1,721,046 research outputs found
Mitotic homologous recombination at engineered repeats in Saccharomyces cerevisiae and in novel transgenic mice
No full textThesis (Ph. D. in Genetic Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.Includes bibliographical references.Although homologous recombination provides an efficient means for repairing and tolerating DNA damage, mitotic recombination between misaligned sequences can lead to loss of genetic information (e.g. deletions, translocations and loss of heterozygosity). Given that such genetic changes may promote tumorigenesis, it is critical to identify those genetic and environmental factors that render cells susceptible to homologous recombination. Our goal is to elucidate the mechanisms of DNA damage-induced recombination and to determine the role of DNA repair enzymes in modulating homologous recombination in eukaryotic cells. Alkylating agents are abundant in our environment and are generated endogenously as normal metabolites. In addition to their mutagenic and cytotoxic effects, alkylating agents stimulate homologous recombination in eukaryotic cells. Removal of alkylated bases by DNA glycosylases, such as the Magl 3-methyladenine (3MeA) DNA glycosylase, initiates the base excision repair (BER) pathway. To investigate the molecular basis for methylation-induced homologous recombination in S. cerevisiae, intrachromosomal recombination was measured under conditions where MAGI expression levels were varied. Cells lacking Magl show increased susceptibility to methylation-induced recombination, suggesting that unrepaired 3MeA lesions induce recombination. Overexpression of M4GI also elevates recombination levels, presumably due to the accumulation of recombinogenic BER intermediates.(cont.) To study the relative importance of specific DNA repair enzymes in modulating recombination in mammals, we have engineered transgenic mice that make it possible to quantify homologous recombination events in primary somatic cells, both in vitro and in vivo. The FYDR (fluorescent yellow direct repeat) mice carry two different mutant copies of an expression cassette for enhanced yellow fluorescent protein (EYFP) arranged in a direct repeat. Homologous recombination between these truncated sequences restores expression of EYFP. Using flow cytometry, spontaneous and DNA damage-induced recombination events were quantified in primary fibroblasts cultured from embryonic and adult tissues. In addition, recombination events that occurred in vivo were detected directly in disaggregated skin cells. Currently, FYDR mice are being crossed with mice carrying engineered defects to determine how specific gene traits modulate susceptibility to mitotic recombination. Ultimately, this tool will help us better understand how environmental agents and specific genes influence cellular susceptibility to cancer-promoting recombination events in mammals.by Carrie A. Hendricks.Ph.D.in Genetic Toxicolog
The impact of age, exposure and genetics on homologous recombination at the engineered repeat sequence in mice
Full text linkThesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references.Mitotic homologous recombination is a critical pathway for the repair of DNA double-strand breaks and broken replication forks. Although homologous recombination is generally error-free, recombination between misaligned sequences can lead to deleterious sequence rearrangements, and conditions that stimulate homologous recombination are associated with an increased risk of cancer. To study homologous recombination in vivo, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which a homologous recombination event at a transgene yields a fluorescent cell. To study homologous recombination using FYDR mice, we developed one- and two-photon in situ imaging techniques that reveal both the frequency and the sizes of isolated recombinant cell clusters within intact pancreatic tissue. We then applied these tools to analyze the effects of cancer risk factors such as exposure, genetic predisposition and age on homologous recombination in vivo. To determine the effect of exposure to exogenous carcinogens on homologous recombination, FYDR mice were treated with two different chemotherapeutic agents, cisplatin and mitomycin-C.(cont.) Results show that exposure to these DNA damaging agents causes an induction of recombinant pancreatic cells in vivo, indicating that homologous recombination is an active repair pathway in adult pancreatic cells and that exposure to certain carcinogens stimulates recombinational repair. As a first step towards exploring the effect of genetic predisposition to genomic instability on homologous recombination in vivo, FYDR mice were crossed with mice carrying a defect in p53, a critical tumor suppressor that is mutated in almost 50% of all human tumors. Although loss of p53 is known to promote genomic instability, results show that p53 status does not significantly affect the spontaneous recombinant cell frequency in the pancreas in vivo or the rate of homologous recombination in cultured fibroblasts in vitro. Age is a risk factor for many types of cancers. Here we examined the effect of age on homologous recombination in two tissues of FYDR mice, pancreas and skin. In the pancreas, a dramatic accumulation of recombinant cells is seen with age, resulting from both de novo recombination events and clonal expansion of recombinant cells. In contrast, the skin shows no increase in recombinant cell frequency with age.