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Molecular mechanisms of Wip1 phosphatase function in DNA damage response
Štorchová, Radka ; Macůrek, Libor (advisor) ; Fajkus, Jiří (referee) ; Líčeníková Hořejší, Zuzana (referee)
Human cells are constantly exposed to diverse factors causing DNA lesions, which activate the DNA damage response (DDR). Depending on the severity of DNA damage, DDR can promote temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). DDR signalling is regulated by a cascade of post- translational modifications, where key mediators are represented by protein kinases ATM, ATR and DNA-PK. Wip1 phosphatase (encoded by PPM1D gene) plays an important role in DDR termination by dephosphorylation of many targets of these kinases. In this thesis, we investigated checkpoint-independent functions of PPM1D in cells and described several new substrates. We discovered, that PPM1D interacts with the shelterin complex and localizes at telomeres. PPM1D dephosphorylates the shelterin component TRF2 at S410. TRF2 S410 phosphorylation enhanced TRF2 interaction with TIN2, indirectly also increasing recruitment of TPP1 to telomeres. Importantly, cells over expressing PPM1D showed increased number of telomeric fusions. These findings might be very relevant for some cancer types, in particular those expressing high levels of PPM1D or carrying C-terminally truncated mutations in PPM1D. To validate the published substrates and to detect possible new targets of...
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Telomeric DNA sequences in beetle taxa vary with species richness
PRUŠÁKOVÁ, Daniela
(TTAGG)n sequence is an ancestral common telomere motif in insects, yet not ubiquitous, especially in beetle lineage. This study indicates that this sequence has been repeatedly lost and replaced by two known alternative telomeric motifs, the (TCAGG)n and (TTAGGG)n, or other unknown alternatives. (TTGGG)n found in one taxa has been proposed as a novel candidate sequence. However, telomeric motifs in many other beetle species remain still undiscovered. Taken together, our extensive mapping of telomeric motifs showed that the diversity is positively related to the species richness of taxa, regardless of the age of the taxa.
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Spontánní regrese rakoviny
RADOVÁ, Lenka
The main goal of this thesis is to find and gather documented cases of spontaneous cancer regression without targeted therapy. Based on the case studies, this thesis aims to categorize individual cases and explain spontaneous regression at the immunological level. According to the findings, we will attempt to evaluate whether the observed cases of cancer regression would find experimental or clinical use.
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Mapování telomerázové aktivity a telomerické sekvence u brouků
PRUŠÁKOVÁ, Daniela
(TTAGG)n sequence is an ancestral common telomere motif in insects, yet not ubiquitous, especially in beetle lineage, where the structure of telomeres varies even between representatives of the same clade. Telomerase activity related to the ancestral sequence was examined using TRAP assay, occurence of (TTAGG)n and (TCAGG)n repeats across beetles was determined using hybridization methods.
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Evolution of karyotypes and sex determination in the turtle family Geoemydidae
Clemente, Lorenzo ; Rovatsos, Michail (advisor) ; Montiel Jimenez, Eugenia Elisabet (referee) ; Castiglia, Riccardo (referee)
(IN ENGLISH) The majority of studied turtles show temperature-dependent sex determination, but genotypic sex determination (i.e. presence of sex chromosomes) was identified sporadically. This thesis aims to investigate and expand our knowledge on the evolution of the karyotype and the sex determination in turtles, particularly focusing on the family Geoemydidae, a group of turtles with previously documented variability in sex determination systems. The presence of sex chromosomes was explored by a combination of conventional and molecular cytogenetic techniques for the analysis of karyotypes, distribution of constitutive heterochromatin (C-banding) and repetitive elements and comparative genome hybridization (FISH, CGH). In total, 49 species of turtles from nine different families were cytogenetically examined in this study. In the family Geoemydidae, a remarkable similarity in karyotypes was identified, consisting of 2n=52 chromosomes (which is suggested to be the ancestral diploid number for all turtles) and a similar topology of rDNA loci and telomeric repeats. Sharma et al. (1975) previously reported ZZ/ZW sex chromosomes in Pangshura smithii. However, in the analysis presented in this thesis, it is suggested a possible misidentification of these sex chromosomes due to erroneous pairing of...
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Non-resonant Raman Spectroscopic Study of Guanine Quadruplex Structures
Golan, Martin ; Mojzeš, Peter (advisor) ; Mašek, Vlastimil (referee)
Parts of human telomere sequences containing at least 4 guanine subsequences show the ability to form intrastrand quadruplexes of remarkable conformational diversity. Former studies using conventional Raman spectroscopy have revealed that the sequence G3(TTAG3)3 at milimolar concentrations in phosphate buffer solution doped with Na+ ions (ionic strength 150 mM) adopts antiparallel conformation regardless of the length of standing at room temperature or annealing, whereas K+ ions cause gradual transition to "3+1" or even parallel conformation. On the other hand, measurements carried out upon sequence AG3(TTAG3)3 at similar concentrations using Photonic Crystal Fibre-enhanced Raman Spectroscopy (PCFRS) suggest that in the respective presence of both Na+ and K+ (ionic strength 100 mM), a parallel structure is adopted. The hereby presented work employs conventional Raman spectroscopy and Drop Coating Deposition Raman spectroscopy to examine the sequence AG3(TTAG3)3 at concentrations ranging from units to hundreds of milimoles in strands. It concludes that the structure adopted in the presence of Na+, resp. K+ ions is antiparallel, resp. "3+1", and doesn't change over time despite both long standing and annealing. Two hypotheses about the cause of the differences between the results obtained by PCFRS and...
