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New possibilities to distinguish monozygotic twins
Letková, Kristína ; Kulichová, Iva (advisor) ; Priehodová, Edita (referee)
The necessity of distinguishing monozygotic twins from a genetic point of view is important not only for determining paternity, when it is decided which of two identical twins is the father, but also, for example, for criminal cases, where it is necessary to identify the perpetrator who is one of the monozygotic twins. In addition to analyzing and evaluating molecular biological methods, the work also deals with monozygotic twins themselves, their emergence and the causes of their emergence. The topic affects both forensic genetics and medical fields.
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Chromatin modifications and small regulatory RNA in plant intergenerational and transgenerational stress memory: experiments in plants subjected to drought
Macek, Jan ; Holá, Dana (advisor) ; Čermák, Vojtěch (referee)
Water is important for many vital processes, and a lack of water in plants causes drought stress. In case plants survive drought, they can be better adapted for subsequent drought periods. This is defined as stress memory, which will last for a certain period after the stress ended, and can even be transmitted to offspring to help it deal with stressful environment. In such case it is called an inter- or transgenerational stress memory and is probably mediated by chromatin modifications or non-coding RNAs. In this thesis, I collected and discussed original papers dealing with the molecular basis of this phenomenon in drought-stressed plants, focusing mostly on various methodical aspects. It is evident that the maintenance of chromatin modifications across generations is quite a complex process. Currently available information is still scarce, concerns mostly DNA methylation, is incomplete and conflicting. To better understand this topic in the future, the methods of analysis of these modifications need to be adjusted (and focus also on histone modifications), the general design of the experiments has to be improved (analysed generations subjected to stress/control conditions, drought verification, number of replicates) and some factors that can affect plant stress memory should be purposefully...
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Regulation of histone post-translational modifications and its use in the diagnosis and treatment of lung cancer
Marada, Kryštof ; Janoštiak, Radoslav (advisor) ; Červená, Klára (referee)
The aim of this bachelor's thesis is to summarize the epigenetic mechanisms, namely DNA methylation, non-coding RNA and post-translational modification of histones, within the scope of which the five currently most researched types are described, i.e. acetylation, methylation, phosphorylation, ubiquitinylation and sumoylation. Furthermore, a summary of the modifying enzymes whose function is to add and remove these epigenetic components. And also the description of the proteins that recognize these components. The following is a description of how epigenetic processes are involved in the course of cancer. The remaining part of the work is devoted to lung cancer, its most common causes, what methods are currently used to treat it, and how post-translational modifications of histones could be used in its treatment. Most of this chapter is devoted to histone deacetylase inhibitors, as these inhibitors represent potential as a new component of lung cancer treatment, which also makes them the target of much research.
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The effect of indole alkaloids on selected cell lines and DNA structures
Dobrovolná, Michaela ; Hoová, Julie (referee) ; Brázda, Václav (advisor)
G-quadruplexes (G4) are secondary DNA structures formed by a cluster of guanines that have been shown to play a role in many biological functions, including regulation of replication timing and repression of oncogene expression. In this work, four plant alkaloids (harmine, harmane, harmaline, and brucine) were tested as potential G4 ligands that could regulate, induce, or convert different G4 topologies. Furthermore, their effect on fibroblast cell line HDF164 and breast cancer cell line MCF-7 was studied, in which case a dose-dependent growth inhibition mechanism was demonstrated. Interaction with the parallel G4 promoter of the proto-oncogene c-Myc was confirmed for harmine, harmaline, and brucine, and with the hybrid G4 of telomeric DNA for harmine, harman, and harmaline. In addition, the ability of the tested compounds to interact with calf thymus double-stranded DNA (ctDNA) was verified by UV-Vis absorption spectroscopy. The methylation assay showed that the DNA of MCF-7 cells treated with harmine and brucine was hypermethylated.
