Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer de esôfago é uma malignidade altamente freqüente e letal. Uma característica específica das áreas de alta incidência de câncer de esôfago é a grande proporção de duplas mutações no gene TP53, sendo, ao menos uma delas, uma transição G para A em sítios CpG. Essas transições resultam de malpareamentos GT causados pela desaminação espontânea da 5-metilcitosina em ilhotas CpG. A enzima de reparo de DNA Timina-DNA Glicosilase (TDG) é responsável pelo primeiro passo na remoção da timina de malpareamentos GT em CpG. A alta proporção de mutações em sítios CpG em câncer de esôfago das áreas de alta incidência sugere que a via de reparo de DNA iniciada pela TDG pode estar prejudicada. A presença de duplas mutações, sendo ao menos uma delas em CpG, levantou a hipótese de que a primeira mutação no TP53 reduz a atividade da via de reparo iniciada pela TDG, que acarretaria a segunda mutação em sítios CpG. Dessa forma, o objetivo desse trabalho foi analisar o efeito da p53 sobre a expressão e atividade da TDG. Os resultados obtidos mostram que a expressão de TDG é regulada transcricionalmente pela p53 numa gama de linhagens celulares e é induzida pelo dano ao DNA, de forma p53-dependente. Além disto, os resultados apontam um possível papel da proteína p53 ativa na migração nuclear e atividade da TDG. Estes resultados ainda nos levam à conclusão de que o silenciamento de TDG aumenta a sensibilidade à morte celular induzida por MMS quando a p53 é encontrada na forma selvagem, mas não quando esta proteína é mutada, e de que o status mutacional de TP53 parece afetar a expressão de TDG em CEE primários. Juntos esses resultados sugerem que a p53 regula o reparo de DNA mediado pela TDG e que a inativação de p53 em células tumorais pode contribuir para a aquisição de um mutator phenotype. / Esophageal squamous cell carcinoma (ESCC) is a highly frequent and fatal malignancy in the world. A peculiar characteristic of the high incidence areas of esophageal cancer is the large proportion of double mutations in TP53 gene, being, at least one of them, a G to A transition at CpG sites. These transitions result from GT mismatches caused by the spontaneous deamination of 5-methylcytosine at CpG sites. The DNA repair enzyme Thymine-DNA Glycosylase (TDG) is responsible for the first step in the removal of the thymidine from the GT mismatches at CpG sites. The high proportion of mutations at CpG sites in esophageal tumors in the high incidence areas suggests that the DNA repair pathway initiated by TDG might be impaired. The large number of double mutations, with one being at a CpG site, raised the possibility that the first mutation in TP53 reduces the activity of the TDG base excision repair pathway, increasing the chance of a second mutation event at a CpG site. In this way, the aim of this work was to analyze the effect of p53 on the expression and activity of TDG. The results achieved show that TDG expression is regulated by p53 in a variety of cells lines at the trancriptional level and induced by DNAdamage in a p53-dependent manner. Furthermore, these results point out a possible role of active p53 in the nuclear migration and activity of TDG. The results further support the notion that TDG silencing increases the sensitivity to cell death induced by Methylmethane sulphonate when p53 is found in a wild-type, but not in a mutant form, and that TP53 mutation seems to affect TDG expression in primary ESCC. Together, these results suggest that p53 regulates TDG-mediated repair and that p53 inactivation in cancer cells may contribute to a mutator phenotype through loss of TDG function.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer de esôfago é uma malignidade altamente freqüente e letal. Uma característica específica das áreas de alta incidência de câncer de esôfago é a grande proporção de duplas mutações no gene TP53, sendo, ao menos uma delas, uma transição G para A em sítios CpG. Essas transições resultam de malpareamentos GT causados pela desaminação espontânea da 5-metilcitosina em ilhotas CpG. A enzima de reparo de DNA Timina-DNA Glicosilase (TDG) é responsável pelo primeiro passo na remoção da timina de malpareamentos GT em CpG. A alta proporção de mutações em sítios CpG em câncer de esôfago das áreas de alta incidência sugere que a via de reparo de DNA iniciada pela TDG pode estar prejudicada. A presença de duplas mutações, sendo ao menos uma delas em CpG, levantou a hipótese de que a primeira mutação no TP53 reduz a atividade da via de reparo iniciada pela TDG, que acarretaria a segunda mutação em sítios CpG. Dessa forma, o objetivo desse trabalho foi analisar o efeito da p53 sobre a expressão e atividade da TDG. Os resultados obtidos mostram que a expressão de TDG é regulada transcricionalmente pela p53 numa gama de linhagens celulares e é induzida pelo dano ao DNA, de forma p53-dependente. Além disto, os resultados apontam um possível papel da proteína p53 ativa na migração nuclear e atividade da TDG. Estes resultados ainda nos levam à conclusão de que o silenciamento de TDG aumenta a sensibilidade à morte celular induzida por MMS quando a p53 é encontrada na forma selvagem, mas não quando esta proteína é mutada, e de que o status mutacional de TP53 parece afetar a expressão de TDG em CEE primários. Juntos esses resultados sugerem que a p53 regula o reparo de DNA mediado pela TDG e que a inativação de p53 em células tumorais pode contribuir para a aquisição de um mutator phenotype. / Esophageal squamous cell carcinoma (ESCC) is a highly frequent and fatal malignancy in the world. A peculiar characteristic of the high incidence areas of esophageal cancer is the large proportion of double mutations in TP53 gene, being, at least one of them, a G to A transition at CpG sites. These transitions result from GT mismatches caused by the spontaneous deamination of 5-methylcytosine at CpG sites. The DNA repair enzyme Thymine-DNA Glycosylase (TDG) is responsible for the first step in the removal of the thymidine from the GT mismatches at CpG sites. The high proportion of mutations at CpG sites in esophageal tumors in the high incidence areas suggests that the DNA repair pathway initiated by TDG might be impaired. The large number of double mutations, with one being at a CpG site, raised the possibility that the first mutation in TP53 reduces the activity of the TDG base excision repair pathway, increasing the chance of a second mutation event at a CpG site. In this way, the aim of this work was to analyze the effect of p53 on the expression and activity of TDG. The results achieved show that TDG expression is regulated by p53 in a variety of cells lines at the trancriptional level and induced by DNAdamage in a p53-dependent manner. Furthermore, these results point out a possible role of active p53 in the nuclear migration and activity of TDG. The results further support the notion that TDG silencing increases the sensitivity to cell death induced by Methylmethane sulphonate when p53 is found in a wild-type, but not in a mutant form, and that TP53 mutation seems to affect TDG expression in primary ESCC. Together, these results suggest that p53 regulates TDG-mediated repair and that p53 inactivation in cancer cells may contribute to a mutator phenotype through loss of TDG function.

