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Influence of makroelements from food on DNA and epigenetic profile
Veselý, Zdeněk ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
The macroelements contained in food have an important function for the human body. They are involved in several of biochemical reactions in the body and their abundance can prevent serious diseases. The theoretical part of the bachelor thesis describes the function of minerals in the human body, the function of DNA and epigenetic mechanisms such as DNA methylation or histone modification. The influence of nutrition and function of selected macroelements – sodium, magnesium, calcium, potassium on epigenetic modifications and on the stability of G-quadruplexes was described. The aim of the experimental part of this work was to study the effect of these substances on DNA structures in vitro and to prepare them experimentally for in vivo studies.
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Polyphenols in nutritions and their effect on DNA
Osorio, Juan ; Černayová, Diana (referee) ; Brázda, Václav (advisor)
Epidemiologické studie prokázaly vliv konzumace rostlinných potravin v prevenci široké škály nemocí. Přírodní antioxidanty přítomné v těchto potravinách, mezi nimiž jsou velmi důležité polyfenoly, mohou být zodpovědné za tuto činnost podporující zdraví. Cílem bakalářské práce je ukázat interakci určitých polyfenolů s genetickým materiálem prostřednictvím různých signálních mechanismů, zejména pokud jde o stabilizaci nekanonické struktury DNA G-kvadruplex a poukázat tak na nejselektivnější látku pro inhibici biochemických procesy. Dále práce obsahuje podrobné informace, které mohou pomoci pochopit, jak mohou polyfenolové sloučeniny interagovat s DNA prostřednictvím epigenetických mechanismů a G4 struktur, a které faktory mohou ovlivnit jejich účinnost. Různé experimenty, biologickým a experimentálním opakováním, byly použity k potvrzení interakce mezi sloučeninami a DNA.
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Analyses of inverted repeats in human patogen genomes
Dobrovolná, Michaela ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
Helminth parasites are highly prevalent in humans in developing countries. According to WHO, approximately 2 billion people are infected worldwide. The etiological agents of parasitic infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms), causing inflammatory responses, malnutrition, and anemia that are the primary cause of mortality. Drug resistance is accelerated by the overuse of human anthelmintics, as well as poor infection prevention and control. The therapeutic potential of small molecule ligands binding G-quadruplexes (G4s) has been demonstrated. For instance, that it can be used to stabilize the quadruplex structures and eliminate drug-resistant pathogens. G4s are secondary structures formed in guanine-rich nucleic acid sequences, which can regulate the process of gene expression, DNA damage repair, transcription, and translation of oncogenes. Here we used the G4Hunter Web Tool to identify and compare G-quadruplex sequences (PQS) in the nuclear and mitochondrial genomes of six Platyhelminth and four Nematode species to identify targets for G4 ligands to predict new drug targets and more effective drugs. We found that PQS are nonrandomly distributed in these genomes. Most of the G-quadruplexes are in the proximity of genes, suggesting their role in genetic regulation. Interestingly, less infective Platyhelminthes were found enriched with PQS, compared to highly infective species with a lower PQS frequency. In contrast, a Nematoda, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This highly infective species can tolerate high-stability G4 structures, which are not counter-selected at all in contrast to Caenorhabditis elegans.
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Infuence of natural and synthetic G4-ligands to p53 transactivatio
Perná, Kristýna ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Secondary DNA structures, such as G-quadruplexes, occur in the promoters of human genes. Dysfunctions of these quadruplex structures have been observed in several cases of cancer, and therefore these structures are the subject of research for the design of new anticancer drugs. The p53 protein is an important regulatory protein in the process of cell cycle control and DNA repair. Mutations in the gene encoding this protein occur in more than 50 % of cancer cases. In this thesis, the influence of natural ligands binding to G-quadruplexes on the binding and activity of the p53 protein was investigated. The theoretical part of the thesis describes the structure and binding properties of p53 protein, the structure and role of G-quadruplexes and describes selected G4-ligands occurring in food - curcumin, quercetin, berberine and ellagic acid. The aim of the experimental part of this thesis was to determine the effect of these substances on the transactivation activity of the p53 protein in vivo based on their interaction with G-quadruplexes using yeast isogenic systems. The interaction between selected G-quadruplex structures and ligands was first verified in vitro using the ThT assay and circular dichroism.
