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Development of methods for determination of authenticity of cosmetic products with a plant component
Langová, Denisa ; Brázda, Václav (referee) ; Vorlová, Lenka (referee) ; Márová, Ivana (advisor)
In the final thesis, methods were developed for determining the authenticity of cosmetic products and food products with a plant component. The presence of natural ingredients in a series of model and commercial cosmetic products was verified and their authenticity was verified, too. The focus of the work was the development of a molecular-biological method for the detection of plant components (herbs, fruits, essential oils) and probiotics present in cosmetic products. Commercially available isolation kits were used for the isolation of DNA from cosmetic products and the method was optimized for the isolation of plant DNA present in cosmetic products. The presence of a plant matrix in cosmetic products was detected by the polymerase chain reaction (PCR) method. Instrumental methods were used to detect the presence of fragrance allergens in cosmetic products. A method of quantifying these allergens using liquid chromatography was introduced and the measurement results were compared with the results of gas chromatography with mass detection. The proposed method of isolation and subsequent amplification of plant DNA is suitable for determining the authenticity of herbal ingredients in cosmetic products. The proposed HPLC method is a simple and inexpensive method suitable for determining the allergens present in the amount necessary to comply with EU analyte legislation and in the required sensitivity (except for hydroxycitronellal). Both methods are suitable to be used for the analysis of food products, too.
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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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The presence and localization of local DNA structures in the genome of Schizosaccharomyces pombe
Kubínová, Michaela ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The thesis focuses on the study of local DNA structures (cruciforms and G quadruplexforming sequence) in the genome of Schizosaccharomyces pombe, a yeast used in the food industry. The analysed local structures are non-randomly distributed within the genome. Based on previous studies, it has been found that they often colocalize with regulatory regions of genes and that the role of these secondary structures in the regulation of basic cellular processes (e.g. replication or transcription) is significant. This analysis was performed using specialized bioinformatics tools (G4Hunter and Palindrome Analyser) that allowed me to identify and analyze these structures in terms of their presence and localization. Many times less IR was found in mtDNA compared to the occurrence of IR in chromosomes. The number and frequency of PQS in mtDNA was also found to be very low. It is very different from the yeast Saccharomyces cerevisiae in this respect. It was also found that the number of IRs found decreases with increasing IR length and about 17% of IRs do not have a loop. A large enrichment of IRs was observed in the repeat_region and rRNA, and in the case of PQS in the rRNA and mRNA regions, i.e. sequences important for cellular processes.
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Interaction of selected natural substances used in food industry with DNA and its structural motifs
Gardošová, Zuzana ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
G-quadruplexes represent secondary DNA structures formed in guanine-rich nucleic acid regions. These structures are involved in many biological processes, including DNA replication, transcription, and telomere maintenance. Several natural substances interacting with G-quadruplex structures have been described. Many of them can be used in the treatment of cancer or other areas of therapeutic practice. G-quadruplexes are dynamic structures whose stability can be affected by a variety of different factors, including chemical modifications to DNA. One of these modifications is DNA methylation, which is an important epigenetic mechanism regulating gene expression. DNA methylation can affect the function and stability of G-quadruplex structures. The theoretical part of the present work focuses on DNA secondary structures, characterization of G-quadruplexes and their ligands and describes the relationship between DNA methylation and G-quadruplex structures. In the experimental part, the binding ability of the natural substances quercetin, berberine, piperine, and caffeine to G-quadruplex structures formed in telomeric oligonucleotide sequences and sequences derived from the proto- oncogene c-Myc was confirmed. Furthermore, the ability of berberine and quercetin was proven to stabilize G-quadruplexes in the aforementioned sequences. Bioinformatics analysis showed that the frequency of G4 is higher in CpG regions than in their surroundings, and the highest frequency of G4 within CpG regions was observed on chromosome 19. Global methylation assays demonstrated that the breast cancer cell line exhibited hypomethylation compared to the non-tumor human dermal fibroblast cell line. After treatment with berberine, the analyzed DNA of both cell lines showed hypermethylation, whereas DNA after interaction with quercetin showed hypomethylation.
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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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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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Development of methods for genetic analysis of plant foods
Fialová, Lenka ; Brázda, Václav (referee) ; Doškař, Jiří (referee) ; Márová, Ivana (advisor)
Multiplex real-time PCR-HRM is an approach which has gained some attention in recent years. It has already found applications in clinical diagnostics and food authenticity and safety control. Compared to its corresponding singleplex PCR assays, an optimized multiplex PCR assay provides the same information in a fraction of time. First part of this work dealt with isolation of DNA from both fresh fruits and processed commercial products. Six different DNA isolation protocols were tested with fresh fruits – three silica column-based kits, two magnetic carrier-based kits and one conventional protocol. One method was chosen as the most suitable and was applied to DNA isolation from commercial products. These experiments also involved optimisation of the chosen method. The second part of this work was focused on the development of a triplex real-time PCR assay for simultaneous detection of blueberry, strawberry and raspberry, and its application on DNA isolated from commercial products. During DNA isolation, calcium chloride was shown to be a promising agent for removal of pectin from samples. In several samples, presence of raspberry DNA was confirmed by singleplex PCR. We found out that for accurate results of food analysis by this assay, further optimization of its parameters would be needed.
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Eucaryotic cells systems and their biotechnologycal applications
Porubiaková, Otília ; Obruča, Stanislav (referee) ; Vorlíčková, Michaela (referee) ; Brázda, Václav (advisor)
The submitted dissertation is divided into several parts. The first part deals with the interactions of the p53 protein and its isoforms with different potential DNA substrates under different experimental conditions. These are predominantly DNA and its non-canonical structural motifs, such as G-quadruplexes or cruciform structures, whose interactions have been studied in yeast isogenic systems or by in vitro methods. The seconds part deals with the bioinformatic analysis of the mentioned secondary DNA structures in different organismal groups, and the result is a set of publications that show their non-random distribution in the genome and their relationship with regulation. The last part of the work contains unpublished results, including the results of testing the effect of natural and synthetic substances on aging in model human cells.
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