The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...

(English) Meiotic homologous recombination, homologous chromosomes synapsis, and F1 hybrid sterility (enabling formation of species) are mutually interconnected phenomenons, one being the prerequisite to the latter. In the present thesis, these phenomenons were investigated on a genetic and mechanistic level using a mouse subspecies as a model. Noncrossovers (NCOs, gene conversions), 90% prevalent resolution of Prdm9- determined meiotic double-strand breaks (DSBs), were uniquely identified and characterized on a chromosome-wide level. The mean gene conversion tract length, based on 94 NCOs events, was calculated to be 32 bp. On a local level, the NCOs overlapped the known hotspots of PRDM9-controlled histone trimethylation and DSB formation, indicating their origin in the standard meiotic DSB repair pathway. On chromosome-wide level, NCO and CO distributions differed, in particular COs being relatively preferred over NCOs in subtelomeric regions. A specific subset of nonparental/asymmetric NCOs and COs was underrepresented in our datasets, proposing their problematic repair, hypothetically enabled by sister chromatids, and thus not contributing to indispensable homologous synapsis. Genome-wide crossover (CO) rates, genetically and mechanistically crucial ~10% of DSB repair, were proven to be...

5 Abstract Genetic mechanisms of regulation of gene expression form the basis for proper development, function of organisms and their responses to variable life conditions. However, they are relatively slow. Life processes that require a fast response to the changing environmental and metabolic conditions are mostly executed on the level of proteins especially their posttranslational modifications and protein- protein interactions.The goal of the experimental work that led to the presented thesis consisted in exploitation of the model organism Caenorhabditis elegans for analysis of regulation of gene expression by transcription factors from the protein family of nuclear receptors. The model system C. elegans enables very efficient experimental procedures in the field of genetics, genomics and functional analysis of phenotypes. In the experimental work connected with this thesis, I studied the regulation of gene expression under specific experimental conditions from the perspective of advanced functional proteomics and I focused on the employment of separation methods and methods of advanced proteomics, especially by mass spectrometry.In the first part of the work, I characterized the nuclear receptor NHR-60 on the protein level. This nuclear receptor is expressed as two protein forms with a mass of 50 kDa...

Introduction: Congenital short femur, or proximal femoral focal deficiency (PFFD), is a rare complex deformity of the lower extremity with femoral dominance. The clinical findings cover wide range of variety, from femoral absence till inconspicuous shortening of the femur. Aim of the study: 1. Molecular analysis of pseudoarthrosis tissue in congenital short femur with focusing on osteogenic and angiogenic gene expression in comparison with physiological bone. The differences in gene expression were expected. 2. Retrospective analysis of femoral healing after prolongation calculating the severity of affection, age, distance of elongation and complication. The extended healing according to severity type and age was expected compared to control group. Material and methods: The RNA from piece of one was isolated and transcription profile of possible 113 genes of osteogenesis and angiogenesis was detected by biochip technology (SuperArray Bioscience Corporation). 10 samples analyses were performed (7 of PFFD, 3 controls). The data of 57 PFFD patients indicated for elongation of the femur with the types Pappas III, IV, VII, VIII and IX and 12 patients in control group were evaluated retrospectively and statistically by GLS method. Results: The expected differences in gene expression in PFFD tissue...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kryštof Dobečka Supervisor: PharmDr. Tomáš Smutný, Ph.D. Advisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Experimental approaches for studying hepatic enzyme induction mediated by pregnane X receptor The thesis focuses on hepatic pregnane X receptor (PXR)-mediated induction of biotransformation enzymes. Emphasis is placed on experimental models and methods which are used for the assessment of enzyme induction. In addition to summarizing its well- established role as a xenobiotic-sensing receptor, PXR is also presented as a transcription factor with an important role in endogenous pathways. Furthermore, cell and animal models are evaluated in terms of expression and function of PXR and its target xenobiotic-metabolising enzymes. Primary human hepatocytes in 2D cultures are considered to be the gold standard of in vitro hepatic models. However, 3D technologies are expected to be increasingly used in the future. The use of animal models is limited due to pronounced interspecies differences in PXR activation. Thus, humanized models have been established to overcome these limitations. Next, this thesis comments screening methods for an assessment of interaction between PXR and...

No description available.

In this study, we examined the immunomodulatory effect of excretory/secretory products, crude adult extracts and crude larvae extracts from Hymenolepis diminuta on the intestinal epithelilal cell line from a rat. For determination of the immunomodulation effect of all H. diminuta extracts was used relative gene expression of TNFa, IL-17re and IL-33 from epithelial cells and it was tested using real-time PCR. Our result showed that excretory/secretory products had the strongest antiinflammatory effect on the epithelial cells. We assume that crude adult extracts play an important role in increase of gene expression of IL-33 and also in the immunomodulatory ability of H. diminuta in the host organism.

This thesis focuses on the importance of the heme catabolic pathway in chronic hepatitis C (HCV). The aim is mainly to investigate, whether expresion/activity of key enzymes of the heme catabolic pathway, heme oxygenase (HMOX) and biliverdin reductase (BLVRA) in the liver and blood (study A) or promoter variations of HMOX1 and UDP- glucuronosyltransferase (UGT1A1) (study B) may be associated with the progression of fibrosis and may also predict antiviral treatment outcome in patients chronically infected with HCV. We set up a new sensitive method to quantify HMOX activity by reduction gas chromatography. We developed and extensively validated RealTime PCR assay for HMOX and BLVRA expression in the liver and peripheral blood leucocytes (PBL). The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. No association was detected between either expression of HMOX/BLVRA or the HMOX1/ UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. A marked difference in BLVRA expression in PBL between the sustained responders (SVR) and patients with treatment failure (NVR) was detected before antiviral treatment and during the follow-up. Our data suggests, that BLVRA basal expression...

5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...

Vysoce výkonné sekvenční technologie produkují obrovské množství dat, která mohou odhalit nové geny, identifikovat splice varianty a kvantifikovat genovou expresi v celém genomu. Objem a složitost dat z RNA-seq experimentů vyžadují škálovatelné metody matematické analýzy založené na robustníchstatistických modelech. Je náročné navrhnout integrované pracovní postupy, které zahrnují různé postupy analýzy. Konkrétně jsou to srovnávací testy transkriptů, které jsou komplikovány několika zdroji variability měření a představují řadu statistických problémů. V tomto výzkumu byla sestavena integrovaná transkripční profilová pipeline k produkci nových reprodukovatelných kódů pro získání biologicky interpretovovatelných výsledků. Počínaje anotací údajů RNA-seq a hodnocení kvality je navržen soubor kódů, který slouží pro vizualizaci hodnocení kvality, potřebné pro zajištění RNA-Seq experimentu s analýzou dat. Dále je provedena komplexní diferenciální analýza genových expresí, která poskytuje popisné metody pro testované RNA-Seq data. Pro implementaci analýzy alternativního sestřihu a diferenciálních exonů jsme zlepšili výkon DEXSeq definováním otevřeného čtecího rámce exonového regionu, který se používá alternativně. Dále je popsána nová metodologie pro analýzu diferenciálně exprimované dlouhé nekódující RNA nalezením funkční korelace této RNA se sousedícími diferenciálně exprimovanými geny kódujícími proteiny. Takto je získán jasnější pohled na regulační mechanismus a poskytnuta hypotéza o úloze dlouhé nekódující RNA v regulaci genové exprese.