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Dynamics and variability of induced transgene silencing in tobacco cell line BY-2
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Lafon Placette, Clément (referee)
RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...
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The use of biochemical methods for adult age-at-death estimation compared to the traditional morphological methods
Miškovský, Jakub ; Kotěrová, Anežka (advisor) ; Šolc, Roman (referee)
The abstract The first mentions of various methods of estimating a person's age-at-death expectancy based on his skeletal remains are as old as forensic science itself. Estimation of a person's lage-at-death estimation is an integral part of his biological profile and many other data about a given individual can be derived from it. The estimate for the remains of adults is much more complex, inaccurate and less reliable than expectancy for the remains of children, as morphological changes are not as progressive as during adolescence. Classical morphological methods allow estimation only in wide age intervals. However, there are several methods based on chemical or molecular basis that can be used to estimate the age of adults. The aim of this bachelor thesis is a general summary of principles and methods based on macroscopic visual evaluation of skeletal indicators, as well as chemical, biochemical or molecular methods and their mutual comparison with an indication of their advantages, limits and disadvantages. The key words: age at death estimation, biological profile, methods of age estimation, DNA methylation, racemization of aspartic acid
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Role of the FTO gene in the genetic determination of common multifactorial diseases
Dlouhá, Dana
Obesity is a risk factor for development of cardiovascular disease, diabetes type 2 and some cancers. Newly detected genetic risk factor for body weight is the FTO gene ("fat mass and obesity associated"). The aim of this thesis was determine 1) whether the presence of risk alleles correlate with BMI in Czech population and to determine 2) whether there is an association between variants in the FTO gene and risk of myocardial infarction/ acute coronary syndrome (MI/ ACS), 3) renal failure (ESRD), or 4) incidence of colorectal cancer (CRC). We analyzed polymorphisms rs17817449 (first intron) and rs17818902 (3rd intron) using by PCR-RFLP and then also RT PCR. We found an association of the first intron variant (but not the 3rd one) and BMI in Czech control population. We have detected an association of 1st intron SNP and BMI changes during the intervention study in obese children, but not in obese females. We found a correlation between the risk allele and increased risk of ACS (OR 1.49) in patients with MI. In patients with ESRD was detected association between the risk allele and the risk of disease (OR 1.37). We didn't confirmed the association between rs17817449 and the development of CRC. Representative selected groups of the Czech populations "MONICA" and "HAPPIE" were used as controls. One...
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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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From adenoma to colorectal cancer: The study of DNA methylation profiles
Fabianová, Ivana ; Vymetálková, Veronika (advisor) ; Šmahel, Michal (referee)
Colorectal cancer (CRC) is a major public health problem worldwide and is one of the most common types of cancer in advanced countries. Recent statistics still present that the Czech Republic has a high incidence of CRC worldwide, especially in Czech men. CRC is known to be a disease that is caused not only by genetic and chromosomal abnormalities but also by epigenetic changes with the best-known DNA methylation. Changes in DNA methylation are the most prominent mechanisms that alter gene expression. Loss of gene function by epigenetic silencing of critical genes plays a key role in the development and progression of sporadic human tumors, including CRC. CRC usually develops from a harmless protrusion, known as an adenoma. However, little is known about the exact timing of DNA methylation changes in the transition from a healthy colon, through an adenoma to a malignant state. This bachelor thesis aims to summarize in detail the aberrant changes in DNA methylation in people with adenoma and in patients with CRC and at the same time to summarize the currently used methods of DNA methylation detection. Keywords: colorectal cancer, DNA methylation, adenoma, CpG island methylation phenotype, hypermethylation, hypomethylation
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Genetic and epigenetic mechanisms (and their cooperation) in the leukemogenesis of acute myeloid leukemia in adults.
