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Molecular composition of constitutive heterochromatin
Pajpach, Filip ; Král, Jiří (advisor) ; Holá, Dana (referee)
Constitutive heterochromatin of eukaryotes includes various types of repetitive DNA and transposons characteristic for given region. DNA of centromeric and telomeric re- gions is usually highly methylated and transcribed to RNA transcripts, which participate in formation, functions and spreading of heterochromatin along with histones, their mod- ifications and non-histone proteins. The most typical histone modification in heterochro- matin is methylation, which forms the binding site for protein HP1. This protein (and his paralogues in other eukaryotes except for S. cerevisiae) participates in formation of com- plexes including other proteins like histone methylases SUV39H and their paralogues. Es- sential are also telosome proteins regulating telomeric heterochromatin, Polycomb group proteins and many others, for instance MBD1, Epe1, SUMO and DNA methylases DNMT. Many proteins form complexes, which partake in mechanisms necessary for heterochro- matin maintenance, for example RDRC and RITS complexes in RNA interference, SHREC complex in heterochromatin spreading, and PRC complexes forming heterochromatin in specific situations. Key words: centromere, DNA, histone, HP1, constitutive heterochromatin, methylation, modification, protein, RNA, specific, telomere
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The use virus derived vectors for the expression of proteins in plants
Kratochvílová, Kateřina ; Moravec, Tomáš (advisor) ; Holá, Dana (referee)
The tobacco mosaic virus (TMV) is one of the most studied virus. Its properties (simplicity, self assembly of viral capsid, the ability to proliferate in a large variety of hosts) offers a lot of applications in various scientific or biological fields. Gene vectors based on the tobacco mosaic virus represent a very good way for expressing proteins in plants. Because the tobacco mosaic virus is well researched and a large number of his properties have already been discovered, it is used for nanotechnologic sectors in this period. Not only for the formation of nanoparticles, but also for example for a potential production of vaccines in plants. In this paper I summarize the known information about the process of self assembly of tobacco mosaic virus and the possibility of using this virus in the emerging fields of biology. Key words Tobacco mosaic virus, self assembly, viral vector, nanotechnology
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Chromatin modifications in association with priming phenomenon in plants
Marková, Hana ; Holá, Dana (advisor) ; Fischer, Lukáš (referee)
Plants are exposed to many stressors to which they must be able to adapt. It has been found that plants can memorize the respective stress response and may respond to a subsequent stress exposure faster and to a greater extent. The mechanism of this so-called priming could be associated with the changes in the levels of chromatin modifications. Chromatin modifications are usually dynamically changing during the stress reaction. Some of these changes could persist for some time, thus the specific stress memory, mitotically and meiotically transmissible, could be established. Such a phenomenon was observed for stress caused by dehydration, salinity, mechanical damage, a combination of various stressors and a systemic resistance to pathogenic bacteria. So far, only a few studies on this topic exist; but even now it is clear that there are differences in the response of specific genes as well as the dependence on the length of the stress stimulus and the duration of the period between the first and second stress. A major disadvantage of existing studies is that they focus solely on histone modifications. Regarding the chromatin modifications studied so far, H3K4me3 could serve as the main mark for such priming. On the other hand, H3K27me3 modification is apparently not used as a memory tag. However, it is still...
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Epigenetic regulation of DQB1 gene in patients with type 1 diabetes mellitus
Gécová, Dominika ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Holá, Dana (referee)
Background: Type 1 diabetes mellitus is a multifactorial disease caused by beta cell destruction of Langerhans pancreatic islets. From the genetic aspect the main predisposition lays on HLA class II genes (40 - 50%), molecules of which present exogenous peptides to CD4+ T lymphocytes. Enviromental factors play a crucial role in the etiopathogenesis of T1DM. Through epigenetic regulation (e.g. DNA methylation) the genetic and enviromental factors communicate. The level of methylation in the regulatory regions can significantly affect expression of these genes. Aims: The aim of the diploma thesis was to define methylation profile of HLA DQB1 alleles in type 1 diabetes mellitus patients and determine their expression. Methods: The genotyping of HLA class II genes (HLA-DRB1, HLA-DQA1, HLA-DQB1) was performed using sequence specific primers. DNA was treated with sodium bisulfite, regulatory region of HLA DQB1 was amplified and cloned into E.coli, strain DH5α/XL1-Blue. Positive clones were sent for sequencing and results analyzed. RNA was transcribed to cDNA by reverse transcription and the level of expression was analyzed by quantitative PCR. Results: Statistically significant differences were found in total methylation of DQB1*0201 and *0302 alleles in the B section of DQB1 gene. Difference in...
