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DNA isolation and identification of nonpathogenic species of clostridia isolated from cheeses
Sedláček, Zbyněk ; Eva, Kvasničková (referee) ; Rittich, Bohuslav (advisor)
In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).
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Hydrogen production by bacteria of genus Clostridium
Sedláček, Zbyněk ; Turková, Kristýna (referee) ; Rittich, Bohuslav (advisor)
The bachelor thesis deals hydrogen production by bacteria of the genus Clostridium. Thesis gives an overview about hydrogen production mentioned microoganism, characterized further biotechnological methods and show examples of industrial production of hydrogen. Also there are describes the quantitative characteristic and metabolism of bacteria genus Clostridium and examples of usable substrates. Polymerase chain reaction (PCR) is used to the detection and identification by species and specific primers. DNA isolated by fenol extraction from bacterial culture Clostridium tyrobutyricum DSM 2637 was amplified using genus-specific PCR to form PCR products size 619 bp.
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SStudy of crystalline structure of polyhydroxybutyrate and nucleating activity of selected additives
Sedláček, Zbyněk ; Tocháček, Jiří (referee) ; Bálková, Radka (advisor)
This diploma thesis deals with study of crystalline structure of polyhydroxybutyrate (PHB), which contains different types of additives for studying of their nucleation activity and which were prepared by mixing. It is about boronitrid (BN), sacharin, hydroxapatit, plasticizer Tegmer a tree types of talc. Crystal structure was analysed by differential scanning calorimetry and x-ray diffraction, supramolecular structure was observed by optical microscopy (polarized and confocal laser scanning). Nucleating activity was evaluated by isothermal and non-isothermal crystallization made on calorimeter and heated table of optical microscope. There is not influence of additives on crystallographic structure, but additives affects number and size of spherulites including crystal domains defects, which can have impact on final mechanical properties. BN and talcs react as nucleating agents, other additives during low and high cooling speeds (vc) inhibit nucleation and in middle cooling speeds are without effect. Nucleating activity is not evaluated by numerically, because decrease of crystallization temperature together with vc is not linear. Results of direct methods are based on picture analysis, which is great benefit for understanding of crystal behaviour of PHB.
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Application of novel genomic techniques in studies of pathogenesis of selected rare inherited disorders
Nosková, Lenka ; Kmoch, Stanislav (advisor) ; Sedláček, Zdeněk (referee) ; Živný, Jan (referee)
Rare diseases are a heterogeneous group of disorders. Knowledge of their molecular basis is poor and till recently there were no appropriate methodical approaches due to a limited number of patients. Novel genomic techniques, especially the DNA array technology and the next generation sequencing emerging in last few years, enabled studies of these diseases even in small families and sporadic cases. This PhD thesis focuses on application of novel genomic techniques in studies of rare inherited diseases. It describes a use of DNA array technology in linkage analysis, analysis of differential gene expression, analysis of copy number variations and homozygous mapping, and a use of next generation sequencing technology. Combination of these methods was used for identification of molecular basis of adult neuronal ceroid lipofuscinosis, Rotor syndrome, isolated defect of ATP synthase and mucopolysaccharidosis type IIIC.
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Association study with the ADA gene and detailed characterization of two chromosome aberrations in autism
Vážna, Alžběta ; Sedláček, Zdeněk (advisor) ; Ferák, Vladimír (referee) ; Korabečná, Marie (referee)
Autism affects up to 1/150 children and represents therefore a serious social problem. It is a complex disorder with a clearly documented genetic component, but so far unexplained aetiology, which is currently a subject of intensive research. In the field of genetics various gene and chromosome defects are examined, as well as other mechanisms, including epigenetics, which could play a role in pathogenesis. In our work we tried to replicate the finding of association between the ADA*2 risk allele and autism in a sample of 385 Czech children. Our sample was larger than those originally published. We also focused on individual endophenotypes (types of autism, degree of mental retardation and co-morbidity). Our results did not confirm the association of autism with the ADA*2 allele in the complete sample or in any of the subsets. Chromosomal changes represent another finding in autistic patients. We performed the analysis of a ring chromosome 17 and a chromosome X deletion in two patients. Our studies represent an up to now unimaginable link between classical cytogenetics and molecular genetics at the DNA sequence level. As the first in the world, we described the structure of a human ring chromosome. Characterization of the defects allowed us to speculate on the impact of the genes involved in the phenotype...
