The green tea is considered a functional food with antioxidant activity, and this delays the process of old age and helps in the prevention of chronicle and degenerative diseases, such as cancer and heart affections. Of catechins, present in green tea, may is extremely important in the inactivation of free radicals, which provoke damages to the DNA molecules and to the cell membranes prevent the cytotoxicity induced by the oxidative stress in different tissues, by actuating in the process of chemoprevention. In this study two crosses, ST and HB aimed at evaluating the protective effects of green tea in three different concentrations (25, 50 e 75mg/mL), combined with the antitumor agent, doxorrubicin (DXR), of the genotoxic action; by the of wing spot test (SMART) of Drosophila melanogaster. The results obtained demonstrated that in MH descendants there was not a statistically significant increase in the frequencies of spots, in the individuals treated with green tea, in standard cross (ST) as well as in the high bioactivation cross (HB - characterized by a high constitutive level of cytochrome P450) . However we could observe a statistically significant decrease in the sum of mutant spots, in MH descendants of both crossings (ST and HB), in all concentrations of green tea associated to DXR. These results suggest that green tea does not present genotoxic effect, but presents protective affect against the mutagenic and recombinagenic action of DXR in the somatic cells of D. melanogaster, in the experimental concentrations used in this study. / O chá verde (CV) é considerado um alimento funcional com atividade antioxidante, que retarda o processo de envelhecimento e auxilia na prevenção de doenças crônicas e degenerativas, como o câncer e doenças cardiovasculares. As catequinas, presentes neste chá, podem inativar os radicais livres que causam danos na molécula de DNA e dessa maneira, prevenir a citotoxicidade induzida pelo estresse oxidativo, em diferentes tecidos, atuando no processo de quimioprevenção. Neste estudo dois diferentes cruzamentos, ST e HB foram utilizados para avaliar o efeito do CV em três diferentes concentrações (25, 50 e 75mg/mL) combinados com um agente tumoral, doxorrubicina (DXR), de comprovada ação genotóxica; através do teste para detecção de manchas (SMART) na asa de Drosophila melanogaster. Os resultados obtidos demonstram que para os descendentes MH não houve aumento, estatisticamente significativo, nas freqüências de manchas, nos indivíduos tratados apenas com CV, tanto no cruzamento padrão (ST) quanto no cruzamento de alta bioativação (HB - caracterizado pelo alto nível constitutivo de citocromo 450). Contudo, foi observada uma redução, estatisticamente significativa, no total de manchas mutantes, nos descendentes MH, de ambos os cruzamentos (ST e HB), em todas as concentrações do CV quando associadas com a DXR. Estes resultados sugerem que o CV não possui efeito genotóxico, e, apresenta efeito protetor contra a ação mutagênica e recombinogênica induzida pela DXR em células somáticas de D. melanogaster, nas concentrações experimentais utilizadas neste estudo. / Mestre em Genética e Bioquímica

Muitos genes têm sido indicados como responsivos ao estresse genotóxico, mas devido à necessidade de validação, a busca por marcadores gênicos continua. Vários genes são relacionados ao sistema ubiquitina-proteassomo (UPS), o qual é responsável pela remoção seletiva de proteínas, sendo que falhas no UPS têm sido relacionadas a doenças neurodegenerativas e ao câncer. Assim, o presente trabalho teve como objetivo a busca e confirmação de marcadores gênicos de resposta ao estresse genotóxico, por meio do estudo da expressão transcricional e protéica dos genes ERN1, EIF2AK3, GADD153 e TRAF2, visando à confirmação das respostas em linhagens de fibroblastos (GM07492A e AS405) e de glioblastoma (U87MG), sob tratamentos com peróxido de hidrogênio (H2O2) e Bleomicina (Blm). Foram utilizados o Ensaio Cometa, a análise de expressão gênica transcricional por qPCR em tempo real e de expressão gênica ao nível protéico (imunofluorescência). Os resultados mostraram que os tratamentos empregados foram capazes de induzir danos no DNA, sendo que a sensibilidade ao tratamento e a capacidade de recuperação das linhagens foi variável dependendo do agente testado. A análise de expressão gênica mostrou que GM07492A apresentou indução dos genes ERN1 e GADD153 após tratamento com H2O2 (resposta precoce, zero e 2 h) e Blm (durante todo pós-tratamento). A linhagem AS405 exibiu indução de ERN1 e GADD153 para H2O2, enquanto que para Blm foram induzidos os genes EIF2AK3 e GADD153. Para U87MG, a indução de EIF2AK3 pelo H2O2 ocorreu de modo tardio, enquanto