Search

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018., Cataloged from PDF version of thesis., Includes bibliographical references., With the advent of combinatorial chemistry, the number of novel synthetic chemicals has skyrocketed over the past three decades, bringing about tremendous advances in medicine and material science. At the same time, the massive libraries of existing chemicals coupled with the unprecedented rate of new chemical generation presents a unique and costly challenge to toxicity testing in the 21 st century. In recent years, the United States has seen large coordinated efforts across governmental agencies to shift from expensive and slow traditional in vivo tests to more affordable and higher throughput in vitro methods. For each human cell, about 100,000 DNA lesions occur every day. Unrepaired DNA damage can lead to deleterious health consequences, including cancer and aging. Therefore, an essential endpoint in cell-based chemical safety testing is the assessment of a compound's genotoxic potential. In this work, we developed a CometChip platform that addresses two major areas that are lacking in genotoxicity testing: 1. rapid and sensitive detection of bulky DNA adducts, and 2. robust and physiologically relevant metabolism of test compounds. The assay uses two DNA repair synthesis inhibitors, hydroxyurea and I-[beta]-D-arabinofuranosyl cytosine, to cause strand-break accumulation and HepaRGTM cells to provide high levels of liver-specific functions. We also conducted extensive validation studies and a small chemical screen to demonstrate the platform's applicability in genotoxicity testing. One of the most important decisions of proliferating cells under stresses is to divide, senesce, or die. Therefore, in vitro measurements of cell survival after a toxic exposure are among the most fundamental and broadly used endpoints in biology. The gold standard for cell survival testing is the colony forming assay, which is exquisitely sensitive but sees limited uses due its low-throughput nature and requirement of large dishes. We have developed MicroColonyChip as a high-throughp, by Le Phuong Ngo., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018., Cataloged from PDF version of thesis., Includes bibliographical references., With the advent of combinatorial chemistry, the number of novel synthetic chemicals has skyrocketed over the past three decades, bringing about tremendous advances in medicine and material science. At the same time, the massive libraries of existing chemicals coupled with the unprecedented rate of new chemical generation presents a unique and costly challenge to toxicity testing in the 21 st century. In recent years, the United States has seen large coordinated efforts across governmental agencies to shift from expensive and slow traditional in vivo tests to more affordable and higher throughput in vitro methods. For each human cell, about 100,000 DNA lesions occur every day. Unrepaired DNA damage can lead to deleterious health consequences, including cancer and aging. Therefore, an essential endpoint in cell-based chemical safety testing is the assessment of a compound's genotoxic potential. In this work, we developed a CometChip platform that addresses two major areas that are lacking in genotoxicity testing: 1. rapid and sensitive detection of bulky DNA adducts, and 2. robust and physiologically relevant metabolism of test compounds. The assay uses two DNA repair synthesis inhibitors, hydroxyurea and I-[beta]-D-arabinofuranosyl cytosine, to cause strand-break accumulation and HepaRGTM cells to provide high levels of liver-specific functions. We also conducted extensive validation studies and a small chemical screen to demonstrate the platform's applicability in genotoxicity testing. One of the most important decisions of proliferating cells under stresses is to divide, senesce, or die. Therefore, in vitro measurements of cell survival after a toxic exposure are among the most fundamental and broadly used endpoints in biology. The gold standard for cell survival testing is the colony forming assay, which is exquisitely sensitive but sees limited uses due its low-throughput nature and requirement of large dishes. We have developed MicroColonyChip as a high-throughp, by Le Phuong Ngo., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018., Cataloged from PDF version of thesis., Includes bibliographical references., With the advent of combinatorial chemistry, the number of novel synthetic chemicals has skyrocketed over the past three decades, bringing about tremendous advances in medicine and material science. At the same time, the massive libraries of existing chemicals coupled with the