The purpose of this thesis was to perform a comprehensive evaluation of proposed sluiceway deflectors in Hells Canyon Dam with the use of Computational Fluid Dynamics (CFD). A CFD model developed and validated by Politano et al. (2010) was used to assess the downstream performance of the deflectors. Relative performance is measured by effects of the deflectors on the flow field, Total Dissolved Gas (TDG) production, and probability of mechanical fish injury. The deflectors evaluated in this model included the deflector with dimensions determined from a physical model as well as three additional deflector geometries that adjusted elevation, length and transition radius based on the physical model deflector. Physical model testing, at a 1:48 scale, of deflectors on Hells Canyon Dam performed by Haug and Weber (2002) provided a baseline deflector for the deflectors modeled in this study. The physical model was built and tested by the IIHR Hydroscience and Engineering. The performance study that this thesis focuses on was performed at two different tailwater elevations, established with two different total river flowrates of 25 kcfs and 45 kcfs. Each deflector was evaluated considering the spillway jet regime, tailrace flow pattern, and total dissolved gas (TDG) production. According to the model, decreasing the deflector length or increasing the transition radius results in more TDG production at all tailwater elevations. At 45 kcfs, the height of the deflector does not appreciably affect the spillway jet regime or the TDG distribution in the tailrace. However, increasing the deflector elevation at this river flow increases the amount of powerhouse entrainment and induces a recirculation in the western region of the tailrace. The baseline deflector performed best because it had the smallest impact on the tailrace flow pattern and produced the least TDG. The performance of the selected deflector was further evaluated for additional river flow rates of 37 kcfs, 45 kcfs and a 7Q10 flow condition of 71.5 Kcfs, with the 7Q10 condition being tested with and without the deflector. Although the deflector was able to prevent the spillway flow from creating a large amount of downstream TDG the 7Q10 flow condition significantly increased the TDG values downstream of the deflector relative to the other tested conditions. With the chosen deflector TDG values returned to forebay levels after 1 and 3.5 miles for the 37 kcfs and 45 kcfs river flowrates, respectively. With the deflector installed the 7Q10 flow condition creates considerable TDG production however the deflectors are able to reduce TDG production by 10% from the test without a deflector installed. For all evaluated river flows, with the chosen deflector, entrainment from the powerhouse is observed in the simulations; this entrainment is caused by the sluiceway surface jets. As powerhouse flow increases there is an observed decrease in entrainment. This is due to the increase of flow velocity in the streamwise direction, or perpendicular to the direction of entrainment. An important western recirculation that is prominent in the 7Q10 flow condition is also caused by the introduction of deflectors onto the spillways. Reversed flows near the fishtrap region and water directed back into the aerated section of the spillway are consequences of this recirculation. The effect causes a 25% percent increase of entrained flow relative to the no deflector 7Q10 flow. Injury of fish traveling over the spillway and through the sluiceway was estimated with the use of inert spherical particles and the computed flow field. Acceleration and strain experienced by the particles was calculated over the length of the spillway region. Numerical results were compared against literature values published by Deng (2005). Including the deflectors in the design increases the probability that fish will be injured. The most extreme cases of fish injury probability were 37 kcfs and the 7Q10 kcfs flowrates. For these cases, injuries experienced by the fish were 10% and 3% for minor and major injuries respectively. With comparison of the 7Q10 flows it appears that the inclusion of the deflector increases the induced minor injury induce from 5% to 10% and the major injury from 1% to 3%. Fish tailrace residence time was calculated using inert particles introduced to the computed fluid flow field. These particles were tracked for 650 feet past the sluiceway inlets and their time to completion was recorded. Particles were released from the sluiceways as well as the powerhouses for the 37 kcfs, 45kcfs and 7Q10 flow conditions. Particles released from the sluiceways reduced in residence time with an increase in sluiceway flowrate. With some amount of powerhouse entrainment increasing the residence time of the particles released from the powerhouse. These particles follow the entrainment to the deep low velocity region in the stilling basin. As the lateral flow increases some of the particles released from the spillway will join the high speed jets produced by the deflectors and their residence time will be reduced. According to the model, deflectors consistently reduce overall residence time and are therefore not expected to increase fish migration time. Water surface elevation near the fishtrap was measured for the 25 kcfs, 37 kcfs, 45 kcfs and 7Q10 flow conditions. The wave height near the fishtrap for the 7Q10 deflector case was predicted to be about one foot above the estimated water surface elevation. According to the model the inclusion of the deflector reduces the wave height.

