During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

Over the course of an organism's life, its genome is exposed to endogenous and exogenous chemical, physical and biological agents - genotoxins. These genotoxins alter its basic structural components - sugar residues, phosphodiester bonds, and nitrogenous bases. Organisms have therefore evolved a plethora of different strategies to both repair DNA lesions and maintain genomic stability. These DNA repair pathways are linked with several other cell pathways, including chromatin remodelling, DNA replication, transcription, cell cycle control, apoptosis - programmed cell death (PCD), thereby providing a coordinated cellular response to DNA damage. Biochemical mechanisms of DNA repair are relatively well understood in yeast and mammals, however, far less so in plants. While these repair mechanisms are evolutionary conserved, significant differences still remain. Therefore, further investigation is required. This thesis summarises the introduction of a novel plant model - the moss, Physcomitrella patens (Physcomitrella). As a haploid gametophyte with unique characteristics of high frequency of homologous recombination (HR), and apical growth of filaments, it is an ideal organism to study DNA repair in plants. Previous research on Physcomitrella regarding mechanisms of DNA lesion repair induced by...

This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.

We studied how distribution of nuclear-spin states of H+ 3 ions or H2 molecules influence rate coefficients of H+ 3 -electron recombination or reactions of H2 with N+ or H+ , with regard to kinetic and internal temperatures of the reactants. Experiments were carried out in plasma environment or in an ensemble of ions in an ion trap. Main diagnostic methods were the Langmuir probe diagnos- tics, laser absorption- and mass spectroscopy. The distribution of nuclear spin states (para and ortho) was varied using a specially constructed para-hydrogen generator. We performed pioneer measurements of the rate coefficients for the nuclear-spin-state-selective binary and ternary H+ 3 -electron recombination in thermalised plasma. We performed studies of N+ + para/ortho-H2 reaction with high accuracy and interpreted the results as dependent on fine structure states of N+ ions. We measured a temperature dependence of the rate coeffi- cients for radiative and ternary channels of H+ + para/ortho-H2 association. 1

A two-dimensional model of the resonant electron-molecule collision processes with one nuclear and one electronic degree of freedom introduced by Houfek, Rescigno and McCurdy [Phys. Rev. A 73, 032721 (2006)] and a similar two- dimensional model of the dissociative recombination with potential proposed by Hamilton [Ph.D. thesis, University of Colorado, (2003)] are formulated within the time-dependent framework and solved numerically using the finite-element method with the discrete variable representation basis, the exterior complex scaling method and the generalized Crank-Nicolson method. On the model of electron-molecule collisions we illustrate how the time-dependent calculations can provide a deep insight into the origin of oscillatory structures in the vibrational excitation cross sections if one evaluates the cross sections not only at sufficiently large time to obtain the final cross sections, but rather at several characteristic times which are given by the evolution of the system. With use of the time- dependent calculations we demonstrate the complex nature of the dissociative recombination model dynamics and we propose the interpretation of the recom- bination process mechanism. We also propose few techniques for the explanation of the sharp structures in the dissociative recombination cross sections...

(English) Meiotic homologous recombination, homologous chromosomes synapsis, and F1 hybrid sterility (enabling formation of species) are mutually interconnected phenomenons, one being the prerequisite to the latter. In the present thesis, these phenomenons were investigated on a genetic and mechanistic level using a mouse subspecies as a model. Noncrossovers (NCOs, gene conversions), 90% prevalent resolution of Prdm9- determined meiotic double-strand breaks (DSBs), were uniquely identified and characterized on a chromosome-wide level. The mean gene conversion tract length, based on 94 NCOs events, was calculated to be 32 bp. On a local level, the NCOs overlapped the known hotspots of PRDM9-controlled histone trimethylation and DSB formation, indicating their origin in the standard meiotic DSB repair pathway. On chromosome-wide level, NCO and CO distributions differed, in particular COs being relatively preferred over NCOs in subtelomeric regions. A specific subset of nonparental/asymmetric NCOs and COs was underrepresented in our datasets, proposing their problematic repair, hypothetically enabled by sister chromatids, and thus not contributing to indispensable homologous synapsis. Genome-wide crossover (CO) rates, genetically and mechanistically crucial ~10% of DSB repair, were proven to be...

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

The purpose of this work and the project under which it was created is to develop, compare and validate several theoretical approaches and computation methods used to calculate the cross sections of dissociative recombination. For the most part it is con- cerned with the indirect dissociative recombination of molecular ions of H+ 2 in the singlet ungerade channels computed with three distinct approaches. First, the fully numerically solvable two-dimensional approach developed at ÚTF MFF UK as a part of my master's thesis. Second, a vibrational frame transformation method based on the work of Chang and Fano [E. S. Chang and U. Fano, Phys. Rev. A 6, 173 (1972)] and then enhanced into a full energy-dependent form by Gao and Greene [H. Gao and C. H. Greene, J. Chem. Phys. 91, 3988 (1989)], [H. Gao and C. H. Greene, Phys. Rev. A 42, 6946 (1990)], fur- ther improved by our own revisions. Third, a two-dimensional R-matrix method based on matching exact 2D solutions from a small interaction region to asymptotic solutions in the non-interacting region. We thoroughly discuss the various advantages and caveats of these methods and, in the later chapters, present our work on employing them for the realistic recombination of HeH+ + e− . Furthermore, we attempt to extend the presented models to the description of the...

Ovarian cancer is serious and one of the most common gynecologic cancers. Carboplatin is the therapeutic agent of the first choice in the ovarian cancer therapy. However, after the primary therapeutic response to carboplatin, the relapse of the disease may occur with developed resistance to carboplatin. Chemoresistance and insufficient therapy response are considered to be the reason of the high mortality rate of ovarian cancer. The DNA damage response pathways play an important role in the therapeutic response and chemoresistance development. Restoration of homologous recombination function in cancers is the key mechanism of resistance development to platinum agents. Based on this knowledge, we formed our hypothesis, that the inhibition of homologous recombination could increase the sensibility to carboplatin. The main goal of this thesis was to define the role of double-strand breaks repair in response to chemotherapy of ovarian cancer. Protein MRE11 is part of the MRN complex, that participates in double-strand breaks repair. Using mirin as a pharmaceutic inhibitor of MRE11 we were aiming to determine the impact of homologous recombination on the effect of carboplatin and its role in resistant development to carboplatin. In the practical part of the thesis, we described the association between...