GADD153 mostrou-se induzido após ambos os tratamentos. A proteína ERN1 apresentou expressão discreta e pontual, inclusive nos pontos onde não houve indução transcricional, indicando uma expressão basal. Essa proteína se expressou em GM07492A no tratamento com Blm em zero hora, diferentemente de AS405. Para U87MG tratada com H2O2 observou-se discreta expressão de ERN1, sendo mais evidente para Blm. Quanto à proteína GADD153, esta foi expressa em fibroblastos nos vários tempos analisados. No entanto, U87MG mostrou expressão nuclear apenas nas células tratadas, sendo mais evidente para H2O2 comparativamente à Blm. Assim, as alterações observadas nos perfis de expressão gênica são compatíveis com a indução de danos no DNA, indicando o envolvimento de genes do UPS nas respostas celulares ao estresse genotóxico. Em conjunto, os resultados estimulam uma avaliação mais detalhada desses genes como marcadores de resposta ao estresse e evidencia a sua importância no cenário da via UPS. / Many genes have been reported as responsive to genotoxic stress, but due to the need of validation, the search for genetic markers still continues. Several genes are related to the ubiquitin-proteasome system (UPS), which is responsible for the selective removal of proteins, and UPS failures have been associated to neurodegenerative diseases and cancer. Thus, this study aimed the search and confirmation of genetic markers that were responsive to genotoxic stress. For this, we evaluated the transcriptional or protein expression of the genes ERN1, EIF2AK3, GADD153 and TRAF2, seeking confirmation of responses in fibroblast cell lines (GM07492A and AS405) and glioblastoma (U87MG) under treatment with hydrogen peroxide (H2O2) and bleomycin (BLM). We used the Comet Assay, the transcriptional analysis of gene expression by quantitative real-time PCR and protein expression byimmunofluorescence. The results showed that the treatments employed were able to induce DNA damage, and that cell sensitivity to treatments and recovery capability of cell lines varied according to the tested agent. The gene expression analysis showed that GM07492A presented induction of ERN1 and GADD153 genes after treatment with H2O2 (early response, zero and 2 h) and Blm (throughout the post-treatment). The cell line AS405 showed induction of GADD153 and ERN1 after H2O2, whereas with Blm the genes induced were EIF2AK3 and GADD153. For U87MG, the induction of EIF2AK3 by H2O2 occurred at a later stage, while GADD153 was promptly induced after both treatments. The protein ERN1 showed discreet and punctual expression, even at time point without transcriptional induction, indicating a basal expression. This protein was expressed in GM07492A by treatment with Blm at zero hour, differently of AS405. For U87MG treated with H2O2, ERN1 showed a slight expression, being more evident for Blm. Regarding GADD153, protein expression was observed in fibroblasts at all time point. However, U87MG showed nuclear expression only in cells treated with H2O2, being more evident that in BLM-treated cells. Thus, the observed changes in gene expression profiles are consistent with the induction of DNA damage, which indicates the participation of UPS genes in cellular responses to genotoxic stress. Together, the results encourage further evaluation of these genes as markers of stress response, demonstrating its importance in the UPS acting scope.

This work is devoted to the study of boron doped diamond as electrode material, its properties and use in electroanalytical methods - in voltammetric and subsequently amperometric methods in combination with high performance liquid chromatography. The series of boron doped diamond films was tested with respect to the effect of boron concentration on their morphology, quality, electrochemical and spectral properties using scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and cyclic voltammetry and differential pulse voltammetry. Further, the effect of boron concentration on the determination of selected substances was investigated, both for their oxidation (2-aminobiphenyl, benzophenone-3) and for their reduction (5-nitroquinoline). Furthermore, a voltammetric and amperometric method was developed for the determination of a mixture of aminobiphenyls and aminonaphthalenes using a boron doped diamond electrode. The effects of activation cleaning programs on the signal of benzophenone-3 were investigated using a boron doped diamond electrode, and the determination of benzophenone-3 on boron doped diamond electrode in the presence of the selected surfactant was studied. Boron doped diamond as carbon-based material was compared with other selected carbon materials such as glassy...