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The function of telomeres and cell-free DNA in the healthy volunteers and patients with chosen pathological condition
Zinková, Alžběta ; Korabečná, Marie (advisor) ; Vodička, Radek (referee) ; Drábek, Jiří (referee)
More than 70 years have passed since the discovery of cell-free DNA (cfDNA), but the greatest interest in this topic and knowledge has undoubtedly occurred in the last thirty years. It is used mainly in oncology and prenatal diagnostics. While it is routinely used diagnostically in these fields, little is known about its physiological functions in the organism. Our research therefore focuses on understanding this role and in experiments works mainly with samples obtained from healthy individuals. The first study focused on the differences between plasma and serum in healthy individuals. We asked the question whether they differ in cfDNA concentration and telomeric sequences abundance. We found that the serum contains significantly more cfDNA than plasma, on the other hand, plasma is relatively richer in telomeric sequences. In stimulation experiments with THP1 cells, samples cultured with DNase-treated serum (without cfDNA) showed a higher expression of mRNA TNF-α (Tumor necrosis factor α) than samples untreated. The same trend was observed when plasma samples were stimulated. A study involving plasma samples from ten patients with celiac disease and ten healthy controls showed significant differences in mRNA TNF-α expression between experiments in which THP1 cells were stimulated by DNase-treated...
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Evolution of sex chromosomes and karyotypes in boas and pythons
Charvát, Tomáš ; Rovatsos, Michail (advisor) ; Majtánová, Zuzana (referee)
- ABSTRACT - Snakes (Serpentes) are a group of squamate reptiles (Squamata) that represents more than one third of the total reptile species diversity. Snake karyotype is generally conserved with the most common chromosome number of 36 (16 macro- and 20 microchromosomes) in diploid state. It is believed that this karyotype was also present in the common ancestor of all snakes. The majority of snake species belong to the group Caenophidia and share homologous ZW sex chromosomes. Snakes from the groups "Scolecophidia" and "Henophidia" have mostly poorly differentiated, homomorphic sex chromosomes, which made them impossible to distinguish from the autosomes in the past. These snakes were for many years assumed to have ZW sex chromosomes as well. However, recent studies demonstrated not only ZW but also two non- homologous XY sex chromosome systems in non-caenophidian snakes and thus the sex determination systems in snakes are much more variable than previously thought. In this thesis, eight species of henophidian snakes (representatives from the genera Eryx, Cylidrophis, Python and Tropidophis) and one caenophidian species (Ophiophagus hannah) were examined using conventional and molecular cytogenetic methods. However, sex chromosomes were not detected in the henophidian species, only in Ophiophagus hannah,...
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Evolution of sex chromosomes in reptiles
Mazzoleni, Sofia ; Rovatsos, Michail (advisor) ; Zrzavá, Magda (referee) ; Liehr, Thomas (referee)
- ABSTRACT - Among vertebrates, reptiles represent the ideal group for the study of sex determination. Reptiles include lineages with environmental sex determination (ESD) as seen in crocodiles and tuatara, lineages with genotypic sex determination (GSD), like e.g. iguanas, chameleons, skinks, lacertid lizards and birds, and few groups which possess variability in sex determination mechanisms, i.e. geckos, dragon lizards and turtles. This thesis is focused on the evolution of sex chromosomes and sex determination in turtles. The majority of turtle species exhibit ESD, which is considered the ancestral sex determination system of this group, while GSD either as male or female heterogamety evolved independently at least five times. We investigated the presence of sex chromosomes in representative species of turtles by cytogenetic analyses. The analyses included the reconstruction of karyotypes, distribution of constitutive heterochromatin (C-banding, methylation analysis) and repetitive elements (fluorescence in situ hybridization) and comparative genome hybridization (CGH), which often characterize the degenerated Y or W and can be helpful in the identification of "cryptic" sex chromosomes. We described XX/XY sex chromosomes in seven previously unstudied Australasian chelids (Pleurodira) from the genera...
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Cell senescence with a focus on telomerase theory
Laštůvková, Viktorie ; Tlapáková, Tereza (advisor) ; Hanus, Robert (referee)
Cell senescence is an irreversible state in which the cell cycle ends. The cell remains metabolically active, but its physiological functions are limited. Cell cycle arrest is a response to the action of a number of factors, most of which affect the secondary structure of DNA in various ways and cause damage to it. Other initiators of cell entry into the senescent state are oncogenes and chemotherapeutics. One of the best studied factors, which is also in a way the "fairest", is the shortening of the telomere. Telomeres are non-coding regions of DNA at the chromosome ends that shorten slightly with each division of the cell. If the cell does not have mechanisms to compensate for this loss, the Hayflick limit is reached after a certain time. In general, senescence can be considered as a protective mechanism to prevent DNA damage pathologies. However, the presence of senescent cells in tissues is often ambivalent, accumulates with age, and can cause many degenerative diseases. The senescent phenotype includes a wide range of signs such as changes in cell size and shape, increased granularity, or altered range of expressed genes. A significant phenomenon of senescent cells is the secretion of biologically active factors contributing to physiological or pathological changes in the organism. Key words:...
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