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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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Sequences forming G-quadruplexes in the amyloid beta precursor human gene and its homologues
Stránská, Anna ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The APP gene encodes the transmembrane protein amyloid beta precursor, which is expressed in many cell types, including neurons. Its functions have not yet been fully described, but it is clear that it is being cleaved before being exported to the extracellular space. It is degraded by various degradation pathways also undergoes homodimerization, which can produce particles with protective neuronal function as well as fragments that are toxic and cause nerve cell death. The formation of harmful amyloid beta plaques and their accumulation among neurons in the brain is closely linked to the onset and progression of Alzheimer's disease, a neurodegenerative brain disease manifested by death and loss of neurons, which leads to dementia, i.e. loss of cognitive functions. There is currently a lot of research that deals with the links between neurodegenerative diseases and the occurrence of G-quadruplexes in genes that are involved in disease manifestations. G-quadruplexes are non-canonical DNA and RNA nucleic acid secondary structures that arise in guanine-rich regions. They are important mainly in terms of their connection with biological processes such as the regulation of gene expression in genes and mainly in oncogenes because they occur in important regions of the gene such as the promoter. It is possible to stabilize them with small molecules, and it is this ability that is used in research into the therapeutic treatment of various diseases. A bioinformatics analysis of both the human gene and 346 other gene homologs was performed to determine the importance of G-quadruplexes localization and conservation in the human APP gene. For this purpose, the G4Hunter program was used, which provided information about the found sequences with the potential to form a G-quadruplex, such as their location or G4Hunter score. In vitro analysis was performed using thioflavin T reagent, which tested the ability of the found sequences to form G-quadruplexes under physiological conditions. The results confirmed the presence and evolutionary importance of G-quadruplexes found in the APP gene of Homo sapiens and their ability to assemble into quadruplex structures in the presence of salts such as sodium and potassium.
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Optimization of G-Quadruplex Identification Algorithm
Labudová, Dominika ; Martínek, Tomáš (referee) ; Hon, Jiří (advisor)
Identification of G-quadruplexes in DNA has been the interest of many studies in recent years. As a result, many identification tools were created. This bachelor's thesis focuses on analysis and optimization of a tool called pqsfinder along with creating a web interface for this tool. The optimization provided significant increase in the algorithm's speed and ability to process long DNA sequences. The web interface was designed and implemented using modern and effective technologies with emphasis on the user friendliness of the web application.
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Presence and localization of local DNA structures in papillomavirus genomes
Vyoralová, Andrea ; Kollerová, Silvia (referee) ; Brázda, Václav (advisor)
Papillomaviruses are sexually transmitted pathogens with a genome length of about 8 kbp. The probability that an adult person will suffer from a papillomavirus infection is up to 80–90%. In most cases, the immune system will eliminate the infection. However, in women it can lead to the development of cervical cancer. That is caused by the high-risk human papillomaviruses, against which vaccination is available. Sequences rich in guanine have been found in the genomes of papillomaviruses, in these sequences the formation of G-quadruplexes occur. They are formed by stacked G-tetrads and are stabilized by monovalent cations, most often K+ and Na+. They are found e.g. in telomeres, oncogene promoters, transcription factor binding sites and recombination sites. Inverted repeats (IR) are also found in the genomes of these viruses. They consist of sequences of nucleotides followed by its reverse complement. Inverted repeats are being referred to as hotspots of genomic instability because they fold into hairpin or cruciform structures that disrupt DNA replication. G4Hunter and Palindrome analyzer were used to analyze the genomes. The analysis revealed that the presence of PQS (Potential Quadruplex-forming Sequence) is higher in papillomaviruses infecting vertebrates than in viruses infecting humans due to the higher content of guanine and cytosine which are connected to the formation of PQS. A higher frequency of PQS presence was found in the genomes of papillomaviruses than in Archaea, Bacteria and Homo sapiens. IR analysis showed that the shortest IRs (6 bases) are the mostly present in the genome and also that IRs formed of 25–30 bases are found in only a few genomes.