Les rythmes biologiques constituent une propriété fondamentale de la vie, permettant aux organismes d’appréhender leur environnement et d’en anticiper les changements. Ces rythmes possèdent une double origine : une horloge moléculaire génère ces rythmes, qui sont ensuite synchronisés par des facteurs environnementaux. Si les organismes terrestres sont essentiellement soumis au rythme d’alternance jour/nuit, les espèces marines côtières et estuariennes occupent un biotope plus changeant encore : ils sont en effet également confrontés au rythme des marées. Pourtant, leurs rythmes biologiques sont à ce jour encore mal connus et les mécanismes moléculaires de(s) (l’) horloge(s) sous-jacente(s) ne sont pas caractérisés. Parallèlement, les efflorescences d’algues toxiques, en constante augmentation depuis 1970, constituent un problème écologique majeur, tant local qu’international. Les objectifs du présent travail consistaient à caractériser les rythmes de référence de l’huître Crassostrea gigas et d’en déterminer l’origine (moléculaire, zeitgebers). Il s’agissait ensuite d’étudier les perturbations potentielles de ces rythmes par l’algue toxique Alexandrium minutum, qui produit des toxines paralytiques et est régulièrement présente dans de nombreuses mers du globe. Les travaux réalisés ont permis de mettre en évidence l’existence d’un rythme d’activité valvaire circadien, faible et dual, et n’a pas permis de supporter l’hypothèse de l’existence d’une horloge circatidale. Nous avons formulé l’hypothèse que, chez C. gigas, le rythme tidal est soit d’origine exogène, soit produit par l’horloge circadienne synchronisée par les marées. Les analyses moléculaires réalisées sur le gène circadien cryptochrome dans le muscle adducteur - effecteur du mouvement des valves - ont montré que ce gène oscille à une fréquence tidale dans le muscle strié, favorisant notre seconde hypothèse. Par ailleurs, au-delà des gènes de l’horloge, l’algue A. minutum réprime l’expression de gènes impliqués dans différentes voies métaboliques importantes : la lutte contre le stress oxydant (cat, gpx), la respiration mitochondriale (cox1), l’immunité (ilk), la détoxification (mdr). Finalement nos analyses ont permis de mettre en évidence un impact génotoxique d’A. minutum chez C. gigas. / Biological rhythms constitute a fundamental property of life, allowing organisms to anticipate and adapt to their changing environment. These rhythms present a double origin: they are generated by a molecular clock and synchronized by environmental cues. Whereas terrestrial organisms are mainly subjected to day/night alternation, coastal and estuarine marine species inhabit an even more cycling biotope. They are indeed also submitted to tides. Nevertheless, biological rhythms in marine species are still unrecognized and molecular mechanisms of the underlying oscillator(s) are to date not determined. At the same time, toxic algae blooms are increasing since the 1970s and represent a major ecological concern, both at local and international levels. An objective of the present work was the characterization of biological rhythms in the oyster Crassotrea gigas and of their origin (molecular mechanism, zeitgebers). Furthermore, the work was designed to study the potential disruption of these rhythms by the toxic algal of worldwide distribution Alexandrium minutum, which produces paralytic toxins. The present results show the existence of a weak and dual circadian rhythm of valve activity in the oyster, and do not provide evidence for the existence of any circatidal clock. We suggested that, in the oyster C. gigas, the tidal rhythm could either be generated exogenously or endogenously by the tidally-synchronized circadian clock. Molecular analyses performed on the circadian gene cryptochrome in the adductor muscle of oyster, the effector of valve movements, revealed that Cgcry oscillates at tidal frequency in the striated muscle. This result supports our second hypothesis. Furthermore, A. minutum represses the expression of genes involved in key metabolic pathways: struggle against oxidative stress (cat, gpx), mitochondrial respiration (cox1), immunity (ilk), detoxification (mdr). Moreover, A. minutum impacts C. gigas at DNA level, being thus genotoxic.