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Genetic variability in growth, reproductive and photosynthetic parameters of plants and its changes by exogenously applied steroids
Rothová, Olga ; Holá, Dana (advisor) ; Motyka, Václav (referee) ; Hradilík, Jan (referee)
While animal steroid hormones are very well known and have been studied for a long time, in plants no steroid substances were known until relatively recently. Only in the second half of the past century brassinosteroids were discovered; later on, their hormonal function in plants was confirmed. Still a lot of unknown remains as regards their function in plant cells. This paper presents in its first part the evidence that brassinosteroids control in maize (Zea mays L.) grown under field conditions not only its morphology and yield but also some developmental/reproduction characteristics like e.g. number of female inflorescences or speed of the development of male inflorescences. Particular response of a plant depends, however, on the type of applied brassinosteroid, its concentration, and last but not least also on a particular maize genotype and developmental stage of the plant during applicatin. Impact of brassinosteroids on primary photosynthetic processes in plants has not been proven under these conditions, neither on the activity of photosystem (PS) I nor on the Hill reaction. No statistically significant differences in the content of photosynthetic pigments have been found either. Another topic dealt with in this thesis is the possible protective influence of brassinosteroids on plants...
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Different response of maize inbred and hybrid genotypes to drought: analysis of leaf proteome and photosynthetic processes
Benešová, Monika ; Holá, Dana (advisor) ; Prášil, Ilja (referee) ; Pospíšilová, Jana (referee)
Drought is one of the most important stress factors affecting plants. Increasing drought resistance via plant breeding is currently the main approach for improvement of plant productivity and reduction of water usage. Plant breeders and scientists search for reliable and easy methods of the determination of drought sensitivity in different genotypes. A precise knowledge about processes underlying plant stress response and defence against limited water availability is necessary for this. The aim of this study was a detailed analysis of photosynthetic processes and changes in leaf proteome (again aimed especially at proteins involved in photosynthesis) during drought stress. This analysis was a part of a complex project focused on the possible causes of genetic variability and drought resistance in agronomically important crops. Another goal was to analyse whether the measurement of selected photosynthetic parameters can be used for the determination of drought sensitivity per se and/or for the prediction of the response of hybrids based on known behaviour of their parents. Young plants of maize inbred lines and their hybrids were used as a model for this analysis, which was made under conditions of moderate and more severe drought. Two inbred lines which were (together with their F1 hybrids) used...
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Targeted next generation sequencing of candidate genes responsible for impaired spermatogenesis and male infertility
Daňková, Michaela ; Liška, František (advisor) ; Holá, Dana (referee)
Infertility is a widespread health problem, caused by the male factor in about half of all cases, and in about a half of the infertile men the cause is unknown. In a significant number of these men, genetic etiology is assumed. Current routine methods of laboratory diagnostics, which include karyotype examination, exclusion of mutations in the CFTR gene, and Y chromosome microdeletions, do not usually reveal the cause of infertility. That is why researchers' efforts aim at detecting mutations in other genes that are causing male infertility. In recent years, animal models have been used to identify many genes necessary for fertility. Based on these findings, 12 candidate genes have been selected (CAPZA3, CDC14B, CDC42, CNTROB, CSNK2A2, GOPC, HOOK1, HRB, OAZ3, ODF1, RIMBP3, SPATA16) that are essential for spermatogenesis. Mouse or rat mutants in these genes are primarily associated with oligoasthenoteratozoospermia, since they are involved in sperm morphogenesis. However, the phenotype spectrum may comprise also azoospermia. The purpose of the thesis was to determine the sequence of the afore mentioned genes in infertile men with impaired spermatogenesis and to reveal presence or absence of pathogenic mutations in these genes, using cDNA and genomic DNA from peripheral blood. The candidate genes were...
