The diploma thesis is focused on optimalization of temperature gradient gel electrophoresis (TGGE) for diagnosing of mutations in the GJB2 gene. This gene encodes the protein connexin 26, which is part of the inner ear. Mutation in the gene GJB2 are the most common cause of congenital deafness, usually with autosomal recessive inheritance. DNA samples with known genotypes, which included the mutation c.35delG, W24X and c.358- 360delGAG were selected for the optimization heteroduplex analysis TGGE. The samples were provided by the Department of Medical Genetics, University Hospital Hradec Králové. TGGE method is used to separate nucleid acids. Compared to conventional electrophoresis, there is created a temperature gradient across the gel. The sample of DNA moves in an electric field at a speed proportional to their size. However, at the specific temperature, the strands begin to separate, which results in slowing down the speed of migration. The melting temperature is highly dependent on the primary structure of the analyzed segment of DNA, which allows to differentiate the same lenght fragment having different base sequences. Optimization was performed in two overlapping sections of the encoding part of the gene GJB2. Useful primers and analytical conditions have been found for both sections of...

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The study analyse presence and distribution of mutation in the LIF and IL-11 genes. The alterations were studied in peripheral blood samples by temperature gradient gel electrophoresis and sequencing.

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The aim of this work was to determine whether there are besides morphogenetic and metabolomic changes in viroid-infected plants also some alterations in the expression of transcription factors (TFs) known from our previous work to be involved in the secondary metabolites production, namely HlMyb1, HlMyb3, HlbHLH, HlbZIPA and HlbZIP2 TFs. Infectious vectors were prepared containing dimers of two closely related viroids- hop stunt viroid (HSVd) and cucumber pale fruit viroid (CPFVd). To achieve infection of hop (Humulus lupulus L.), biolistic inoculation of young shoots was performed. Hop cv. Admiral was chosen as a model for our experiments, because of its high flavonoid content in leaves. Infection of hop with both of these viroid species was proven by means of Northern hybridization and dot-blot techniques. Plants infected with HSVd showed serious symptoms such as stunted growth, epinasty and rugosity of leaves. Interestingly, decoloration of the petioles of the plants infected with HSVd was observed, maybe as a result of lower anthocyanins production. These symptoms were similar but milder in CPFVd-infected hops. On plants bearing symptoms, HPLC analyses were performed and compared to controls to detect changes in the levels of flavonol glycosides, phenolic acids, bitter acids and xanthohumol. In HSVd-infected hop leaves and petioles, significant decrease in the contents of flavonol glycosides and phenolic acids was observed. On the other hand, an increase in the amount of xanthohumol and bitter acids was detected in HSVd-infected tissues compared to healthy controls. Unlike to HSVd infection, the decrease of all analyzed secondary metabolites was observed in CPFVd-infected material. This difference suggests an alternative response of metabolome pathways to CPFVd-caused pathogenesis in comparison to HSVd. Semiquantitative RT PCR was performed to assay levels of TFs in healthy and infected hop tissues. Quantitative RealTime analyses of putative hop transcription factors HlbZIPA and HlbZIP2 were carried out using RNA isolated from HSVd-infected petioles. Increased mRNA levels of bZIP TFs were detected in infected material, suggesting an involvement of these factors in the response of the host plants to HSVd infection. Using thermodynamic methods of TGGE and heteroduplex analysis, several sequence variants of HlbZIP2 were idetified. According to aminoacid sequence alignment, this putative factor belongs to a group of bZIP proteins known for ABA/stress signalling in A. thaliana.

Nitrification is the biological process whereby ammonia is converted first to nitrite by ammonia-oxidizing bacteria, and then the nitrite is subsequently converted to nitrate by nitrite-oxidizing bacteria. Ammonia and nitrite levels are closely monitored during treatment of wastewater due to their toxicity to other biological processes. Sybron Chemicals, Inc., is a company that manufactures a nitrifying bioaugmentation culture (1010N) that is used to enhance the naturally occurring levels of biological nitrification. The microbial population of the 1010N product has been examined using a combination of conventional bacteriological methods and modern molecular techniques, with the goal of developing nucleic acid probes that can be used to detect the product in an environmental sample. Small regions of the 16S rRNA genes of the bacteria in 1010N (and two new nitrifying enrichment cultures) were amplified via the polymerase chain reaction (PCR) and analyzed via temperature gradient gel electrophoresis (TGGE). TGGE is a procedure that allows for separation and visualization of individual PCR products that are the same size, based on differences in their sequence. Two of the predominant PCR products in 1010N were purified from the TGGE gel matrix, reamplified via PCR, and sequenced to allow for phylogenetic analysis and nucleic acid probe design. Coincidentally, two strains (NS500-9 and MPN2) that had been isolated from the 1010N mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the 1010N product, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter, respectively, in the existing database. Using the nucleic acid sequences of the cloned DNA, organism-specific DNA probes were designed for both NS500-9 and MPN2. Unfortunately, difficulties were encountered in using the probes to monitor 1010N activity levels via quantitative dot blot hybridizations (rRNA-DNA). Therefore, efforts were redirected to using the TGGE semi-quantitatively with an internal PCR standard (Brüggeman, et al., 2000) to estimate original cell numbers of 1010N within a mixed consortium. This method was not applicable to our system due to substantial preferential binding of the primers to template other than the standard. Samples from a laboratory-scale bioreactor, bioaugmented with 1010N, were used in an attempt to correlate an increase in activity with a detectable shift in population via TGGE. No detectable shift in population was detected in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when 1010N was present in activated sludge. In both whole cell spiking experiments and genomic DNA spiking experiments, it was found that 1010N must be present at a level of at least 5% of the total population in order to be detected. While this provides some information about microbial populations, in order to evaluate the biological activity of a system, nucleic acid probes should be used in a rRNA based study. / Master of Science

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"...