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Kvalita spermatu ovlivňuje oplození u sladkovodních ryb
CHENG, Yu
Short-term storage of sperm is a widely used technique during artificial fertilization in hatchery practice due to its convenient, inexpensive and practical value. Chapter 2, three egg incubation techniques were compared to supply one simple and efficiency method to incubate fertilized sterlet eggs. The percentage of cleavage, neurulation and hatching rates of embryos and larvae were examined to compare the effects of incubation systems consisting of placing Petri dishes 1) in the hatchery tank, 2) on a table in small incubation box in an air-conditioned room or 3) in a large thermostatic incubator box. No significant differences were found among these egg incubation systems. The experience gained allowed us to practice this simple method of egg incubation in Petri dishes on other fish species such as carp or zebrafish. Chapter 3, attention was paid to processes of egg activation and fertilization, which are highly important issues in fish reproductive biology. In the sterlet, multiple treatment groups with different gradient of spermatozoa number and volume of activation solution were used to fertilize eggs and further determine the minimum number of spermatozoa with optimal volume of activation water. The artificial fertilization experiments revealed that a total of 50,000 spermatozoa in 8 ml or 32 ml of activation water was sufficient for successful fertilization. It was obtained that a very low concentration of 100-1,300 spermatozoa per ml in activation water can achieve efficient fertilization with good quality sperm. Chapter 4, sperm managements including prevent sperm spontaneous movement, artificial seminal plasma composition, use of natural seminal plasma from good sperm were explored. The fertilization optimizations with different methods, spermatozoa: egg ratio and minimum sperm concentration were also examined in zebrafish. Results indicated that extender E400 was efficient during zebrafish sperm storage; a test tube with a well-defined amount of 6,000,000 zebrafish spermatozoa in E400 extender per 100 eggs and 100 ?L of activation solution has proven to be more successful than using a Petri dish. Sperm of European catfish can be collected and stored in immobilization solution for as long as 1 week for successful fertilization. Sterlet sperm with bad quality can be revitalized by incubation with seminal plasma from good sperm. The results help to establishing a sperm in vitro storage method and fertilization procedures in freshwater fishes. Chapter 5, activation solution is one of the key factors providing sperm and egg performance during fertilization. Different activation solutions were employed to test the practical application efficiency in a hatchery jar with sperm that was aged but diluted in artificial seminal plasma. The large-scale artificial fertilization trials indicated that fertilization and hatching rate were high (78-87%) after one week of sperm storage in a carp artificial seminal plasma in activation solutions as hatchery water and Perchec solution. Results offered the practical basic for the further application of aging sperm in a hatchery. Chapter 6, in this study, aged sperm in common carp was used to evaluate the sperm motility and kinematics, sperm viability, osmolality and pH of seminal plasma sperm short-term in vitro storage. DNA methylation analysis based on the whole-genome bisulfite sequencing was also performed to reveal the molecular changes of aging sperm. The results showed that aging spermatozoa in common carp has a considerable negative impact on their performance and as well as artificial fertilization success. the level of methylation at the CpG sites increased significantly with 24 hours post stripping spermatozoa compared to the fresh group, but then reduced significantly at 96 hours post stripping. The results contribute to investigate the sperm phenotype and functional changes in common carp and providing clues for understanding the molecular changes during sperm storage
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Epigenetic regulation of HLA class II genes in relation to senescence of organism
Říhová, Adéla ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Slavčev, Antonij (referee)
Introduction: Glycoproteins of the major histocompatibility complex (MHC) are an irreplaceable part of immune response regulation and immune homeostasis maintenance. The regulation of the expression plays an important role in adaptive immune response. Recently, DNA methylation in regulatory areas, crucial for DNA availability to transcription factors, is one of the most researched mechanisms of this type of regulation. The DNA methylation is, among others, related to the aging processes. Increased predisposition age-related immunosenescence in higher age could result from the changes in methylation status of regulatory areas of MHC class II genes. Aims: The aim of this thesis is to analyze the methylation status of regulatory areas of DQB1 gene and to compare the differences between generations and specific alleles. The differences in the levels of DQB1 gene mRNA transcription between generations and specific alleles is also compared. Methods: Both DNA and RNA were isolated from blood samples obtained from donors of three different age groups. DNA was genotypized and modified by bisulfite conversion. The regulatory areas of DQB1 genes were then amplified and subcloned into bacteria. The positive clones were selected and subjected to DNA methylation analysis. RNA was reverse transcribed into cDNA...
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Epigenetické regulace u autoimunitních onemocnění se zaměřením na revmatoidní artritidu
Horková, Veronika ; Daňková, Pavlína (advisor) ; Hušáková, Markéta (referee)
Exact cause of rheumatoid arthritis, as well as other autoimmune diseases has not been identified yet. In last twenty years, epigenetics showed a new face of immune system. DNA methylation, modification of histones - proteins around which DNA is wrapped, or interference of small RNA sequences - microRNAs, these all are heritable changes outside the DNA sequence that provide another component involved in autoimmunity. Presented epigenetic mechanisms alter gene expression and thus facilitate production of pro- inflammatory factors leading to autoimmune reactions. Moreover, genes regulating apoptosis are also frequently targeted by epigenetic modifications. Not only these mechanisms provide another level of immune defense, they also explain higher female susceptibility to autoimmune diseases and the influence of environment on pathogenesis of these diseases.
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Role of the FTO gene in the genetic determination of common multifactorial diseases
Dlouhá, Dana ; Hubáček, Jaroslav (advisor) ; Černá, Marie (referee) ; Rossmeislová, Lenka (referee)
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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The study of epigenetic regulation of gene HLA II. Clas within family relationships
Chmel, Martin ; Černá, Marie (advisor) ; Urbanová, Jana (referee)
Introduction: At our post-genomic era the studies of epigenetic regulation constitutes one of the tools for understanding the function of genes. Epigenetic regulation can directly control the temporal and spatial gene activity or silencing. The molecular basis of these regulations are DNA bases modifications, chromatin remodeling and RNA interference. At the same time, these mechanisms have a special way of transferring genetic information to subsequent generations called epigenetic inheritance. It has been proven epigenetic deregulation of certain genes as cause for many disease. For this reason, the study of epigenome HLA genes seems particularly important because these genes play a fundamental role in regulating the immune system. Aims: The aim of this work is to create a description of epigenetic modifications within families. It is an analysis of histone modifications and DNA methylation in the promoter region of the gene HLA DQA1. The aim was also to compare the differences in epigenetic modifications between alleles and compared the differences in these modifications between generations. The results will be compared with the analysis of the level of expression of the gene HLA DQA1. Methods: From collected peripheral blood of donors were isolated DNA, RNA, and leukocytes. DNA was used for...
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