unprecedented rate of new chemical generation presents a unique and costly challenge to toxicity testing in the 21 st century. In recent years, the United States has seen large coordinated efforts across governmental agencies to shift from expensive and slow traditional in vivo tests to more affordable and higher throughput in vitro methods. For each human cell, about 100,000 DNA lesions occur every day. Unrepaired DNA damage can lead to deleterious health consequences, including cancer and aging. Therefore, an essential endpoint in cell-based chemical safety testing is the assessment of a compound's genotoxic potential. In this work, we developed a CometChip platform that addresses two major areas that are lacking in genotoxicity testing: 1. rapid and sensitive detection of bulky DNA adducts, and 2. robust and physiologically relevant metabolism of test compounds. The assay uses two DNA repair synthesis inhibitors, hydroxyurea and I-[beta]-D-arabinofuranosyl cytosine, to cause strand-break accumulation and HepaRGTM cells to provide high levels of liver-specific functions. We also conducted extensive validation studies and a small chemical screen to demonstrate the platform's applicability in genotoxicity testing. One of the most important decisions of proliferating cells under stresses is to divide, senesce, or die. Therefore, in vitro measurements of cell survival after a toxic exposure are among the most fundamental and broadly used endpoints in biology. The gold standard for cell survival testing is the colony forming assay, which is exquisitely sensitive but sees limited uses due its low-throughput nature and requirement of large dishes. We have developed MicroColonyChip as a high-throughp, by Le Phuong Ngo., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016., Cataloged from PDF version of thesis., Includes bibliographical references., Methylating agents are ubiquitous in our internal and external environments and can cause damage to all cellular components, including our DNA. If left unrepaired, methylated DNA can cause mutations, cell death, and disease, such as cancer and neurodegeneration. The majority of DNA lesions caused by methylating agents are repaired by the base excision repair (BER) pathway, which is initiated by the lesion-specific alkyladenine glycosylase (Aag). Loss of Aag in embryonic stem (ES) cells renders them sensitive to the methylating agent MMS (methyl methanesulfonate) compared to wild-type (WT). Surprisingly, this phenotype is reversed in hematopoietic myeloid progenitors and cerebellar granule neurons (CGNs) where Aag' cells are resistant to MMS induced killing compared to WT. In this study, we investigated how Aag can cause cell-specific responses to alkylating agents. We generated new WT, Aag-/-, and Aag overexpressing (mAagTg) 129 and C57B1/6 ES cells and showed that inbred genetic background did not affect sensitivity to MMS, indicating this to be a cell-intrinsic response. Moreover, we found that cells overexpressing Aag were even more sensitive to MMS than Aag-/- cells, suggesting that ES cells endure methylation treatment best when they express Aag within an optimal range. To study Aag-dependent neural sensitivity to methylating agents, we optimized protocols for the isolation and culture of primary cerebellar granule neurons and determination of cell death after drug treatment by high-throughput imaging. CGNs isolated from WT, Aag-/-, and mAagTg mice exhibited cell sensitivity to MMS treatment that was dependent on Aag and Parp activity, thus recapitulating in vivo results and proving that CGN death is cell-intrinsic. Cell death was independent of caspases, mitochondrial depolarization, and AIF translocation. We did observe the formation of enlarged mitochondria and are investigating whether mitochondrial dynamics are causative of cell death in an Aag-dependent m, by Carrie Marie Margulies., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016., Cataloged from PDF version of thesis., Includes bibliographical references., The integrity of our DNA is challenged by roughly 100,000 lesions per cell on a daily basis. Failure to cope with DNA damage can lead to cancer, immunodeficiency and degenerative disease. Quantitating and understanding an individual's DNA repair capacity may enable us to predict and prevent disease in a personalized manner. Base Excision Repair (BER) is known for the recognition and repair of endogenous and exogenous mutagenic non-helix-distorting lesions produced by DNA base alkylation, deamination and oxidation. BER is initiated by the action of one of eleven DNA glycosylases known-to-date. Many studies have shown that levels of these glycosylases can vary between individuals, suggesting