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Tolerance of DNA damage by novel biologically active platinum complexes
Vystrčilová, Jana ; Vrána, Oldřich (referee) ; Nováková,, Olga (advisor)
The anti-tumor activity of platinum based drugs is mediated by their ability to attack DNA. Platinum complexes can alter the structure of DNA by modifying the bases, mainly guanines. The biological consequnces of such interactions are compromising replication and transcription. RNA polymerase complex can stall at a damaged site in DNA and mark the lesion for repair by proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Main goal of this thesis was to study RNA polymerases tolerance of DNA damage by novel, biologically active platinum (II) complexes involving derivatives of aromatic cytokinines as the ligands; cis-[Pt(2-chloro-6-(4-methoxybenzylamino)-9-isopropylpurin)2Cl2](PR-001), cis-[Pt(2-chloro-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-002 )and cis-[Pt(2-(3-hydroxypropylamino)-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-005). DNA templates (constructs) that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II and bacteriophage T7 RNA polymerase were prepared. The method is making use of polymerase chain reaction (PCR) and biotin-streptavidin interactions and paramagnetic particles to purify the final product. Synthetic oligomers duplexes (75-mer, 56-mer and 15-mer) are ligated to 5´-biotin pCI-neo-G-lessT7 PCR fragment, the 15-mer is either unmodified or modified with a site-specific lesion of PR-005 and cisplatin. We also studied the inhibition of RNA polymerases activity on globally modified plasmid pCI-neo and pUC 19 by novel platinum complexes and cisplatin. We found that bifunctional adducts of complex PR-005 contrary to adducts of PR-001 and PR-002 effectively decrease amount of full lenght transcripts produced by both, human and bacterial RNA polymerases. This result can be explained by a sterical block, induced to DNA by intrastrand cross-link of PR-005 with bulky aromatic ligands.
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Topic Identification from Spoken TED-Talks
Vašš, Adam ; Ondel, Lucas Antoine Francois (referee) ; Kesiraju, Santosh (advisor)
Táto práca sa zaoberá problémom spracovania prirodzeného jazyka a následnej klasifikácie. Použité systémy boli modelované na TED-LIUM korpuse. Systém automatického spracovania jazyka bol modelovaný s použitím sady nástrojov Kaldi. Vo výsledku bol dosiahnutý WER s hodnotou 16.6\%. Problém klasifikácie textu bol adresovaný s pomocou metód na lineárnu klasifikáciu, konkrétne Multinomial Naive Bayes a Linear Support Vector Machines, kde druhá technika dosiahla vyššiu presnosť klasifikácie.
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Role of the WIP1 phosphatase in the nucleolus
Palková, Natálie ; Macůrek, Libor (advisor) ; Sztacho, Martin (referee)
Protein phosphatase 2C delta (known as WIP1) is an important negative regulator of the DNA damage response (DDR) signalling. As a chromatin-bound protein, it dephosphorylates and thus inactivates ATM kinase and the transcription factor p53. Increased expression of WIP1 suppresses the function of the tumour suppressor p53 and contributes to the development of several cancer types, including breast and brain tumours. In recent years, it has been shown that WIP1 can also regulate cellular processes that are not directly linked to DDR, such as ensuring telomere stability. However, alternative functions of the WIP1 protein have not yet been fully understood. In this work, we described a novel role of the WIP1 phosphatase in the nucleolus, the organelle responsible for ribosome biogenesis. We found that WIP1 associates with many nuclear proteins, and using deletion mutants, we identified a nucleolar localisation sequence (NoLS) at the C-terminus of the protein. Using super-resolution microscopy, we detected the localisation of WIP1 phosphatase in the fibrillar centres of the nucleolus. We employed an inducible Cas9 system for generating double-strand breaks in ribosomal DNA and found that WIP1 has an impact on recruitment of DNA repair factors to the nucleolar caps. Analysis based on quantitative...
