|
|
Optimizing heteroduplex analysis, TGGE, for diagnosing inherited diseases.
Rutová, Martina ; Fikrová, Petra (advisor) ; Beránek, Martin (referee)
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...
|
|
|
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
Křížan, Jiří ; Šíma, Petr (advisor) ; Jonáková, Věra (referee) ; Kročová, Zuzana (referee)
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"...
|
|
|
Expression analysis of selected regulation factors in hop with relation to symptoms of viroid pathogenesis
FÜSSY, Zoltán
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.
|
|
| |
|
| |
guest ::