a basis for inter-individual differences in DNA repair capacity. Moreover, the methods for measuring DNA repair capacity used so far are cumbersome, time consuming, low throughput and only allow for the analysis of one glycosylase at a time. We have taken a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several DNA glycosylases and their immediate downstream endonuclease (APE1) can be tested simultaneously, at single-cell resolution, under physiological conditions. Taking advantage of the transcriptional properties of several DNA lesions we have designed and engineered specific fluorescent reporter plasmids for OGG1, AAG, MUTYH, UNG and APE1. Inter-individual differences in DNA repair capacity of a panel of cell lines derived from healthy individuals have been measured. Regression models that incorporate these measurements have been developed in order to predict cellular sensitivity to the chemotherapeutic and DNA damaging agents 5-FU, H₂O₂ and MMS, with the interest of understanding the contributions that these differences can have on personalized disease prevention and treatment. Finally, we have conducted a pilot population study with 56 healthy subjects where we implemented all the methods developed in order to determine the f, by Isaac Alexander Chaim., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016., Cataloged from PDF version of thesis., Includes bibliographical references., Methylating agents are ubiquitous in our internal and external environments and can cause damage to all cellular components, including our DNA. If left unrepaired, methylated DNA can cause mutations, cell death, and disease, such as cancer and neurodegeneration. The majority of DNA lesions caused by methylating agents are repaired by the base excision repair (BER) pathway, which is initiated by the lesion-specific alkyladenine glycosylase (Aag). Loss of Aag in embryonic stem (ES) cells renders them sensitive to the methylating agent MMS (methyl methanesulfonate) compared to wild-type (WT). Surprisingly, this phenotype is reversed in hematopoietic myeloid progenitors and cerebellar granule neurons (CGNs) where Aag' cells are resistant to MMS induced killing compared to WT. In this study, we investigated how Aag can cause cell-specific responses to alkylating agents. We generated new WT, Aag-/-, and Aag overexpressing (mAagTg) 129 and C57B1/6 ES cells and showed that inbred genetic background did not affect sensitivity to MMS, indicating this to be a cell-intrinsic response. Moreover, we found that cells overexpressing Aag were even more sensitive to MMS than Aag-/- cells, suggesting that ES cells endure methylation treatment best when they express Aag within an optimal range. To study Aag-dependent neural sensitivity to methylating agents, we optimized protocols for the isolation and culture of primary cerebellar granule neurons and determination of cell death after drug treatment by high-throughput imaging. CGNs isolated from WT, Aag-/-, and mAagTg mice exhibited cell sensitivity to MMS treatment that was dependent on Aag and Parp activity, thus recapitulating in vivo results and proving that CGN death is cell-intrinsic. Cell death was independent of caspases, mitochondrial depolarization, and AIF translocation. We did observe the formation of enlarged mitochondria and are investigating whether mitochondrial dynamics are causative of cell death in an Aag-dependent m, by Carrie Marie Margulies., Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011., Cataloged from PDF version of thesis., Includes bibliographical references., The stability of the genome is constantly challenged by both endogenous and exogenous DNA damaging agents. DNA damage, if left unrepaired, can give rise to permanent genetic alterations that ultimately increase our risk of cancer and other diseases. To combat these threats, eukaryotic cells activate a DNA damage response (DDR) that coordinates a wide variety of cellular processes including cell cycle progression, DNA repair, or in the case of severe/irreparable damage, apoptotic cell death. In addition to its role in cancer prevention, the DDR is fundamental in cancer treatment as noted by the numerous DNA damage-based chemotherapies. Thus, understanding how cell fate is determined by consequences of DDR is important for basic biological science and for medical applications. Alkylating agents comprise a major class of DNA damaging chemotherapeutics. The 0⁶MeG DNA lesion is a highly mutagenic, carcinogenic, and cytotoxic lesion produced by SNl methylating agents. Additionally, persistent 0⁶MeG lesions induce apoptosis in an 0⁶MeG DNA methyltransferase (MGMT) repair- and mismatch repair (MMR)-dependent manner. Here, we examine