(cont.) In vitro studies using primary fibroblasts indicate that the ability to undergo homologous recombination in response to endogenous and exogenous DNA damage does not significantly change with age, suggesting that these skin cells are able to undergo de novo homologous recombination events in aged mice. Thus, we propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of these cells to persist and clonally expand within the tissue. To further characterize the FYDR mice as a tool for studying homologous recombination, we exploited positive control FYDR-Recombined mice in which all cells carry the full-length coding sequence for enhanced yellow fluorescent protein. Studies show that expression of the FYDR transgene varies among mice, among tissues, and even among cells within a tissue. However, the variation in FYDR expression does not significantly change with age or exposure to exogenous carcinogens. Furthermore, positive control mice reveal that several tissues, in addition to the pancreas and skin, may be amenable for studying homologous recombination in the FYDR mice.(cont.) Thus, our studies demonstrate that FYDR mice combined with in situ imaging technology provide powerful tools to study the effects of cancer risk factors on homologous recombination in vivo. Ultimately, by applying these techniques to study additional cancer risk factors, we may better understand the relationship between DNA damage, homologous recombination and cancer.by Dominika M. Wiktor-Brown.Ph.D
Glycosaminoglycan mediated differentiation in stem cells
Full text linkIncludes bibliographical references.Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005."February 2005." Vita.(cont.) Such therapeutic approaches require an understanding of the mechanisms regulating stem cell differentiation. The second part of this thesis investigates the role of HSGAGs in embryonic stem cell differentiation into endothelial cells. Differentiation of stem cells was accompanied by increases in the transcript levels of key HSGAG-biosynthetic enzymes, and the quantity of cell surface HSGAGs. Differentiation into endothelial cells was inhibited by ablation of the HSGAG-biosynthetic machinery by chlorate treatment, or by the enzymatic degradation of the HSGAGs. Exogenous addition of heparin to chlorate-treated cells partially restored differentiation into endothelial cells. These effects were mirrored in phospho-ERK levels, suggesting the involvement of the MAPK pathway. These results suggest that stem cell differentiation can be regulated by modulating the HSGAG moiety and this opens up new treatment modalities for cancer therapy and regenerative medicine.The new paradigm is that cancers may originate from stem cells, and that terminal differentiation of stem cells is a possible treatment for cancers. Understanding the origin of cancers requires elucidation of factors causing genetic rearrangements. The first part of this thesis explores the effects of NO on homologous recombination in embryonic stem cells. Using terminal differentiation of stem cells as a therapy for cancer necessitates an understanding of factors governing their differentiation. The second part of this thesis explores the effects of heparan sulfate glycosaminoglycans (HSGAGs) on stem cell differentiation. Inflammation is increasingly recognized as an important risk factor for cancer. During inflammation, macrophages secrete NO, which reacts with superoxide or oxygen to produce ONOO⁻ or N₂O₃, respectively. Although ONOO⁻ and N₂O₃ are potent DNA damaging agents, little was known about the ability of these agents to induce homologous recombination in mammalian cells. Homologous recombination events are a significant source of mutations that are likely to contribute to initiation and progression of some cancers. In the first part of this thesis, the recombinogenic potential of ONOO⁻ and N₂O₃ was characterized by sister chromatid exchanges, chromosomal direct repeat substrate and interplasmid recombination assays. Our results show that on a per lesion basis, ONOO⁻ -induced oxidative base lesions and single strand breaks are more recombinogenic than N₂O₃-induced base deamination products. These results are in accordance with the model that ONOO⁻ -induced recombination may contribute to inflammation-induced cancer. Directed differentiation of stem cells holds an immense potential for regenerative medicine as well as cancer therapy.by Tanyel Kiziltepe.Ph.D
Leveraging cell micropatterning technology for rapid cell-based assessment of chemical toxicity and population variation in toxicity susceptibility
Full text linkThesis: 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-throughput platform that can directly measure a cell's ability to divide and has the potential to provide highly sensitive and rapid toxicity assessment of chemicals of interest. The technology is based on the use of a microcolony array where the size distributions for different conditions provide a direct measure of cell survival. We have results showing that MicroColonyChip is as sensitive as the gold standard assay, reduces ~80% incubation time, and requires ~250x less surface area for cell growth. In addition to detecting genotoxic agents, it is also important to understand how an individual responds to internal and external assaults to DNA as a necessary first step for assessment of human health outcomes. There is a high variability in DNA repair capacity among people, and more studies are needed to elucidate whether a causal relationship between DNA repair capacity and clinical outcomes exists. We applied CometChip to study repair kinetics in human primary lymphocytes. In order to account for the extensive crosstalk and competition between different repair pathways, repair of different types of DNA damage was measured. To test the assay's sensitivity and reproducibility, a small population of 56 healthy volunteers were recruited to give blood samples. Isolated lymphocytes from different individuals show significant differences in repair kinetics of oxidative damage and a sevenfold variation in repair rates. Taken together, the work described here represents significant technological advances in addressing a number of major challenges in chemical toxicity testing as well as in the evaluation of health outcome variability across populations. The technologies also open doors to exciting opportunities in personalized strategies for disease prevention and intervention.by Le Phuong Ngo.Ph. D