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Where transcription meets translation
Hegrová, Karolína ; Krásný, Libor (advisor) ; Mašek, Tomáš (referee)
Transcription and translation are key steps in gene expression. The RNA polymerase (RNAP) plays a major role in the transcription process, while the ribosome is involved in translation. In bacteria, these two processes are not separated. RNAP and the ribosome interact, and its called transcription- translation coupling. In this thesis, I discuss the mechanism of transcription and translation, with the main focus on transcription-translation interactions. I divide these interactions into indirect, which are caused by regulátory molecules, and direct, where the ribosome directly binds with RNAP. When physical binding occurs, either a tight junction between these molecules occurs or a bridge is formed by transcription factors. Then I describe regulatory function of this connection and explain the exceptions where transcription and translation don't link. In the last part of the thesis, I focus on elongation factor Tu (EF-Tu), its important role in metabolism, its interactions with MreB protein, and how this factor is used by some bacteriophages. Finally, I mention its possible role in transcription-translation interactions. Key words: transcription, translation, transcription-translation coupling, RNA polymerase, ribosome, transcription factors, EF-Tu
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Roles of histone H3 lysine methylation in the gene expression regulation
Čizmazia, Viliam ; Veverka, Václav (advisor) ; Ormsby, Tereza (referee)
Viliam Čizmazia Roles of histone H3 lysine methylation in the gene expression regulation Abstract Histone post-translational modifications play a key role in epigenetic regulation of chromatin land- scape and various cellular functions. They can directly mediate interactions between DNA and histones, but also represent recognition signals for specific reader proteins. A particular type of these modifications, lysine methylation, marks a number of specific sites within the terminal as well or globular regions of histone proteins and encodes for various instructions for DNA-related processes such as gene transcription. The profiles of individual lysine methylations are regulated by specific methyltransferases (writers) and antagonizing demethylases (erasers). Their deregula- tion is often associated with diseases such as developmental abnormalities or cancer. For this rea- son, a number of histone-modifying enzymes are considered attractive therapeutic targets. This review is focused on key players and mechanisms underlying the deposition of the most important histone H3 lysine methylations, their genomic distribution and contextual roles in transcriptional events. In addition, it highlights the importance of structure-based approaches in exploring the molecular details behind the activity of histone...
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Bioorthogonal reactions on DNA for regulation of transcription
Chakrapani, Aswathi ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Vrábel, Milan (referee)
This PhD thesis describes the design and synthesis of photocaged or glucosylated derivatives of epigenetic 5-(hydroxymethyl)pyrimidine-modified nucleotides and DNA using chemical and enzymatic methods and the studies on their regulation of gene expression in bacterial (Escherichia coli RNA polymerase) in vitro transcription level. In the first part of the thesis, the design and syntheses of 5-(nitrobenzyloxymethyl)-2'-deoxyuridine (dUNB) and -cytidine (dCNB) phosphoramidites are described. These photocaged nucleoside phosphoramidite building blocks were used in the automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as forward primers in a polymerase chain reaction (PCR) to construct DNA templates modified at specific sites of the promoter region. The specific site photocaged DNA was then irradiated with light to result in the corresponding specific site 5-(hydroxymethyl)-modified DNA. Bacterial in vitro transcription studies of both the specific site photocaged and uncaged DNA were carried out. The incorporation of the photocaged epigenetic pyrimidine nucleotides at the -35 region of the promoter region of the template DNA inhibited transcription partially while the presence of the same outside the -35 region did not have any significant...
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The omega subunit of Bacillus subtilis RNA polymerase.