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Self-regulating mechanisms of photosynthetic systems
Semerák, Matěj ; Mančal, Tomáš (advisor) ; Holá, Dana (referee)
Photosynthesis, a process utilising energy of arriving photons for driving electron transport chain creating transmembrane pH gradient, is a widespread way of subsistence in the nature. However, the intensity of sunlight can exceed the rate which the organisms are able to manage by their gadgetry. In this situation, essential protective mechanisms, safely draining the excess energy away, take a turn. Many theories about the principle and regulation of these functions have been developed and new still arise. It appears that the attention focuses mainly on the antenna complex LHCII. It is possible to state that with high probability, the protective processes are assured by several mechanisms, and quite a stable opinion prevails that crucial role in their activation is played by decreasing pH. That is logical since the more photons come, the more intensively the transport of protons across the membrane happens, thus ΔpH reflects the balance between ATP usage and the membrane apparatus illumination. Generally, the phenomenon is called NPQ (non-photochemical quenching), because it weakens the chlorophyll fluorescence. An important task is probably handled by carotenoids, mainly zeaxanthin, created by violaxanthin deepoxidase at low pH; it provides chlorophylls with energetical trap during excitation...
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Photosystem I: from bacteria to green plants
Palovská, Markéta ; Holá, Dana (advisor) ; Matyska Lišková, Petra (referee)
Type I Photosystems (PS) are characterized by a Fe-S cluster that serves as the terminal electron acceptor. They are present in 4 prokaryotic groups and all photosynthetic eukaryotes. This work summarizes the knowledge on the structure and function of PS I subunits. The simplest PS (homodimeric; only 2 subunits) is present in Heliobacteria. PS of green sulphur bacteria is also homodimeric and consists of 4 proteins including a membrane-bound cytochrome. The homodimeric PS of Candidatus Chloracidobacterium thermophilum is functional in aerobic conditions. The reaction center of the well-characterized PS I of Cyanobacteria has a heterodimeric structure; the same applies for the eukaryotic PS I. Cyanobacterial PS consists of 12 proteins and forms trimers under low light conditions. The trimer formation is completely suppressed in green plants. Their PS I (at least 15 proteins) is adapted to bind light-harvesting membrane proteins. The PS I of Glaukophyta and Rhodophyta is slightly different from green plants. Various hypotheses about the evolutionary origin of PS I exist. The last common ancestor of all PSs probably shared features of both Type I and Type II. The purpose and the origin of a PS I heterodimer is also discussed in this work. A further understanding of PS I evolutionary history would be helped by...
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Expression of candidate genes for prostate cancer
Krupicová, Daniela ; Mareš, Jaroslav (advisor) ; Holá, Dana (referee)
4 Abstract Prostate cancer is one of the major medical problems within the male population in the Czech Republic and in the world. It is on second place among cancer illnesses with respect to mortality in czech male population. Its incidence strongly increases with age. Prostate cells have a unique ability to accumulate zinc in high concentrations compared to other tissues of human body. It is necessary for the proper physiological function of the prostate. There was detected loss of this accumulation ability in prostate cancer cells, which seems to be a condition to carcinogenesis in prostate cells. In this thesis was investigated the expression of four genes involved in the maintenance of homeostasis of zinc in prostate cells. Genes ZIP1 and ZIP7 encode zinc transporters, genes MT1-F and MT2 encode metallothioneins. There was collected 90 biopsy specimens from patients with prostate cancer or with benign prostatic hyperplasia. mRNA was isolated from these samples, cDNA was obtained by RT-PCR. This cDNA was detected by gel electrophoresis and the results were statistically evaluated. Several correlations was found between gene expression and the clinical data of patients. The most important result, there was found lower levels of expression of genes MT1- F and ZIP1 in samples of patients with cancer...
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