Today, fastening articles such as screws and nails are treated with different surface coatings to withstand corrosion. The Swedish distributor ESSVE® Produkter AB uses a nano coating called CorrSeal™ for high corrosion protection of their screws. Thermal diffusion galvanization (TDG) is a more environmentally friendly method that the company seeks to use as replacement for the current treatment. This process of zinc diffusion is carried out at around 400 °C for several hours. The aim of the project is to investigate the possibility to surface treat a wood screw using TDG. The elevated temperature is suspected to decrease the hardness of the hardened screw. Therefore, a hardened and tempered screw without surface treatment is sent to a TDG facility. Industrial furnaces are used for similar heat treatments of screws with different hardenings. Both processes are analyzed by evaluating the results of hardness, bending, and microscopy. No immediate correlation between the TDG process and heat treatment in the industrial furnaces is found. Results show that the tested screws softened to a higher degree in the TDG process compared to treatment in the industrial furnaces. The mechanical properties of the tested screws, after the TDG process, are not acceptable. The zinc layer thickness on the screws is uneven yet believed to meet the required demands on corrosion resistance. Results also show that incorporating the TDG process in the tempering step is essential to meet the demands on hardness. Additionally, changing the composition of the material can lead to higher resistance against softening at the elevated temperatures. Further research is however needed to present a screw with sufficient corrosion resistance from the TDG process that will meet the demandson hardness and bending.

Chez les mammifères, la méthylation est une marque épigénétique ciblant la cytosine principalement dans un contexte CpG pour produire une 5mC. 5mC est très sensible à une déamination spontanée ou enzymatique, conduisant à la formation d'un mésappariement G/T. La 5mCpeut également être oxydée pour former successivement la 5hmC, la 5fC et la 5caC. Ces modifications de la 5mC participent aux processus actifs de déméthylation de l’ADN. Chez les mammifères, la thymine, dans le mésappariement G/T, est clivée par TDG et MBD4. TDG est également en mesure d'exciser 5fC et 5caC. Cette thèse avait pour but de clarifier la fonction de TDG et MBD4 dans la dynamique de la 5mC. Nous avons montré que MBD4 est associée aux protéines de réparation des mésappariements. Les tests enzymatiques, in vitro, montrent que le complexe MBD4/MMR a une activité bifonctionnelle (glycosylase/lyase) spécifique pour G/T, qui est régulée par la méthylation. Pour TDG, nous avons ciblé cette enzyme dans les cellules MEF et caractérisé la distribution des cytosines modifiées. Les résultats montrent des profils de méthylation/oxydation d'ADN qui sont régulés par TDG et surviennent principalement au niveau des répétitions de CA et dans les rétroéléments spécifiques de la lignée souris. / In mammals, methylation is an epigenetic mark targeting cytosine mainly in a CpG context, producing 5mC. 5mC is highly sensitive to a spontaneous or enzymatic deamination leading to G/Tmismatch. 5mC can also be oxidized to 5- 5hmC, 5fC and 5caC. These modifications of 5mC participate in the active demethylation processes. In mammals, the thymine in G/T mismatch is cleaved by TDG and MBD4 glycosylases. TDG is able also to excise the 5fC and 5caC.This thesis was to clarify the function of TDG and MBD4 in the dynamics of 5mC. We showed that MBD4 is associated with PMS2, MLH1, MSH2 and MSH6 proteins, four proteins involved in DNA mismatch repair (MMR). The in vitro enzymatic tests show that MBD4/MMR complex has a bifunctional glycosylase/lyase activity specific for G/T and is regulated by methylation.For TDG, we targeted this enzyme in MEF cells and characterized the distribution of modified cytosines. The results show that DNA methylation/oxidation patterns are regulated by TDG and occur mainly at CA repeats and at the mouse-lineage specific retro-elements.

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