Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.

The Effect of using online homework exercises on student achievement in a Quantitative methods course

Abstract: Quantitative Methods (QM) is a compulsory course in the Social Science program in CEGEP. Many QM instructors assign a number of homework exercises to give students the opportunity to practice the statistical methods, which enhances their learning. However, traditional written exercises have two significant disadvantages. The first is that the feedback process is often very slow. The second disadvantage is that written exercises can generate a large amount of correcting for the instructor. WeBWorK is an open-source system that allows instructors to write exercises which students answer online. Although originally designed to write exercises for math and science students, WeBWorK programming allows for the creation of a variety of questions which can be used in the Quantitative Methods course. Because many statistical exercises generate objective and quantitative answers, the system is able to instantly assess students’ responses and tell them whether they are right or wrong. This immediate feedback has been shown to be theoretically conducive to positive learning outcomes. In addition, the system can be set up to allow students to re-try the problem if they got it wrong. This has benefits both in terms of student motivation and reinforcing learning. Through the use of a quasi-experiment, this research project measured and analysed the effects of using WeBWorK exercises in the Quantitative Methods course at Vanier College. Three specific research questions were addressed. First, we looked at whether students who did the WeBWorK exercises got better grades than students who did written exercises. Second, we looked at whether students who completed more of the WeBWorK exercises got better grades than students who completed fewer of the WeBWorK exercises. Finally, we used a self-report survey to find out what students’ perceptions and opinions were of the WeBWorK and the written exercises. For the first research question, a crossover design was used in order to compare whether the group that did WeBWorK problems during one unit would score significantly higher on that unit test than the other group that did the written problems. We found no significant difference in grades between students who did the WeBWorK exercises and students who did the written exercises. The second research question looked at whether students who completed more of the WeBWorK exercises would get significantly higher grades than students who completed fewer of the WeBWorK exercises. The straight-line relationship between number of WeBWorK exercises completed and grades was positive in both groups. However, the correlation coefficients for these two variables showed no real pattern. Our third research question was investigated by using a survey to elicit students’ perceptions and opinions regarding the WeBWorK and written exercises. Students reported no difference in the amount of effort put into completing each type of exercise. Students were also asked to rate each type of exercise along six dimensions and a composite score was calculated. Overall, students gave a significantly higher score to the written exercises, and reported that they found the written exercises were better for understanding the basic statistical concepts and for learning the basic statistical methods. However, when presented with the choice of having only written or only WeBWorK exercises, slightly more students preferred or strongly preferred having only WeBWorK exercises. The results of this research suggest that the advantages of using WeBWorK to teach Quantitative Methods are variable. The WeBWorK system offers immediate feedback, which often seems to motivate students to try again if they do not have the correct answer. However, this does not necessarily translate into better performance on the written tests and on the final exam. What has been learned is that the WeBWorK system can be used by interested instructors to enhance student learning in the Quantitative Methods course. Further research may examine more specifically how this system can be used more effectively.

Alterations of the epigenome in brain cancer: loss of 5-hydroxymethylcytosine

Abstract : 5-Methylcytosine is an epigenetic mark, which can be oxidized to 5-hydroxymethylcytosine (5hmC) in DNA by ten-eleven translocation (TET) oxygenases. It is an initial step in the demethylation of 5mC. Levels of 5hmC is relatively high in the brain compared to other organs, but these levels are known to be significantly reduced during the development of a brain tumor, especially in glioblastoma multiforme (GBM). However, no known mechanisms may fully explain this abnormality. The objectives of my project were to (1) understand the implications of the demethylation pathway mediated by TET, and (2) gain a deeper insight in the epigenetic make-up of brain tumors. (1) U87 cells were incubated with 5mC, 5hmC, 5-formylcytosine (5fC) or co-incubated of 5hmC with 3,4,5,6-tetrahydro-2’-deoxyuridine (dTHU) over a timeline of 0, 24, 48 and 96 hours. (2) 130 brain tumors (GBM= 79; grade II/III= 51) were obtained directly from surgery and immediately suspended in DNA extraction buffer. Both cell samples and tumor tissues underwent DNA extraction and DNA digestion protocols. The percent per cytosine (%/C) was obtained by quantification of 5mC, 5hmC, 5fC, 5-hydroxymethyluracil (5hmU) and 5formyluracil (5fU) using LC-MS/MS. (1) Cellular incubations showed that it is possible to increase levels of 5hmC in DNA, but also a slight increase in 5mC levels throughout the experiment. 5HmC levels dramatically increased by 1.9-fold after 96h. On the other hand, no increase was observed in 5fC levels. Both 5hmC and 5fC incubations were accompanied by high increases in 5hmU and 5fU levels respectively. The addition of dTHU to the 5hmC incubation decreased 5hmU incorporation by 65%. (2) The average levels of 5mC, 5hmC and 5fC, in brain tumors, were 4.0, 0.15 and 0.021 %/C respectively. 5HmU and 5fU levels were present at comparable levels of 5hmC and 5fC. Levels of 5hmC, 5hmU and 5fU were significantly lower in the DNA of GBM specimens. There was a strong correlation between 5mC with 5hmC and 5fC in GBM, but this was absent in low grade tumors. The presence of 5hmU and 5fU in brain tumor and the increase in their levels during cell incubations indicate a deamination activity in these cancerous cells, which may impinge on the cellular levels of 5hmC, in particular. Furthermore, upon the incubations with 5hmC, downstream levels of 5fC did not increase suggesting a TET malfunction. TET activity is maintained in GBMs, but impaired in low grade tumors due to isocitrate dehydrogenase-1 (IDH1) mutations. Therefore, in brain tumors, a strong deamination activity and TET impairment may lead to epigenetic reduction of 5hmC.