The promyelocytic leukemia protein (PML) is a tumour suppressor. It has been reported that PML interaction with the p53 protein is involved in the activation of cell cycle checkpoints and, when persistent, may lead to the premature onset of cellular senescence. Cellular senescence is a state of permanent cell growth arrest that is associated with characteristic morphological and metabolic changes and persistent DNA damage signalling. Importantly, PML nuclear bodies coassociate with persistent DNA damage foci in senescent cells; however, the role of this interaction is still obscure. My goal was to characterize the role of PML in DNA damage response (DDR) and the induction of premature cellular senescence after genotoxic stress, namely X-radiation, using both siRNA-mediated PML knock down (PML KD) and complete PML knock out (PML KO) in human cells. The dynamics of DNA damage foci, levels of various proteins involved in DDR, and proliferation rate were measured in both PML KD and KO cells. No significant changes in the formation of DNA damage foci, activated DDR (p53 and Chk2), activated p21CIP1/WAF1 cyclin-dependent kinase inhibitor, senescent morphology, and SA-β-galactosidase activity in PML KO cells were observed. However, PML KO cells displayed higher levels of retinoblastoma protein (Rb) and...

The occupational exposures of sewage workers are complex and variable, and include a great variety of biological and chemical agents. Previous research has focused mostly on infections and various symptoms among sewage workers, e.g. abdominal and respiratory symptoms. At several sewage plants in Sweden, concern arose about occupational cancer, specifically cancer of the stomach, the kidney, and the lung. The aim of this study was to study the cancer incidence among municipal sewage workers, some exposures that might be connected with cancer risk, and self reported abdominal and respiratory symptoms. In a cohort of municipal sewage workers there was no increase in the overall incidence of cancer when compared with the general population. However, there was a slight increase in the incidence of prostate cancer, but not in the sites of original concern among the workers. Infection by the gastric carcinogen Helicobacter pylori (determined from the presence of IgG antibodies in serum against H pylori) was no more prevalent in sewage workers than in comparable referents. Neither were sewage workers more exposed to genotoxic agents than comparable referents, as measured by the alkaline single cell gel electrophoresis (SCG) assay performed on peripheral lymphocytes. There was no increase in the three-month prevalence of abdominal symptoms when compared with other municipal workers. Specifically, there was no difference in prevalence of the common disorders dyspepsia and irritable bowel syndrome. Sewage workers reported adult bronchial asthma significantly more than the referents.

L'oligomérisation enzymatique de la rutine et esculine a donné lieu à cinq fractions d'oligomères de masse moléculaire moyenne entre 2127,42 et 8331,85 g/mol pour la rutine et 688,12 et 6973 g/mol pour l'esculine. L'analyse de ces fractions par FTIR montre que les fractions d'oligorutines sont obtenues à travers des liaisons C-C, C-O et C=O. Les fractions d'oligoesculines sont obtenues à travers des liaisons C-C. Une meilleure solubilité des oligorutines et des oligoesculines dans l'eau et une plus faible solubilité de ces oligomères dans l'éthanol comparé à leurs monomères a été mis en évidence. Une diminution de l'activité antiradicalaire vis-à-vis de DPPH., ABTS+. et OH. proportionnelle à la masse moléculaire moyenne des fractions d'oligorutines a été observé, contrairement aux fractions d'oligoesculines qui montrent un important pouvoir chélateur de ces mêmes radicaux comparé à leurs monomère. Une augmentation du pouvoir chélateur de fer, inhibiteur de la xanthine oxydase, réducteur du cuivre (CUPRAC), de l'activité antigénotoxique, ainsi que de l'activité stimulatrice de la prolifération des splénocytes, et des lymphocytes (B et T) proportionnelle au degré d'oligomérisation des oligomères étudiées a été noté. L'effet des fractions d'oligorutines et oligoesculines étudiées sur les macrophages en suivant la production de monoxyde d'azote (NO) montre un pouvoir anti-inflammatoire comparé à leurs monomères. L'étude de l'activité lysosomale induite par les fractions d'oligorutine révèle un pouvoir immunostimulateur proportionnelle à la masse moléculaire moyenne des oligorutines, et inversement proportionnelle à celle-ci pour les oligoesculines / Rutin and esculin have been polymerized by laccase. Five fractions with between 2127.42 and 8331.85 g/mol for oligorutins, and