Šestáková, Šárka ; Šálek, Cyril (advisor) ; Vymetálková, Veronika (referee) ; Kubričanová Žaliová, Markéta (referee)
Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by great heterogeneity and clonal nature. In recent years, rapidly evolving next-generation sequencing methods provided a deep insight into the mutational background of AML. It was shown that ~ 44 % of AML patients harbor mutations in genes that regulate DNA methylation. So far, many researchers have tried to evaluate the prognostic significance of DNA methylation changes in AML, however, due to a great inconsistency in these studies, none of the reported markers were implemented into clinical practice. The aim of this work was to further investigate the DNA methylation changes in AML patients with specific mutations and their prognostic effect. Next, we wanted to develop a new approach for a complex evaluation of prognostically significant DNA methylation aberrations. In our first project, we assessed the overall DNA methylation, hydroxymethylation, and gene expression in AML patients with mutations in either DNMT3A or IDH1/2 or their combinations. We discovered that each genetic aberration is connected with a distinct pattern of DNA hydroxy-/methylation changes that are not entirely reflected in altered gene expression. Patients with mutations in both genes exhibited a mixed DNA methylation profile most similar to healthy...
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Age estimation from human biological material based on DNA methylation
Matoušková, Laura ; Kulichová, Iva (advisor) ; Priehodová, Edita (referee)
DNA methylation is one of the epigenetics modifications which performs many functions in human body by silencing genes and maintaining genomic stability. Changes in DNA methylation accumulate during life and current studies indicate that the level of methylation in certain loci (CpG dinucletotides) correlates with chronological age. However, the methylation pattern changes not only with increasing age but it can also be affected by our lifestyle choices and biogeographical origin. Therefore, the key for succesful age estimation is finding such CpG loci that are independent (or as little dependent as possible) on other variables. This bachelor thesis describes the issue of age estimation based on DNA analysis of specific methylated DNA loci with focus on its application in forensic practice.
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Importance of epigenetic variability in evolution of clonal plants
Mareš, Štěpán ; Pinc, Jan (advisor) ; Latzel, Vít (referee)
Because of their low genetic variability and limited ability to respond to the changing environment, clonal plants are often considered an evolutionary dead end. On the other hand, numerous recent studies showed that clonal plants can react to the changing environment through epigenetic mechanisms, especially through DNA methylation. Moreover, epigenetic information in clonal plants can be transferred to future generations (so-called transgenerational memory). As a result, epigenetics can ensure better fitness of the next clonal generation. The aim of this work is to summarize the knowledge about the role of epigenetic variability in the life of clonal plants performing limited genetic variability. Key words: epigenetics, DNA methylation, epigenetics variation, clonal plants, transgeneration memory
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Molecular mechanisms of carcinogenic effects of alcohol
Vepřková, Jana ; Kábelová, Adéla (advisor) ; Novotná, Božena (referee)
Alcohol (ethanol) enters the human body mainly through ingestion of alcoholic beverages and its chronic consumption is considered a worldwide socio-economic problem. Besides others, alcohol consumption increases the risk of development of breast, liver, colorectal and upper aerodigestive tract cancer. In the liver, ethanol is metabolised into toxic acetaldehyde which is the main cause of DNA damage leading to cancer development. Acetaldehyde covalently interacts with nucleotides in the DNA forming DNA adducts such as N2 -ethylidene-2'-deoxyguanosine or S- and R-α-methyl-γ-hydroxy-1,N2 - propano-2′-deoxyguanosine. Acetaldehyde can also interact with proteins and disrupt their function. Ethanol metabolism by cytochrome P450 2E1 leads to production of reactive oxygen species, that subsequently damage cellular molecules such as lipides and DNA. Ethanol also initiates carcinogenesis through aberant DNA methylation or interference with retinoic acid metabolism. In cancer development, alcohol interacts with other environmental and genetic factors, which can increase the risk of developing cancer in predisposed individuals.
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Improved demethylation in ecological epigenetic experiments: Testing a simple and harmless foliar demethylation application
DVOŘÁKOVÁ, Hana
Spray application of 5azacytidine on established plant seedlings was tested for its demethylating efficiency. It represents a novel method for plant experimental demethylation with a potentially lower negative impact on plant development compared to the traditional application of the demethylating agent through germination of seeds in its solution. Further, the 5azacytidine spray application was used in practice to erase the epigenetic memory in offspring of Taraxacum brevicorniculatum plants from different competitive conditions. The impact of parental competition on the juvenile phenotype was estimated by measuring growth related traits, while the experimental demethylation allowed for evaluating the significance of DNA methylation marks in bioticaly induced transgenerational effects in T. brevirorniculatum.
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