Exploring the mutagenic consequences of inflammation and DNA damage
Full text linkThesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.Cataloged from PDF version of thesis.Includes bibliographical references.Inflammation is a major risk factor for many types of cancer, and the physiological processes involved in inflammation can contribute to many aspects of cancer development. Inflammation entails reprogramming of cell behaviors that resemble cancer, such as increased proliferation and signals for survival and migration, and it also entails production of reactive chemical species, which can damage DNA to promote genetic instability, another hallmark of cancer. While much research has been dedicated to studying the relationships between inflammation and cancer, it has been difficult to distinguish the relative contributions of modified cell behavior and de novo mutagenesis to the development of cancer. Furthermore, few studies have addressed the role(s) inflammation plays in cancer initiation versus promotion. Here, we utilized a transgenic mouse for detecting mutations in a variety of models of inflammation to parse the mechanisms by which inflammation contributes to mutations and cancer. The RaDR mouse, developed in the Engelward lab, contains a ubiquitously expressed transgene that enables detection of sequence rearrangement mutations following aberrant homologous recombination (HR). These mice also contain the Gpt-[delta] transgene for detecting point mutations and deletions, enabling unprecedented breadth and depth of possible mutation analyses in a single tissue. Our studies began by querying whether elements that regulate inflammation protect against mutagenesis in RaDR animals. We then studied RaDR mutagenesis in several models of intestinal inflammation and cancer. Together, these experiments showed that inflammation does not significantly induce de novo sequence rearrangement mutations, but it greatly increases the overall burden of mutant cells in a tissue as a result of heightened proliferation and clonal expansion. We also used the RaDR mouse model to expand upon studies of DNA repair pathway balance. DNA damage is addressed by a network of pathways, each designed to identify and repair specific types of lesions. One of the most important repair pathways for DNA damage caused by inflammation is the Base Excision Repair (BER) pathway, and we have previously found that BER intermediates can increase the frequency of mutagenic HR. Here, we expand upon that information, showing that acceleration of the BER pathway by increased expression of an initiating enzyme does not increase sequence rearrangement mutations, provided the downstream pathway can be resolved efficiently. Together, the studies described herein demonstrate that inflammation is unlikely to initiate cancer via sequence rearrangement mutations, but inflammation is a strong promoter of cancer in part through increased clonal expansion of mutant cells.by Jennifer Elizabeth Kay.Ph. D
Fluorescent detection of homologous recombination reveals the impact of genetic, physiological, and environmental factors on genomic stability
No full textThesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013.Page 200 blank. Cataloged from PDF version of thesis.Includes bibliographical references.Unless repaired correctly, DNA double strand breaks (DSBs) can cause the loss of millions of base pairs of information and can induce cellular toxicity. DSBs are repaired via mitotic homologous recombination (HR), non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ). Here we use the Fluorescent Yellow Direct Repeat (FYDR) mouse to examine these pathways. Specifically, we crossed FYDR mice with mice lacking an essential NHEJ protein. Consistent with in vitro studies, we observed an increase in HR in the NHEJ deficient mice, indicating a shift from one pathway to another. Additionally, FYDR mice deficient in ERCC1, a protein involved in several pathways including nucleotide excision repair and MMEJ, showed an increase in HR. We describe a possible model for this observation. HR is presumed to be largely limited to replicating cells; however, little is known about differences in HR rates between tissues. Thus, we engineered the Rosa26 Direct Repeat-GFP (raDR-GFP) mouse that enables study of HR in many tissues in response to endogenous and exogenous factors. The raDR-GFP mouse harbors two truncated EGFP genes integrated at the ROSA26 locus. HR at the locus yields a full-length EGFP gene and a fluorescent cell. In adult raDR-GFP mice, differences in frequency of recombinant cells among tissues of challenged and unchallenged mice demonstrate the utility of raDR-GFP mice in measuring exposure-induced HR and the importance of multi-tissue studies. We also observed the progressive accumulation of recombinant cells in the pancreas, liver, and colon with age. These data are consistent with the finding that cancer is an age-related disease requiring time to accumulate tumorigenic mutations. To test the hypothesis that chronic inflammation promotes the induction of DSBs, we bred raDR-GFP mice deficient in an anti-inflammatory cytokine. These mice showed an increase in spontaneous HR in the pancreas. Interestingly, 10 week-infection of RAG2-/- raDR-GFP mice with H. hepaticus, and longer-term 20-week infection with H. trogontum did not have the same effect on HR in the pancreas, liver, or colon. Further studies of large-scale sequence rearrangements, point mutations, and small deletions in multiple tissues in response to environmentally-induced inflammation are planned.by Michelle R. Sukup Jackson.Ph.D