between 688.12 and 6973 g/mol for oligoesculins, were obtained. Fourier transformed infrared analysis showed that oligorutins were formed through C-C, C-O and C=O linkages, while oligoesculins were obtained through C-C linkages. Oligorutins and oligoesculins show a higher solubility in water and a lower solubility in ethanol compared to their monomers. The oligomerization of rutin decrease its antiradical capacity, while oligoesculin fractions demonstrated a high antiradical activity compared to monomeric esculin. Oligomer fractions showed a better iron chelating power, xanthine oxidase inhibition, copper reducing power (CUPRAC), antigenotoxic activity, and splenocytes stimulator activity compared to their monomers. Oligorutin and oligoesculin exhibited an important anti-inflammatory capacity through the nitric oxide inhibition. Moreover, oligorutin fractions demonstrated an immunostimulatory effect proportional to their degree of oligomerization, while oligoesculin fractions showed an immunostimulatory effect inversely proportional to their degree of oligomerization

Nous avons identifié une nouvelle forme de phospho-S27-S33-Ku70 constitutivement surexprimée dans des cellules issues de la leucémie lymphocytaire chronique résistante à la chimiothérapie basée sur des agents alkylants de l’ADN et/ou analogues nucléotidiques. La protéine Ku70 est une protéine essentielle du maintien de la stabilité génomique par son rôle dans la réparation non-homologue (système NHEJ) des cassures double brin de l’ADN (CDB) et par sa fonction télomérique. Le laboratoire d’accueil a déjà démontré, in vitro et in vivo, dans les cellules LLC résistantes une altération de la réparation par le système NHEJ et un dysfonctionnement télomérique. Le travail de thèse a porté sur la caractérisation fonctionnelle de cette nouvelle forme phospho-S27-S33-Ku70. Pour ceci, nous avons utilisé des vecteurs d’expression permettant simultanément d’inhiber l’expression du Ku70 endogène (shRNA) et d’exprimer de façon épisomale différentes formes de Ku70 exogène. Ainsi, nous avons démontré : i) une stricte colocalisation de pS27-pS33-Ku70 avec les foyers γ-H2AX; ii) des cassures double brin (DSB) induisent la phosphorylation de S27-S33-Ku70 sous forme hétérodimère avec Ku80. Cette phosphorylation a lieu quelques minutes après le stress génotoxique et implique l'activité et l'interaction physique avec pS2056-DNA-PKcs, reliant ainsi pS27-pS33-Ku70 au système NHEJ ; iii) les cellules exprimant la forme sauvage exogène S27-S33-Ku70 ou la forme phosphomimétique E27-E33-Ku70 présentent une cinétique de réparation de l’ADN plus rapide que celle des cellules exprimant la forme mutée A27-A33-Ku70. Cependant, iv) la forme sauvage de Ku70 contribue à un niveau plus élevé d'aberrations structurales chromosomiques après la première division cellulaire suite à un stress génotoxique indiquant une infidélité lors de la réparation des dommages de l’ADN. En outre, les cellules exprimant A27-A33-Ku70 possèdent un index cellulaire plus élevé qui est corrélé avec une activation de la voie β-caténine. En adéquation avec sa surexpression dans la forme résistante de la LLC, l’ensemble de ces résultats suggère un rôle oncogénique de la forme phosphorylée de Ku70. Nous avons ensuite testé l’effet des nanodiamants hydrogénés (ND-H) dans des lignées exprimant différentes formes de Ku70. Grâce à leurs propriétés physico-chimiques les ND-H sont capables de potentialiser sous irradiation la production intracellulaire des espèces réactives de l’oxygène (ROS) et ainsi augmenter le taux des cassures (simple et double brin de l’ADN) et solliciter d’avantage le système de réparation de l’ADN. Nous observons que indépendamment de la forme exprimée de Ku70, ce double traitement induisait la sénescence cellulaire ; une découverte d’un intérêt à la fois fondamental (compréhension des voies apoptotiques vs senescence) et d’utilité pharmacologique potentielle. / We have identified a new form of phospho-S27-S33-Ku70 constitutively overexpressed in a subset of chronic lymphocytic leukemia (CLL) B cells resistant to apoptosis induced by DNA double strand breaks (DSB). Ku70 is one of the essential proteins involved in the maintenance of genomic stability through its role in DNA double strand break repair (non-homologous end-joining, NHEJ) and in telomeric protection.Laboratory previously established that resistant CLL cells disclose an upregulated NHEJ DNA repair and an impaired structure of telomeres. The goal of this thesis was to characterize the biological function(s) of this new form of Ku70. For this purpose we have constructed specific EBV-based vectors (siRNA / cDNA) enabling a simultaneous inhibition of