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Study on the relation between G-quadruplexes and p53-driven regulation
Holotová, Paulína ; Vodička, Juraj (referee) ; Brázda, Václav (advisor)
Táto práca sa zaoberá úlohou štruktúr G-kvadruplexov, ich stabilizáciou pomocou ligandov a úlohou p53 v regulácii transkripcie. G-kvadruplex je typ sekundárnej štruktúry nukleovej kyseliny zloženej z 2 – 4 tetrád. Každá tetráda je tvorená štyrmi guanínmi spojenými prostredníctvom Hoogstenovho párovania báz. Táto práca pozostáva z teoretickej a experimentálnej časti. V teoretickej časti bola opísaná podstata G-kvadruplexových štruktúr a ich potenciál pri liečbe rakoviny, úloha p53 v bunkovom cykle a jeho regulácia a ligandy kurkumín a TMPyP4 použité na stabilizáciu G4. V experimentálnej časti sa študovali interakcie ligandov kurkumínu a TMPyP4 so štruktúrami nukleových kyselín. Reportérové kmene kvasiniek obsahujúce PUMA, KSHV aich kombinácie boli transformované plazmidmi kódujúcimi iba selekčné markery a plazmidmi kódujúcimi divokú formu génu pre proteín p53. V teste životaschopnosti, optimálna koncentrácia ligandov (kurkumínu a TMPyP4) bola stanovená pre reportérový kmeň PUMA, ktorá sa ďalej použila v Luciferázovom teste. Test vytesnenia fluorescenčného indikátora s tioflavínom T ako fluorescenčným farbivom dokázal interakciu medzi oligonukleotidmi tvoriacimi G4 a kurkumínom a TMPyP4. Luciferázový test sa použil na vyhodnotenie interakcie medzi transformovanými reportérovými kmeňmi kvasiniek a oboma ligandmi. Výsledky po 24 hodinách ukázali štatistickú významnosť pre každý kmeň v rovnakom prostredí, pričom kmeň PUMA vykazoval najvyššiu transaktivačnú aktivitu - insert PUMA je cieľovým génom pre p53. Kmene s PUMA aj KSHV vykazovali významne nižšiu transaktivačnú aktivitu, keďže prítomné ligandy mohli stabilizovať štruktúru G4 prítomnú v KSHV, a tým znížiť transkripciu. Prítomnosť G4 v sekvencii KSHV sa potvrdila aj pomocou programu G4Hunter, pričom G4Skóre bolo 3,182. Transaktivácia bola výrazne nižšia aj v kmeňoch PUMA prítomných v prostredí kurkumínu a TMPyP4, aj napriek tomu, že táto sekvencia netvorí G4, čo potvrdil aj program G4Hunter. To naznačuje, že študované ligandy kurkumín a TMPyP4 môžu regulovať metabolizmus buniek iným spôsobom, ako sa doteraz predpokladalo.
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Analysis of IFI16 protein binding to DNA
Kratochvilová, Libuše ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
This diploma thesis deals with the binding of interferon gamma-inducible protein 16 (IFI16) to DNA with the potential of G-quadruplex formation. The IFI16 protein contains two tandemly located DNA-binding HIN domains showing differential binding to DNA structures. IFI16 protein has been shown to preferentially bind G-quadruplex structures over other nucleic acid secondary structures. G-quadruplexes are secondary local structures of DNA (or RNA) that are easily formed under physiological conditions in a number of important regulatory regions of the genome, or are part of the genomes of a number of viruses and pathogens. The ability to recognize, specifically bind and stabilize G-quadruplex structures explains the involvement of the IFI16 protein in the cellular processes of replication, transcription and translation and the establishment of innate immune responses. In the first part of the thesis, the sequences of synthetic oligonucleotides with the potential for G-quadruplex formation were characterized by selected biophysical methods and the full-length IFI16 protein was isolated, which was subsequently used for in vitro binding and competitive binding experiments with characterized oligonucleotides. In the last part of the work, isogenic yeast strains differing in the sequences of the responsive element were transformed with plasmid vectors for the expression of p53 and IFI16 proteins with constitutive and GAL inducible promoters, and the one-hybrid yeast system model was optimized for the study of IFI16 protein interactions in vivo. The results show that most of the analyzed sequences are able to form G-quadruplex structures in vitro, even in the presence of only one run of three or more G-bases. While the presence of several G-runs separated by a single nucleotide spacer led to the formation of intermolecular G-quadruplex structures, mutation in the original G-quadruplex sequence induced the formation of intramolecular structures with different conformations. In vitro binding and competitive binding experiments demonstrated specific binding of the IFI16 protein to G-quadruplex structures without differences in protein binding preference to a particular G-quadruplex conformation. Stabilization of G-quadruplex structures in vivo behind the transcription factor responsive element (p53) in the gene promoter induced repression of the transcription of the given gene. In the absence of any binding site of the IFI16 protein, a protein-protein interaction between the IFI16 and p53 proteins occurred, which led to an increase in the transactivation potential of the p53 protein, while the binding of the p53 protein and initiation of reporter gene transcription was influenced not only by the presence of the G-quadruplex motif and its stabilization, but and the DNA sequence adjacent to the p53 responsive element.
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