Thesis (S.M. in Toxicology)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 50-54). / One and a half million new cancer cases are reported each year in the United States. Despite this overwhelming burden of disease, current preventative treatments and early detection techniques are inadequate. With cancers, as with many aggressive diseases, time is of the essence; earlier detection begets better patient prognoses. While contemporary technology offers physicians the ability to battle cancers via stage-one detection, few reliable biomarkers have been developed to assist in detection of upstream tumorigenesis or interpretation of early genomic assault. The objective of the work described here is to identify early biomarkers specific to tumorigenesis, which correlate with initial genomic assault and subsequent mutation in the gpt-delta (B6C3F1) mouse model. The test compound used in this work is aflatoxin B₁, a known human carcinogen produced by fungal spoilage of food materials. Aflatoxin B₁ was one of the carcinogens used in the "training set" of compounds that defined the B6C3F1 animal model. My work had three goals with regard to upgrading this model as a tool for contemporary toxicology. In the first part of the thesis, a kinetic profile was generated of AFB 1-DNA adduct formation and removal. In the second part of the work, adduct levels and specific DNA damage patterns of males and females were compared (males are generally more sensitive than females to this and other toxins). Third, nursing mother mice were treated with two chemo-preventive agents in an attempt to determine if chemoprevention of the dam would lead to protection of her children. This thesis documents generation of a 48-hour time-course assessing adductburden in four-day-old B6C3F1 neonates. These burdens are measured in adducts per mega-base of genomic DNA (based on a single 6mg/kg dose of AFB₁). As previous studies show that 6mg/kg at this age results in near 100% liver tumorigenesis, this timecourse provides significant intuition for the onset and persistence of DNA damage. The results showed that AFB₁-N7-Guanine adduct formation maximized at two hours post dosing and then decreased rapidly; its FAPY derivative proved to be much more stable with time. A slight excess adduct burden was observed in males from 2-48 hours post dosing. Systematic differences in gene expression were observed in nursing female mother mice that were either treated or not with R,S-sulforaphane or D3T (3H-1,2- dithiole-3-thione). While minimal gene expression changes were observed in pups nursed by a 2mg R,S-sulforaphane treated dam, those nursing from a 5mg R,S-sulforaphane treated mother experienced much greater effects. At 300 umol/kg doses of D3T to the mother, no statistically significant gene expression profile alterations were observed in the pups. The work described here did not identify conditions in which a chemopreventive pattern of gene expression in the mother could be transferred efficiently to her offspring. / by Jason T. Bouhenguel. / S.M.in Toxicology

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Title as it appears in MIT Commencement Exercises program, June 2013: Modulation of the ERK1/2 and p53 signaling pathways by nitric oxide during the response to cisplatin in melanoma cells. Vita. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 51-58). / Malignant melanoma causes the highest mortality rate in skin cancers. Although cisplatin has proved efficacious in the treatment of various solid tumors, melanoma seems particularly resistant to this chemotherapeutic drug. Reports show that melanoma patients whose tumors express nitric oxide (NO) synthase and/or nitrotyrosine are often faced with poor prognosis. Moreover, it has been shown that NO produced by melanoma cells sustains lower sensitivity to cisplatin toxicity in vitro. Because inflammatory products such as NO and reactive oxygen species (ROS) are associated with the genesis and evolution of cancer, we hypothesized that these oxidative species may regulate key components of the response of melanoma to cisplatin. Using a system for controlled delivery of NO to simulate the NO levels believed to occur during inflammation, we showed that human melanoma (A375) cells pre-exposed to submicromolar NO concentrations were protected from a subsequent challenge with cisplatin. This protection was strongly associated with increased activity of the MAP-kinase cascade leading to activation of ERK1/2, as well as with downstream modulation of the apoptotic factors Bax and Bcl-2, and the transcription factors p53 and MiTF. Although NO favored increased expression and phosphorylation of p53, it also increased the expression of the p53 inhibitor MDM2, which may have counteracted p53-induced apoptosis upon cisplatin treatment. Also, likely via ERK1/2 activation, NO favored phosphorylation of MiTF, which is associated with survival signals. Furthermore, NO displayed the remarkable ability to overcome the effect of U0126, a MEK inhibitor, and promoted continuous phosphorylation of ERK1/2 (and hence cell survival), in contrast to cells not exposed to NO. Results also demonstrated that cisplatin-induced apoptosis was substantially decreased by the antioxidant precursor N-acetylcysteine (NAC). Unlike exogenous NO, cisplatin-induced ROS were linked to lower activation of ERK1/2, which was reversed by NAC. During the treatment with cisplatin, NAC led to lower levels of p53, which may have partially contributed to increased cell survival. However, in contrast to NO, NAC did not significantly alter the effects of cisplatin upon MiTF and apoptotic proteins studied. Altogether, our findings illustrate the complexity of the regulation of signaling components by oxidative species of distinct natures. / by Chase Thaddeus Maceo Anderson. / S.M.