the DNA damage response induced by the 0⁶MeG lesion at both the molecular and cellular level after treatment with the SN1 methylating agent N-methyl-N'-nitro-Nnitrosoguanidine (MNNG). A systems-level approach combining various experimental techniques was used to quantitatively monitor the temporal regulation of DDR network proteins and, in parallel, phenotypic responses (cell cycle arrest, DNA replication, and apoptosis) induced by 0⁶MeG lesions. Through this approach, we have shown that TK6 human lymphoblastoid cells undergo cell cycle delay through both the first and second cell cycle post treatment. Furthermore, we demonstrate that 0⁶MeG triggers an intra-S-phase arrest in the second S-phase that ultimately leads to cell cycle progression and survival in cells with low/repairable amounts of damage or apoptosis in cells with high/irreparable a, by Ericka Marie Noonan., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011., Cataloged from PDF version of thesis., Includes bibliographical references., The stability of the genome is constantly challenged by both endogenous and exogenous DNA damaging agents. DNA damage, if left unrepaired, can give rise to permanent genetic alterations that ultimately increase our risk of cancer and other diseases. To combat these threats, eukaryotic cells activate a DNA damage response (DDR) that coordinates a wide variety of cellular processes including cell cycle progression, DNA repair, or in the case of severe/irreparable damage, apoptotic cell death. In addition to its role in cancer prevention, the DDR is fundamental in cancer treatment as noted by the numerous DNA damage-based chemotherapies. Thus, understanding how cell fate is determined by consequences of DDR is important for basic biological science and for medical applications. Alkylating agents comprise a major class of DNA damaging chemotherapeutics. The 0⁶MeG DNA lesion is a highly mutagenic, carcinogenic, and cytotoxic lesion produced by SNl methylating agents. Additionally, persistent 0⁶MeG lesions induce apoptosis in an 0⁶MeG DNA methyltransferase (MGMT) repair- and mismatch repair (MMR)-dependent manner. Here, we examine the DNA damage response induced by the 0⁶MeG lesion at both the molecular and cellular level after treatment with the SN1 methylating agent N-methyl-N'-nitro-Nnitrosoguanidine (MNNG). A systems-level approach combining various experimental techniques was used to quantitatively monitor the temporal regulation of DDR network proteins and, in parallel, phenotypic responses (cell cycle arrest, DNA replication, and apoptosis) induced by 0⁶MeG lesions. Through this approach, we have shown that TK6 human lymphoblastoid cells undergo cell cycle delay through both the first and second cell cycle post treatment. Furthermore, we demonstrate that 0⁶MeG triggers an intra-S-phase arrest in the second S-phase that ultimately leads to cell cycle progression and survival in cells with low/repairable amounts of damage or apoptosis in cells with high/irreparable a, by Ericka Marie Noonan., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009., Includes bibliographical references., Our DNA is constantly under the assault of DNA damaging agents that are ubiquitous in nature and unavoidable. Fortunately, our cells have evolved DNA repair mechanisms to maintain genomic integrity against this constant attack. An important type of DNA damage is alkylation damage, which has been the focus of this thesis, the major goal of which is to explore the biological role of a set of alkylation repair proteins, the E. coli AlkB and two human AlkB homologs (ABH2 and ABH3), and how they relate to the 3methyladenine DNA glycosylase (AAG). AAG is a base excision repair (BER) protein that has been well-studied and is known to be involved in the repair of a wide variety of substrates. On the other hand, the direct reversal protein AlkB, and its human homologs, have not been so extensively characterized, but it is known that they can repair not only DNA, but also RNA. Although there are eight human AlkB homologs, attention was focused on ABH2 and ABH3 since they are the more well-characterized homologs and recently implicated in DNA repair.In order to investigate the role of the AlkB proteins, particularly in mammalian cells, I expressed ABH2 and ABH3 in established human cell lines and investigated whether their expression would enhance cell survival after alkylation treatment. However, no detectable phenotype was observed in the cell lines upon treatment with the alkylating agent methyl methanesulfonate (MMS). This is possibly due to endogenous ABH levels being sufficient for repair. We therefore turned to characterization of the Abh2 and Abh3 null mice, as