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The application of AFLP fingerprinting in breeding of Brassica napus
CUŘÍNOVÁ, Petra
AFLP markers are widely used in breeding in some other crops, but their utilization in breeding of Brassica crops is not so frequent. AFLP markers are used for molecular characterization of particular varieties or genotypes and for evaluation of genetic diversity. The aim of this thesis was the application of this method in breeding of rapeseed and in comparative study of genetic variability of different oil seed rape cultivars of Czech, Czechoslovak and German origin. AFLP is based on selective amplification of genomic DNA. Technique consists of three basic steps: 1. restriction of genomic DNA on smaller fragments and ligation of adaptors; 2. selective amplification of fragments with specific primers and 3. electrophoresis of products and computer analysis (Vos et al., 1995). In this thesis 16 Czech (CZ) and Czechoslovak (CZS) and 16 German (G) cultivars of oilseed rape were compared. Two specific primers, EcoRI AGC and fluorescence labeled primer MseI ACC, were chosen for AFLP method. It was possible to distinguish group of CZ/CZS and G cultivars, which have origin in gene resources from Czech Republic and Germany. AFLP technique allows detection of very small differences in Brassica genome. The differences between studied cultivars exist, but they are not very distinct. They show on certain relationship and reduction of genetic basis of genetic resources, which are used in Central Europe.
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Study of molecular markers correlating with prognosis and result of therapy colorectal carcinoma.
Protivová, Martina ; Souček, Pavel (advisor) ; Hromadníková, Ilona (referee)
Colorectal cancer is one of the most common cancer in this country and abroad. A major problem in the treatment of this disease is interindividual variability in response to treatment, since a large proportion of patients show resistance or adverse toxicity to the drug. The cause of this variability can be an individual's genetic makeup. From this perspective a need to find molecular markers for prognosis of the disease and markers through which we can predict response to therapy is growing. The main aim of this study was to find differences in gene expression between healthy and tumor tissue from patients well and poorly responding to treatment based on 5-fluorouracil and compare the results to clinical data. This study aimed to evaluate gene expression of 14 potential molecular markers involved in 5-FU pathways involving metabolism, transport, and objectives of the drug. Patients selection for the study was based on 5-FU regimens treatement. Expression was evaluated in two independent sets consisting of patients with indicated palliative or adjuvant chemotherapy. For each patient malignant and paired nonmalignant tumor tissue was available. Gene expression in these samples was measured using real-time RT-PCR with relative quantification using the preamplified cDNA. In the first phase the gene...
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Advances in the production of antibodies in plants
Angelis, Karel ; Šmídková, Markéta
Plants are the oldest biotechnological production system and as eukaryots have advantage over bacteria to modify expressed proteins. Laboratory model plants like tobacco along with crop plants with established logistics for harvest and storage like potatoes or rice are used for production of desired proteins. Current trend is to replace soil grown plants with liquid cultures of plant cells grown under controlled conditions. In this respect the moss Physcomitrella patens besides ability to grow in liquid culture enables due to efficient homologous recombination to modify posttranslational glycosylation to correspond human. For heterologous expression in plants, recombinant, single-chain molecules composed of only variable VL and VH regions of the original antibody forming binding pocket are constructed and expressed. Recombinant scFv antibodies are then used as a part of therapeutic or diagnostic drugs in oncology.
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Expression analysis of new follicullar B cell populations characterized by absence of CD27 molecule and down-modulation of CD38 molecule.
Kerdíková, Zuzana ; Růžičková, Šárka (advisor) ; Drda Morávková, Alena (referee)
Two novel B cell populations were characterized in peripheral blood of patients with common variable immunodeficiency and healthy controls were observed using flow cytometry in the study supported by the grant IGA MZ ČR NKT11414-3. These B cell populations were defined as CD19+ CD27- CD21+ CD38low CD24+ IgM+ FO I and CD19+ CD27- CD21+ CD38low CD24++ IgM++ cells. Since none of found populations has ever been described, the aim of this thesis was to characterize these populations with focus on analysis of variable regions of the heavy chains of immunoglobulins and genes coding proteins participating in the process of VHDHJH formation (Rag 1, Rag 2, and TdT) produced by cells of these populations. Flow cytometry, single cell sorting, single-cell RT-PCR, IgVH, Rag 1, Rag 2, and TdT specific PCR amplification and cycle sequencing were employed to perform the molecular analysis in individual B lymphocytes. Both populations in two patients with common variable immunodeficiency, two healthy controls, and in two patients with autoimmune diseases - rheumatoid arthritis and systemic lupus erythematosus (as the disease control) - were examined. Finally, the statistical analysis was used to evaluate the differences in expression of variable regions of the heavy chains of immunoglobulins and in Rag1 and 2, and...