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Genotype-phenotype correlation in selected rare disorders using molecular analysis of genome and gene variants
Vlčková, Markéta ; Sedláček, Zdeněk (advisor) ; Gaillyová, Renata (referee) ; Baxová, Alice (referee)
The work was focused on detailed analysis of patients with rare genomic and gene variants. We studied the impact of these variants on the phenotype of the patients. As the majority of our patients, both syndromic and non-syndromic, were reffered to the detailed analysis due to intellectual disability and/or autism spectrum disorder, the work was focused on these two clinical diagnoses. At the beginning we analyzed patients with aberrations detected using cytogenetic analysis, and the extent, gene content and mechanism of origin of the aberrations were refined using molecular genetic methods, most often high-resolution array CGH. Later we analyzed patients with rare or unique submicroscopic aberrations detected using aCGH or SNP array. Using these methodes we analysed in the project patients with deletions of Xp22.1-p22.3, 6q11-q13, 6q14-q16, Xq25, 1q21.1, Xp21.2-p21.3, 2p14-p15, 17q21.31, 9q21.3 a 2p15- p16.1, and a patient with an Xp21.2-p21.3 duplication. In the last years we proceeded to the analysis of syndromic cases using next generation sequencing. This led to the identification of point mutations in the HCFC1, KAT6B, SOS2 and KMT2D genes, which were further studied. The work contributed to the knowledge about the impact of the genome and gene variants identified on the phenotype of the...
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The alterations of MLL (mixed-lineage leukemia) gene and their clinical importance in pediatric patients with acute leukemia
Řezníčková, Leona ; Trka, Jan (advisor) ; Sedláček, Zdeněk (referee)
Presence of the MLL gene rearrangement at 11q23 is an important prognostic feature. Moreover, the rearrangements represent a suitable target for the minimal residual disease (MRD) monitoring in some subtypes of childhood acute leukaemias (AL). Currently more than 80 different translocations involving the MLL gene and more than 50 of those are characterised at the molecular leve\. Using multiplex-reverse transcriptase polymerase chain reaction (multiplex RT -PCR) and - in some cases - its combination with DNA analysis of the translocation breakpoint we examined a group of infants «1 year of age) diagnosed with acute lymphoblastic leukaemia (ALL), children with M4 and M5 subtypes of acute myeloid leukaemia (AML) and patients with secondary leukaemias. Moreover, we exaITŮned children with B-cell precursor fulfilling at least one of the ťollowing criteria: proB immunophenotype, cytogenetically confirmed MLL rearrangement and/or expression oťNG2 molecule shown by f10w cytometry. MultiplexRT PCR technique enables fast detection of the most frequent fusion partners of the :MLL gene (AF4, AF6, AF9, AFlO, ENL a ELL). We screened almost 80 patients diagnosed and treated in the Czech Republic between 1997 and 2007 and we found an MLL-fusion gene in 51 of them. Vast majority of rearrangements (92%) was detected by the...
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Analysis of copy number variant (CNV) in genomes of patiens with mental retardation
Hančárová, Miroslava ; Sedláček, Zdeněk (advisor) ; Gaillyová, Renata (referee) ; Michalová, Kyra (referee)
Mental retardation (MR) is a very heterogeneous common neurodevelopmental disorder with a population prevalence of 2.5-3 %. The importance of genetic factors in the development of MR is high but in a significant number of cases the etiology remains unexplained. Recent studies using array methods pointed to frequent occurrence of copy number variants (CNVs) in patients with MR. Pathogenic CNVs were identified in 10-15 % patients with idiopathic MR and normal karyotype. The aim of our work was the analysis of genome-wide gains and losses of genetic material in a group of Czech patients with MR and a thorough bioinformatic analysis of the genetic changes identified aiming at the assessment of their clinical significance. We performed whole genome analysis using the HumanCytoSNP-12 BeadChips (Illumina) in 183 patients with idiopathic MR, normal karyotype and no FMR1 gene expansion. Data analysis was carried out using two independent programmes, GenomeStudio and QuantiSNP. The findings were subjected to two rounds of thorough bioinformatic analysis. Based on this analysis we classified the CNVs into 4 categories: pathogenic CNVs, probably pathogenic CNVs, CNVs with uncertain clinical significance and benign CNVs. With the exception of the benign variants, all CNVs were confirmed using an independent laboratory...
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Molecular Basis of Familial Hyperuricemic Nephropathies
Živná, Martina ; Kmoch, Stanislav (advisor) ; Jirsa, Milan (referee) ; Sedláček, Zdeněk (referee)
In 1960 Duncan and Dixon described family whth chronic tubulointerstitial kidney disease associated with juvenile onset of hyperuricemia and gout. Based on combination of these clinical symptoms they named the disease familial juvenile hyperuricemic nephropathy (FJHN) [1]. Disease with very similar clinical presentation but different age of onset and kidney histology was described as a medullary cystic kidney disease (MCKD) in 1977 [2]. Until recently the molecular basis and pathogenesis of this syndrome remained unknown. The long term aim of our research group is to elucidate the genetic basis of the disease and to solve pathogenetic mechanisms leading to the individual clinical and biochemical symptoms (e.g. hyperuricemia) and kidney damage in general. We systematically identify patients with this disease and healthy family members and collect relevant clinical information and samples for classification (urine, blood, tissue biopsies) and subsequent clinical, biochemical, molecular biology and cell pathology correlations. We [3, 4] and others [5-7] proved genetic heterogeneity of FJHN and defined four FJHN loci on chromosomes 1q21, 1q41, 16p11.2. and 17q21.3. Further research defined disease causing mutations in three genes - uromodulin (UMOD) [8], hepatonuclear factor 1-beta (HNF-1) [9] and renin (REN)...
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