compared to wildtype and to another alkylation repair deficient animal, Aag null mice. In addition to the primary substrates 1methyladenine and 3-methylcytosine, AlkB, ABH2, and ABH3 can also repair an important class of damage, the etheno base DNA lesions, which can also be repaired by AAG., (cont.) Here we have shown in a mouse model that Abh2 and Abh3 overlap with Aag in protecting mice from sensitivity in response to chemically induced chronic inflammation, in which etheno base lesions are readily generated. In addition, we also employed a biochemical approach using a comprehensive library of lesion-containing DNA oligonucleotides to study the redundancy in repair activity between AAG and AlkB. In doing so, we have found new substrates for AAG and in particular, 1-methylguanine, is a new substrate shared between AAG and AlkB. Thus, although these two proteins employ different mechanisms for repair, our studies established further evidence of the interplay between these proteins and the different repair pathways they represent, underscoring the importance of alkylation damage repair for proper cell homeostasis., by Chun-Yue Lee., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009., Includes bibliographical references., Our DNA is constantly under the assault of DNA damaging agents that are ubiquitous in nature and unavoidable. Fortunately, our cells have evolved DNA repair mechanisms to maintain genomic integrity against this constant attack. An important type of DNA damage is alkylation damage, which has been the focus of this thesis, the major goal of which is to explore the biological role of a set of alkylation repair proteins, the E. coli AlkB and two human AlkB homologs (ABH2 and ABH3), and how they relate to the 3methyladenine DNA glycosylase (AAG). AAG is a base excision repair (BER) protein that has been well-studied and is known to be involved in the repair of a wide variety of substrates. On the other hand, the direct reversal protein AlkB, and its human homologs, have not been so extensively characterized, but it is known that they can repair not only DNA, but also RNA. Although there are eight human AlkB homologs, attention was focused on ABH2 and ABH3 since they are the more well-characterized homologs and recently implicated in DNA repair.In order to investigate the role of the AlkB proteins, particularly in mammalian cells, I expressed ABH2 and ABH3 in established human cell lines and investigated whether their expression would enhance cell survival after alkylation treatment. However, no detectable phenotype was observed in the cell lines upon treatment with the alkylating agent methyl methanesulfonate (MMS). This is possibly due to endogenous ABH levels being sufficient for repair. We therefore turned to characterization of the Abh2 and Abh3 null mice, as compared to wildtype and to another alkylation repair deficient animal, Aag null mice. In addition to the primary substrates 1methyladenine and 3-methylcytosine, AlkB, ABH2, and ABH3 can also repair an important class of damage, the etheno base DNA lesions, which can also be repaired by AAG., (cont.) Here we have shown in a mouse model that Abh2 and Abh3 overlap with Aag in protecting mice from sensitivity in response to chemically induced chronic inflammation, in which etheno base lesions are readily generated. In addition, we also employed a biochemical approach using a comprehensive library of lesion-containing DNA oligonucleotides to study the redundancy in repair activity between AAG and AlkB. In doing so, we have found new substrates for AAG and in particular, 1-methylguanine, is a new substrate shared between AAG and AlkB. Thus, although these two proteins employ different mechanisms for repair, our studies established further evidence of the interplay between these proteins and the different repair pathways they represent, underscoring the importance of alkylation damage repair for proper cell homeostasis., by Chun-Yue Lee., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009., Includes bibliographical references., Our DNA is constantly under the assault of DNA damaging agents that are ubiquitous in nature and unavoidable. Fortunately, our cells have evolved DNA repair mechanisms to maintain genomic integrity against this constant attack. An important type of DNA damage is alkylation damage, which has been the focus of this thesis, the major goal of which is to explore the biological role of a set of alkylation repair proteins, the E. coli AlkB and two human AlkB homologs (ABH2 and ABH3), and how they relate to the 3methyladenine DNA glycosylase (AAG). AAG is a base excision repair (BER) protein that has been well-studied and is known to be involved in the repair