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Regulation of gene expression in anaerobic parasitic protist and practical application of knowledge
Brzoň, Ondřej ; Zubáčová, Zuzana (advisor) ; Horváthová, Lenka (referee)
Because of its importance for every single cell, regulation of gene expression is often studied at all levels today. On the other hand, rather sparse amount of information is available for protists and our understanding of their gene expression is nearly limited to several model organisms. Three anaerobic human parasites have been studied in details: Trichomonas vaginalis, Entamoeba histolytica and Giardia intestinalis. Most recently, some others anaerobic protists (e.g. Entamoeba invadens or some species of Spironucleus genus) have been researched as well. This work is focused on the structure of promoter regions in protein coding genes and application of this knowledge in construction of transfection systems as one of the most effective and powerful tools in molecular and cell biology.
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Financial education mechanism of handicapped pupils at secondary schools
Filip, Josef ; Krutská, Dagmar (advisor) ; Svoboda, Petr (referee)
The thesis deals with problems of disabled pupils'education financing at secondary schools. Its aim is to create the integral survey about the disabled pupils'schooling, to compare and analyse normative financing of the most expanded educational specialization in accordance with regions. Theoretical part includes national educational concepts and programs directed at handicapped pupils. Besides detailed characterization of the health troubles'single types there are also described institutial schooling and normative financing of handicapped pupils. There are the analysed reports of MŠMT (Ministry of education and youth's psychical education) about handicapped pupils at the research part. Charts and graphs contain datas of pupils in the Czech republic according to health trouble's type, number of pupils at special classrooms and of integrated pupils during 2007-2012. The inseparable component of the research there is survey of pupils'schooling in accordance with the schools'promoters. Conclusion is devoted to detailed comparison of normative financing of the most expanded educational specialization 65-51-E/01 Stravovací a ubytovací služby (The board and accommodational service) in the first place for mental disabled pupils and pupils suffering with developmental disorders of learning and behaviour.
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Regulatory mechanisms of ornithin transcarbamylase and beta-glucocerebrosidase gene expression and their relevance to diagnostics
Lukšan, Ondřej ; Jirsa, Milan (advisor) ; Kožich, Viktor (referee) ; Kříž, Vítězslav (referee)
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...
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Characterization of promoter regions of HGSNAT and GBA genes, and a contribution to the study of pathogenesis of MPS IIIC and Gaucher disease
Richtrová, Eva ; Hřebíček, Martin (advisor) ; Macek, Milan (referee) ; Adam, Tomáš (referee)
Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Expression of genes for the conversion of nitriles and amides in Rhodococcus erythropolis
Kracík, Martin ; Pátek, Miroslav (advisor) ; Mikušová, Gabriela (referee)
The strain Rhodococcus erythropolis A4 is a source of enzymes nitrilhydratase and amidase, that catalyse conversion of nitriles and amides. These enzymes are used in industrial biotransformation and bioremediation. Since it was difficult to carry out genetic manipulations aimed at increasing the production of these enzymes in the strain A4, the corresponding genes (ami and nha1 + nha2) of a related strain R. erythropolis CCM2595, in which both plasmid and chromosome manipulations can be routinely performed, were identified and analyzed in this diploma theses. The ami and nha1 + nha2 genes from the strain R. erythropolis CCM2595 were isolated and sequenced together with the flanking regions (5.5 kb in total). The organization of these genes and the expected regulatory genes was described in the strain CCM2595 and mechanisms of regulation of expression of these genes were studied. For the analysis of transcription of amidase and nitrilhydratase genes from both strains of R. erythropolis, the promoter-probe vector pEPR1 replicating in Escherichia coli and R. erythropolis was used. Transcriptional fusion of Pami promoters of the strains A4 and CCM2595 and the reporter gfp gene were constructed. The activity of the Pami promoter was measured by means of fluorescence of gfp gene product (green fluorescent...
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases.
Čepek, Pavel ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Horníková, Lenka (referee)
Abstract Background: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Its incidence in Europe is continuously rising. The highest T1D risk is associated with HLA (human leukocyte antigen) class II genes. HLA class II molecules play a key role in regulation of immune response. They contribute to the selection of T cell repertoire by presenting antigenic peptides to the CD4+ T lymphocytes. HLA class II expression is controlled by regulatory module that is situated 150 - 300 base pairs upstream of the transcription- initiation site in all HLA class II genes. Polymorphisms in this region are linked to some autoimmune diseases. There were identified several promoter alleles (named QAP) in the HLA DQA1 gene promoter region. Most of the polymorphisms appear to be conserved within haplotype. Individual QAP alleles may have a different promoter strength by which they influence expression of HLA DQA1 gene alleles. Promoter strength can be modulated by DNA methylation. Aims:Our aim was to define methylation profile of HLA DQA1 promoters and determine the mRNA expression of individual alleles of HLA DQA1 gene in T1D patients. The mRNA expression level of HLA DQA1 gene alleles was determined using quantitative PCR. Methods: 30 diabetic pacients (age range 21 to 76 years), were included in this pilot...
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Preparation of the constructs for analysis of expression of nuclear receptor nhr-97 by using transgenic techniques in the model system Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Hudeček, Jiří (referee)
The aim of this work was to prepare two constructs of the promoter of a gene coding for nuclear hormone receptor nhr-97 in C. elegans. Nuclear receptors belong to a large group of genes sharing homologous sequences in some vertebrate nuclear receptors. The first part of the work describes the structure of nuclear hormone receptors, their function and significance in the nematodes C. elegans. The model organism C. elegans, its anatomy, life cycle and genome were also described. The work also discusses the structure and use of green fluorescent protein (GFP), which serves to localize the expression of the nhr-97 gene in C. elegans. In the practical part of the work, the preparation of two constructs of the promoter is described. Isolation of genomic DNA of C. elegans, PCR amplification of the promoters and their subsequent cloning into vector pPD95.67 containing a gene coding for green fluorescent protein were performed. To verify the successful cloning of the promoter constructs, sequencing DNA was performed. Cloned promoters of nhr-97 will be used for microinjetions to C. elegans gonads and the expression of this gene regulated from particular promoters will be subsequently monitored using expression of green fluorescent protein in progeny.
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Cis and trans elements in plant response to drought (with a particular focus on photosynthetic genes)
Panchártek, Daniel ; Holá, Dana (advisor) ; Feketová, Zuzana (referee)
Abiotic stresses, such as drought, high salinity and cold can strongly affect plant development and growth. Cis and trans elements are one of the options how plants regulate their own metabolism in those cases. That's the way how the expression of many target genes is induced. The products of these genes function not only in stress tolerance but also in general stress response. Many transcription factors and regulatory proteins (trans elements) are involved in these adaptions; those often interact with specific sequences in gene promoters (cis elements). Recenly, a progress has been made in analyzing the signal paths and complex cascades of gene expression regulation, although a little is still known about this regulation during drought conditions. Some of these target genes code products participating in photosyntesis and the regulation of their expression can significantly affect this process. This essay briefly describes main cis and trans elements of plant response to drought (with a particular focus on the regulation of photosyntetic genes). Keywords: drought, gene expression, cis-element, transcription factor, abcisic acid, photosyntesis, promoter.
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Molecular genetic and biochemical studies of selected inherited metabolic disorders, development and applications of new methods
Mušálková, Dita ; Hřebíček, Martin (advisor) ; Adam, Tomáš (referee) ; Macek, Milan (referee)
Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...
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