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Emissions of greenhouse gases (N2O and CO2) from pasture soil as a result of activity of soil microbial community - preliminary results
Šimek, Miloslav ; Hynšt, Jaroslav ; Brůček, Petr ; Čuhel, Jiří
Cattle overwintering area was shown an important point source of N2O and CO2. The emissions of N2O were mostly directly related to the rate of animal impact as the greatest fluxes of N2O and the highest total cumulative N2O emissions as well were found at the most impacted site. The fluxes of N2O were however very variable in time and most of N2O was emitted during several short events in spring or in winter. Laboratory experiments suggested that total amount of nitrogen escaping from soil from impacted sites is much greater than indicated by field measurements of N2O fluxes and molecular nitrogen (N2) was shown as the main nitrogen gas. It is hypothesized that soil pH is a crucial soil factor controlling the mole fraction of N2O: under slightly alkaline conditions in severely impacted soils, denitrification is completed and most of nitrogen escapes from the soil as N2; under more acidic conditions, however, the activity of nitrous oxide reductase is partly reduced.
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Blastocystis subtypes in pigs
LAKATOSOVÁ, Lucie
Blastocystis is an anaerobic single-celled protozoan that commonly occurs in the intestinal tract of animals and humans. It is a genetically very variable organism. Blastocystis can be found both in healthy specimens and in patients with gastrointestinal disorders. In 2011/2012, I examinated already isolated DNA samples from domestic and wild pigs by molecular methods in the laboratory of the ASCR Parasitology Institute. In total, 110 samples were examined. The domestic pigs were positive in 91 % (51/56) and wild pigs in 67 % (36/54). The overall prevalence of domestic pigs was higher by 24 %. Several sequences of SSU rRNA gene obtained during the work suggest that pigs may serve as host for atypical Blastocystis subtypes.
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Genetic variability of \kur{Isaria} genus in Czech Republic
ČÁPOVÁ, Aneta
My diploma thesis deals with genetic variability of entomopathogenic fungi of the Isaria genus encountered in the Czech Republic. Individual representative of the genus can be found in soil where they attack all developmental stages of insects, giving preference to larvae and pupae. The Isaria fungi find application first and foremost where plants have to be provided biological protection. In case of mitosporic fungi is the precise identification very difficult, taxonomy is often unclear in many genera, including the genus Paecilomyces/Isaria to demonstrate their polyphyletic nature. The fungi are classified primarily with reliance on morphological studies. The most common markers used to identify fungi are the shapes and sizes of their conidia and the biological properties (germination of spores, tests of biological efficiency). Identification made in consideration of the morphological markers is inaccurate and very variable. To overcome those accuracies, there are very useful molecular DNA markers, which can be relevant in ecology, biology and in fungi genetics. This paper relies on applying the ITS region (Internal Transcribed Spacer) as a molecular marker. ITS regions are partial constituent rDNA carrying no code - that is why the regions are likely to accumulate evolutionary changes in the DNA sequence, which makes them suitable for extensive use in taxonomic analyses of many organisms. The study results in a phylogenetic trees constructed by comparing different sequences of ITS regions obtained from the samples of entomopathogenic fungi of the Isaria genus gathered in the Czech Republic during the monitoring stage 2013 to 2014. Thereunder detection of Isaria sp. occurring in the Czech Republic.
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Bacteria deactivation and removal from Wastewater and polluted Air
Kimmer, D. ; Vincent, I. ; Dudák, J. ; Bergerová, E. ; Petras, D. ; Lev, J. ; Holba, Marek ; Kalhotka, L. ; Mikula, Přemysl ; Kořínková, R. ; Kubáč, L.
Procedures permitting to prepare homogeneous functionalized nanofibre structures based on polyurethanes modified by phthalocyanines (PCs) by employing a suitable combination of variables during the electrospinning process are presented. Compared are filtration and bacteria deactivation properties of nanostructures without PCs, modified in bulk and with PCs embedded into polyurethane chain by a covalent bond protecting the release of active organic compound during the filtration process. Finding that the functionalized nanofibre structures have an effect on bacterial growth was confirmed by microbiological, physico-chemical and molecular biological analyses, such as the inoculation in a nutrient agar culture medium, flow cytometry and real-time polymerase chain reaction.
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