of a wide variety of substrates. On the other hand, the direct reversal protein AlkB, and its human homologs, have not been so extensively characterized, but it is known that they can repair not only DNA, but also RNA. Although there are eight human AlkB homologs, attention was focused on ABH2 and ABH3 since they are the more well-characterized homologs and recently implicated in DNA repair.In order to investigate the role of the AlkB proteins, particularly in mammalian cells, I expressed ABH2 and ABH3 in established human cell lines and investigated whether their expression would enhance cell survival after alkylation treatment. However, no detectable phenotype was observed in the cell lines upon treatment with the alkylating agent methyl methanesulfonate (MMS). This is possibly due to endogenous ABH levels being sufficient for repair. We therefore turned to characterization of the Abh2 and Abh3 null mice, as compared to wildtype and to another alkylation repair deficient animal, Aag null mice. In addition to the primary substrates 1methyladenine and 3-methylcytosine, AlkB, ABH2, and ABH3 can also repair an important class of damage, the etheno base DNA lesions, which can also be repaired by AAG., (cont.) Here we have shown in a mouse model that Abh2 and Abh3 overlap with Aag in protecting mice from sensitivity in response to chemically induced chronic inflammation, in which etheno base lesions are readily generated. In addition, we also employed a biochemical approach using a comprehensive library of lesion-containing DNA oligonucleotides to study the redundancy in repair activity between AAG and AlkB. In doing so, we have found new substrates for AAG and in particular, 1-methylguanine, is a new substrate shared between AAG and AlkB. Thus, although these two proteins employ different mechanisms for repair, our studies established further evidence of the interplay between these proteins and the different repair pathways they represent, underscoring the importance of alkylation damage repair for proper cell homeostasis., by Chun-Yue Lee., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007., Includes bibliographical references., The continuity of living organisms depends on their ability to protect their genomes from a constant assault by internal and external sources of damage. To this end, cells have developed a variety of mechanisms to avoid and repair damage to their genetic material. In this thesis, we analyze a yeast gene that has a previously uncharacterized role in the cell's ability to survive after DNA damage. This gene, ANC1, is interesting for several reasons. First, ANC1 is the only common member of seven different multiprotein complexes that all function in general RNA polymerase II-mediated transcription. Second, ANC1 is evolutionarily well-conserved between yeast and humans, suggesting that its function is critically important for survival. And finally, three out of four human homologs of ANC1 have a role in the MLL gene fusions associated with human acute leukemias. Here, we show that ANC1 falls into the same genetic pathway as several members of the postreplication repair (PRR) pathway, but has additive or synergistic relationships with other members of the pathway. Based on our epistasis data and our analysis of ANC1's role in mutagenesis, ANC1 functions in the error-free branch of PRR. Genetically, however, ANC1 is not in the same pathway as several canonical error-free branch members, and thus defines a new error-free branch of PRR. Similar to other genes involved in error-free PRR, ANC1 was found to have a role in suppressing the expansion of the Huntington's Disease-associated CAG triplet repeat. Additionally, we demonstrate a role for ANC1 in the global transcriptional response to MMS treatment: expression changes in transcripts regulated in response to environmental stress are significantly abrogated in ANC1 cells., (cont.) The regulation of this transcriptional response to environmental stress has previously been attributed to the Mec1 signaling pathway. To determine if ANC1's effect on global transcription is linked to Mec1signaling, we assayed the role of ANC1 in mediating the protein-level DNA damage response of Sml1, a downstream member of the Mec1 pathway. We observed that in the presence of MMS the Sml1 protein is abnormally degraded in ANC1 cells, indicating a possible role for ANC1 in this pathway., by Rachel L. Erlich., Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006., Includes bibliographical references (leaves 92-93)., The two most common forms of inflammatory bowel disease (IBD) are ulcerative colitis (UC) and Crohn's Disease (CD), which affect more than 1 million Americans. Recently the incidence