leukemia blasts. These are members of MHC complex HLA-E, molecules derived from MHC class I (MICA, MICB), UL16-binding proteins (ULBP-1, ULBP -2, ULBP -3, ULBP -4) and also Hsp70 protein according to the newest observation. We also atended to detection of expression inducible heat shock...

The increased frequencies of two novel B cell populations defined as IgM+ CD19+ CD27- CD21low CD38low CD24+ and IgM+ CD19+ CD27- CD21low CD38low CD24- in peripheral blood of patients with common variable immunodeficiency (CVID) compared to healthy donors were found. The aim was to search for such B cells in patients with rheumatoid arthritis (RA) and their further characterization. The production of immunoglobulin (Ig) mRNA in single B cells was analyzed using flow cytometry, single cell sorting and RT-PCR, IgVH-specific PCR, cycle sequencing and statistical analysis. The study was focused on analysis of variable regions of the heavy chains of Igs and significant differences in the usage of VH, DH and JH gene segments, mutational frequencies, distribution of silent and replacement mutations, length and composition of CDR3 regions, clonal relation and RAG gene expression in above mentioned B cell populations were found. Because of lack of the surface CD27 molecule being regarded as marker of B cells that have undergone antigen-driven germinal reactions, analyzed populations were considered as naive. However, the pattern and type of mutations suggested that these cells could represent a new type of differentiated memory/antigen- experienced B lymphocytes (in CVID less maturated) with the likely role in...

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...

Amoebae of the genus Acanthamoeba spp. are free-living unicellular organisms found in disparate ecosystems all over the world. Due to their ability to invade human body, evade its defensive mechanisms and cause extensive tissue damage, Acanthamoeba infection can lead to serious, if rare, diseases, affecting most commonly the eye and the central nervous system. Specific therapy for Acanthamoeba infections is not available. A major reason for therapeutic failure in ameobiasis is the ability of the protist to differentiate into resistant stages. These are cysts, known to be formed under prolonged unfavorable conditions, both in the environment and the infected tissues, and the pseudocysts, less durable but rapidly formed under acute stress. The present thesis focuses on as yet unexplored mechanisms of resistance of cysts and pseudocysts. Moreover, further characteristics distinguishing cysts and pseudocysts as well as the processes involved in their formation are investigated. One of the issues addressed is a presence of protective carbohydrate compounds mannitol and trehalose that participate in defensive reactions against abiotic stress in many organisms. Although putative genes for enzymes of the trehalose and mannitol synthetic pathways are present in the genome of Acanthamoeba, only one of the...

Charles University in Prague Faculty of Natural Sciences Summary of Ph.D. thesis The role of LIF gene mutations and the relative distribution of NK cells, NKT and T lymphocytes in follicular fluid and blood of women with different history of infertility Jiří Křížan Prague 2010 1 | P a g e Doctoral degree programs in biomedicine Charles University in Prague and the Academy of Sciences of the Czech Republic Programme: Biomedicine Chairman of the Subject Board: Doc. RNDr. Vladimír Holáň, Dr.Sc. Place of study: Institute of Microbiology, v.v.i., Academy of the Czech Sciences Vídeňská 1083, 142 20 Prague 4 phone: +420 296 442 318 Autor: Mgr. Jiří Křížan Supervisor: RNDr. Petr Šíma, CSc. The dissertation can be found at Dean's Office Faculty of Charles University in Prague 2 | P a g e CONTENTS Contents 2 Summary 3 1. Introduction 5 2. Hypotheses and aims 6 3. Material and methods 7 Material: 7 Methods: 8 4. Results 10 5. Discussion 12 6. Conclusion 15 7. References 16 Bibliography of Autor: 18 1. papers in extenso (thesis background) 18 2. papers in extenso (without regard to thesis) 19 3 | P a g e SUMMARY The aim of the dissertation thesis "The role of LIF gene mutations and the relative distribution of NK cells, NKT, and T lymphocytes in follicular fluid and blood of women with different history of infertility"...