Mikesková, Klára ; Krásný, Libor (advisor) ; Nešvera, Jan (referee)
Transcription is catalysed by the enzyme RNA polymerase (RNAP). RNAP contains a core made up of two α subunits, one of each β, β'and ω. These subunits are conserved in all bacteria. The ω subunit is a small subunit with a molecular weight of 7.6 kDa that binds β'. ω is important for the folding and integrity of RNAP and promoter selection. This was shown by experiments performed with Gram-negative bacteria but the knowledge about in Gram-positive bacteria is minimal. In my Diploma Thesis, I characterized from the model Gram-positive bacterium from the phylum Firmicutes, Bacillus subtilis. First, I prepared various expression strains for isolation of Bacillus subtilis ω. Then, I successfully isolated the ω subunit, which was the main initial aim of this Diploma Thesis. Subsequently, I tested the influence of the ω subunit on in vitro transcription by RNAP associated with the primary σA factor and alternative σF and σE factors that regulate sporulation in Bacillus subtilis. I also evaluated the effect of , a small RNAP subunit found in Firmicutes, both alone and in combination with . The experiments revealed that ω stimulated transcription both from vegetative promoters and sporulation-related promoters. Moreover, this stimulation was synergistically amplified by the δ subunit. This nicely...
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The role of the AGO-hook domain of histone chaperone SPT6L in regulation of gene expression
Kašpar, Tomáš ; Čermák, Vojtěch (advisor) ; Převorovský, Martin (referee)
AGO-hook domains present in some eucaryotic proteins are crucial for a binding family of ARGONAUTE proteins (AGO). These AGO proteins are essential in many biological processes regulating gene expression by small RNA (sRNA), which is complementary to the gene that is supposed to be influenced. This thesis claims to find the function of the putative AGO-hook domain of the protein SPT6L. SPT6L is an elongation factor and histone chaperon of a complex of RNA polymerase II (Pol II) where it is acting in the epigenetic marking of histones. SPT6L of Arabidopsis thaliana is one of two paralogues of SPT6 proteins, that is characteristic of the presence of the AGO-hook domain. This is a plant specificity of this protein. What's more, the function of this domain remains unknown. Despite this, it could be assumed that this domain is necessary for binding of AGO proteins in the complex of Pol II, and by these interactions, it can enable guidance of regulation of chromatin modification, or it can co-transcriptionally influence nascent transcripts of Pol II by the sRNA. This thesis casts light on the function of the AGO-hook domain of SPT6L in A. thaliana in processes of gene regulation and protein interactions. It claims to confirm the interaction of the AGO-hook domain of SPT6L protein with AGO proteins and...
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Annotated translation: Old China (H. Bögeholz: Anders reisen: China, 1989)
Drašnarová, Kateřina ; Žárská, Monika (advisor) ; Drahotová, Markéta (referee)
The subject of this bachelor thesis is the annotated translation of the chapter Das alte China, which is a part of the German travel guide book Anders reisen: China. The translation was carried out on the basis of described translation analysis followed by establishment of the translation strategy. At the conclusion of the thesis the concrete translation difficulties and solutions in the area of grammar, stylistics and semantics are being discussed. A constitutive part is also a consideration of the translator's legitimacy of correcting errors in the source text.
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The delta subunit of RNA polymerase from gram positive bacteria
Matějčková, Jitka ; Krásný, Libor (advisor) ; Beranová, Jana (referee)
1 Abstrakt Aby bakteriální buňka přežila neustále se měnící podmínky, musí se na ně adaptovat. Tato adaptace je podmíněna změnou genové exprese. Klíčovým krokem genové exprese je transkripce. Hlavním enzymem bakteriální transkripce je RNA polymerasa (RNAP), což je esenciální vícepodjednotkový enzym. RNAP je nejvíce prostudována u Escherichia coli, modelového organismu gram negativních bakterií. Porovnala jsem E. coli a Bacillus subtilis (zástupce gram pozitivních bakterií) a shrnula jsem rozdíly v RNAP a transkripci. Jejich RNA polymerasy se liší přítomností podjednotky δ u gram pozitivních bakterií. Tato podjednotka zvyšuje promotorovou selektivitu, recykluje jádro RNAP a celkově stimuluje syntézu RNA. Podjednotka δ ovlivňuje sporulaci a virulenci některých bakterií. V této práci jsem shromáždila současné poznatky o jednotlivých částech genové exprese, zejména o regulaci iniciace transkripce a o podjednotce δ RNAP.
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