of IBD has been rising in Japan, Europe and North America.' Colorectal cancer is a very serious complication of IBD, and a patient's risk increases with increasing extent and duration of disease.2 There is no cure for CD, and the only cure for UC is removal of the entire colon and rectum. It is thought that cancer risk is based on chronic inflammation of the gastrointestinal mucosa. There have been many studies, which have supported this idea and have made progress toward understanding the link between chronic inflammation and cancer. In both UC and CD, it is known that there are increased levels of EA, cG, and eC, which are potentially miscoding lesions, in the DNA of affected tissues.3 Also, 3-methyladenine DNA glycosylase (Aag in mice), an initiator of the Base Excision Repair pathway, shows adaptively increasing activity in response to increased inflammation in UC colon epithelium.4 This thesis demonstrates the importance of Aag in protecting against the effects of chronic inflammation., (cont.) It was found that Aag deficient mice, treated with 5 cycles of dextran sulfate sodium (DSS) to induce chronic inflammation, showed significant signs of increased disease including decreased colon length, increased spleen weight, and increase in epithelial defects. Also, when treated with a tumor initiator, azoxymethane, prior to DSS exposure, Aag deficient mice show a 2.95 fold (p<0.0001) increase in tumor multiplicity compared to wild type treated animals, as well as decreased colon length, increased spleen weight, increased dysplasia/neoplasia, and increased area affected by dysplasia/neoplasia. If UC patients had a deficiency in 3-methyladenine-DNA-glycosylase activity, they would likely be more susceptible to mutations and cancer because of their inability to repair DNA damage caused by inflammatory cytokines and reactive oxygen and nitrogen species. In future studies, it would be beneficial to determine if transgenic Aag over-expresser mice show protection against the damage induced by chronic inflammation. This would make intestinal gene therapy a possible approach to finding the first cure for IBD and inflammation associated colorectal cancer., by Stephanie Lauren Green., S.M.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006., Includes bibliographical references (leaves 92-93)., The two most common forms of inflammatory bowel disease (IBD) are ulcerative colitis (UC) and Crohn's Disease (CD), which affect more than 1 million Americans. Recently the incidence of IBD has been rising in Japan, Europe and North America.' Colorectal cancer is a very serious complication of IBD, and a patient's risk increases with increasing extent and duration of disease.2 There is no cure for CD, and the only cure for UC is removal of the entire colon and rectum. It is thought that cancer risk is based on chronic inflammation of the gastrointestinal mucosa. There have been many studies, which have supported this idea and have made progress toward understanding the link between chronic inflammation and cancer. In both UC and CD, it is known that there are increased levels of EA, cG, and eC, which are potentially miscoding lesions, in the DNA of affected tissues.3 Also, 3-methyladenine DNA glycosylase (Aag in mice), an initiator of the Base Excision Repair pathway, shows adaptively increasing activity in response to increased inflammation in UC colon epithelium.4 This thesis demonstrates the importance of Aag in protecting against the effects of chronic inflammation., (cont.) It was found that Aag deficient mice, treated with 5 cycles of dextran sulfate sodium (DSS) to induce chronic inflammation, showed significant signs of increased disease including decreased colon length, increased spleen weight, and increase in epithelial defects. Also, when treated with a tumor initiator, azoxymethane, prior to DSS exposure, Aag deficient mice show a 2.95 fold (p<0.0001) increase in tumor multiplicity compared to wild type treated animals, as well as decreased colon length, increased spleen weight, increased dysplasia/neoplasia, and increased area affected by dysplasia/neoplasia. If UC patients had a deficiency in 3-methyladenine-DNA-glycosylase activity, they would likely be more susceptible to mutations and cancer because of their inability to repair DNA damage caused by inflammatory cytokines and reactive oxygen and nitrogen species. In future studies, it would be beneficial to determine if transgenic Aag over-expresser mice show protection against the damage induced by chronic inflammation. This would make intestinal gene therapy a possible approach to finding the first cure for IBD and inflammation associated colorectal cancer., by Stephanie Lauren Green., S.M.