endogenous Ku70 and an expression of different forms (mutated, wild, phosphomimetic at ser27-33) of Ku70 resistant to siRNA. Thus, we showed: i) a strict colocalisation of phospho-Ku70 with γ-H2AX foci; ii) that DSB induces the phosphorylation of Ku70 within minutes after genotoxic stress in heterodimer complex Ku70/Ku80. This phosphorylation necessitates both the physical interaction and the activity of pS2056-DNA-PKcs and/or ATM, linking phospho-Ku70 to NHEJ-mediated DNA DSB repair; iii) cells expressing mutated A27-A33-Ku70 exhibit a delayed G2/M cell cycle arrest, slower kinetic of DNA repair, lower level of genotoxic stress-induced chromosomal aberrations, and a higher cellular impedance correlated with translocation of transcriptional factor β-catenin from cytoplasmic membrane to the nucleus. Together, these data unveil an involvement of phospho-Ku70 in fast and inaccurate DNA repair; new paradigm for NHEJ regulation and to the control of resistance and maintenance of malignant cells.In parallel, we have initiated experimental approaches to explore other potential roles of phospho-Ku70. Especially, we were interested to determine whether it could play a role in an initiation of cell senescence induced by combined cells’ treatment by hydrogenated nanodiamonds (H-NDs) particles and ionizing irradiation. H-NDs exhibit positive surface charge in aqueous solutions allowing, when irradiated by photons, electrons’ emission and the release of reactive oxygen species (ROS) causing DNA damage. Effectively, we have established an intracellular increase of ROS that drive cell cycle arrest in G1/S in addition to the G2 arrest activated by irradiation alone. Finally, cells underwent the senescence process characterized byγ-galactosidaze activity, persistent large γ-H2AX foci and senescence-associated heterochromatinisation. Noteworthy, the senescence induced in this way occurred independently of Ku70 (ser27-ser33) status and irrespectively of cell resistance to genotoxic agents administrated alone; a finding of potential use in clinical trials.

Promyelocytic leukemia protein (PML) is a tumour suppressor which is frequently downregulated in human tumours. PML plays a role in many cellular processes including DNA damage response, senescence and apoptosis and is mainly localized in special structures called PML nuclear bodies (PML NBs). The nucleolus is a key nuclear compartment, where transcription of ribosomal DNA and biogenesis of ribosomes take place. The nucleolus is also called a stress sensor because of its role, for instance, in stabilization of tumour suppressor p53. Localization of PML to the nucleolar periphery appears to be prominent after disturbance of nucleolar functions - for example inhibition of rRNA transcription or processing. Thus the relationship between the nucleolus and PML nuclear bodies may be important for cellular response to stress. However, the role of PML nucleolar associations in nucleolar function including mechanism of formation of these structures remain unclear. Here we characterised PML nucleolar structures and mechanism of their formation. We showed that formation of PML nucleolar structures is not caused by replication stress, is not dependent on any specific phase of cell cycle and is not caused by DNA damage response but is induced by topological stress due to inhibition of toposiomerase function....

Cbf11 and Cbf12 proteins, the members of the CSL transcription factors family, are involved in a wide range of cellular processes in the fission yeast Schizosaccharomyces pombe - among other things they regulate cell adhesion and they have also been implicated in maintenance of genome integrity. At the level of the whole genome we previously identified target loci bound by CSL proteins in vivo. Many of them do not contain any consensus CSL-binding element. There are probably different DNA binding modes of the Cbf11/12 proteins and it has not been known what specific biological function is associated with the particular way of DNA binding. For the purpose of studying CSL DNA binding modes we have worked in this project on the implementation of the DNA binding mutation (DBM), which prevents direct DNA binding of CSL proteins to canonical motif in vitro, into the chromosomal locus of the cbf11 and cbf12 genes. Using the "ura4 selection system" we have successfully constructed the scar-less Cbf12-TAP and Cbf12DBM-TAP knock-ins, i.e. the strains without/with DBM in the open reading frame of Cbf12 where Cbf12 is C- terminally TAP-tagged and contains the intact 3'UTR. In our laboratory we have established the CRISPR/Cas9 system by which we have been able to prepare the Cbf11- TAP strain. We have failed to...