Investigation into the role of deoxyribonucleic acid damage and repair during influenza infection and inflammation
No full textThesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.Cataloged from PDF version of thesis.Includes bibliographical references.The DNA in every cell accrues nearly 100,000 lesions daily from both endogenous and exogenous sources. The accumulated damage, e.g. strand breaks and base lesions, can lead to mutations, cell death, and cancer if not repaired efficiently. To protect genome integrity, organisms have evolved multiple DNA repair processes. A deeper comprehension of DNA damage and repair during disease pathogenesis can aid the development of novel therapeutics to reduce the damage and ameliorate the disease. Here, we studied DNA damage and repair in two inflammatory contexts. First, we investigated the role of DNA damage and repair during influenza infection, a common viral respiratory disease with an active inflammatory response. Second, we examined the effects of S-nitrosation, a post-translational modification that is common in inflammatory regions, on repair of alkylation damage. Influenza induces an excessive inflammatory response in the host and a reduction in inflammation reduces morbidity. While inflammation can cause DNA damage and induce DNA repair in other inflammatory contexts, there has been minimal analysis on the existence and function of DNA damage and repair during influenza infection. Utilizing immuno-fluorescent analysis of double strand break markers, we observed an increase in strand breaks both in vitro and in vivo. Influenza infected mice also displayed a significant increase in homologous recombination (HR) gene and protein expression during the recovery phase of infection in multiple virus and mouse backgrounds. Moreover, influenza infected mice deficient in DNA repair proteins AAG, ALKBH2, and ALKBH3, displayed increased morbidity and HR protein expression when compared to wild type. Together, these results raise the possibility of a role for DNA repair and more specifically HR during influenza infection. To study the effects of inflammation on DNA repair protein function, we analyzed the capacity of cells treated with S-nitrosoglutathione (GSNO), a nitrosating agent, to repair alkylation damage. GSNO-exposed cells displayed dysregulation in the activities base excision repair (BER) proteins. Following challenge with an alkylating agent, GSNO-exposed cells had an increase in repair intermediates and reduced viability, suggesting that GSNO exposure inhibits BER completion. The knowledge gained from these studies lays the groundwork for new prevention strategies and novel therapeutics.by Marcus Curtis Parrish.Ph. D
Direct and indirect modulation of inflammation-induced DNA damage
No full textThesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 164-183).Cancer causes 13% of all deaths worldwide. Inflammation-mediated cancer accounts for ~15% of all malignancies, strongly necessitating investigation of the molecular interactions at play. Inflammatory reactive oxygen and nitrogen species (RONs), including peroxynitrite and nitric oxide (NO'), may potentiate malignancy. We hypothesize that the base excision repair (BER) pathway modulates susceptibility to malignancy, by modulating the BER-intermediate levels, large scale genomic rearrangements and toxicity following exposure to RONs. We further hypothesize that DNA methyltransferases are responsible for the memory of genotoxic insult, and the epigenetic propagation of genomic instability, following exposure to genotoxins. Here, we exploited cell lines engineered to carry deficiencies in BER to study repair of DNA damage induced by RONs. Toxicity and BER-intermediate levels were evaluated in XRCC1 proficient and deficient cells, following exposure to the peroxynitrite donor, SIN-1 and to NO*. Using the alkaline comet assay, we find that while XRCC1 proficient and deficient CHO cells incur equivalent levels of SIN-1 induced BER-intermediates, the XRCC1 null cells are more sensitive to killing by SIN-1, as assessed by clonogenic survival. Furthermore, using bioreactors to expose CHO cells to NO', we found that the BER-intermediate levels measured in XRCC1 null cells were lower than in WI cells. We found that while XRCC1 can facilitate AAG-mediated excision of the inflammation-associated base lesions ethenoadenine and hypoxanthine, in vitro; XRCC1 deficient human cells were no more susceptible to NO' than WT cells. However, in live glioblastoma cells, XRCC1 is acting predominantly downstream of AAG glycosylase. This work is some of the first to assess the functional role of XRCC1, in response to RONs and suggests complexities in the role of XRCC1. We also demonstrate that the underlying basis for the memory of a genotoxic insult and the subsequent propagation of genomic instability is dependent on the DNA methyltransferases, Dnmtl and Dnmt3a. We found that a single exposure led to long-term genome destabilizing effects that spread from cell to cell, and therefore provided a molecular mechanism for these persistent bystander effects. Collectively, our findings impact current understanding of cancer risk and suggest mechanisms for suppressing genomic instability, following exposure to inflammatory genotoxins.by James T. Mutamba.Ph.D
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