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 amounts of damage. Based on the signaling and phenotypic data acquired, we developed a conceptual model for MMR's role in triggering cell cycle arrest and cell death. In addition, exploration of the global transcriptional response provided an unbiased approach to further elucidate previously unrecognized biological processes involved in the response to MNNG-induced damage. Taken together, our results have enhanced our understanding of the cellular response to alkylation-induced damage and will contribute to the development of personalized chemotherapeutic treatments in the future. / 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 (p. 217-232). / The critical first step in the host infection by influenza A virus is the binding of the viral surface glycoprotein hemagglutinin (HA) to the sialylated glycan receptors terminated by N-acetyineuraminic acid (Neu5Ac) expressed on the host cell surface. Glycans terminating in Neu5Ac that is a2-6 and a2-3 linked to the penultimate galactose serve as receptors for human- and avian- adapted influenza A virus respectively. This thesis focuses on studying HA, glycan receptors and their interactions both in a biochemical and physiological context to understand the role of these interactions in influenza A virus pathogenesis. The first Specific Aim of this thesis deals with understanding the molecular determinants of glycan receptor-binding specificity and affinity of HA (or avidity in the context of the whole virus) and how these properties govern antigenic drift and efficiency of airborne transmission. This approach contributed to uncovering the relationship between receptor-binding affinity and efficiency of transmission of the 2009 H1N1 pandemic influenza A virus and also predicting the evolution of this virus into a more transmissible strain. The second Specific Aim of this thesis focuses on understanding the distribution of the glycan receptors for human-adapted HA (going beyond a2-3/aX2-6 linkages), in ferret (animal model for influenza research) and in human respiratory tracts. Based on this understanding, this part of the thesis contributed to developing new anti-viral strategies based on targeting the host glycan receptors (instead of the common strategies that directly target the viral proteins) Overall, this thesis has provided functional insights into the role of HA-glycan interaction in viral pathogenesis. As part of this research, various tools and methods were developed. Further, such an approach paves way for elucidating the functional significance of important protein-glycan interactions in other disease models. / by Akila Jayaraman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references. / Due to its common dysregulation in epithelial-based cancers and the extensive characterization of its role in tumor growth, epidermal growth factor receptor (EGFR) has long been an attractive target for monoclonal antibodies. Intense research has culminated in the approval of two antibody-based drugs against EGFR for cancer treatment, with numerous others in clinical trials. However, therapeutic efficacy of these drugs has been disappointingly low due to autocrine signaling, receptor mutation, and transport limitations, necessitating novel antibody designs and mechanisms of action. Recently, it was reported that treatment with combinations of antibodies can induce receptor clustering, leading to synergistic receptor downregulation and anti-tumor activity. The aim of this thesis is to elucidate the details of this phenomenon and to exploit this mechanism to design more effective therapeutic antibodies targeting EGFR. We first illuminate several key aspects of combination antibody-induced clustering. By screening a panel of pairwise combinations, we show that the most potently downregulating pairs consist of two non-competitive antibodies that target EGFR extracellular domain 3. We further find the mechanism underlying downregulation to be consistent with recycling inhibition. Lastly, in contrast to the agonism associated with ligand-induced downregulation, we demonstrate that combination mAb-induced downregulation does not activate EGFR or its downstream effectors and it leads to synergistic reduction in migration and proliferation of cells that secrete autocrine ligand. To enhance antibody binding and induced receptor clustering, we design multispecific antibodybased constructs that engage up to four distinct epitopes on EGFR. We engineer two classes of constructs: one consisting of a full EGFR-specific antibody fused to the variable domain of a second anti-EGFR antibody and the other consisting of a full EGFR-specific antibody fused to one or more EGFR-targeted tenth type three domains of human fibronectin. Both classes of constructs induce robust receptor clustering and downregulation in the absence of signal activation. In vitro downregulation correlates well with in vivo inhibition of tumor growth in several mouse xenograft tumor models and mutational analysis demonstrates that the efficacy of our fusions is attributable to both signaling effects and antibody-dependent cell-mediated cytotoxicity. Our multi-epitopic strategy may be readily applied to other receptor systems to form the basis for a new category of antibody-based therapeutics. / by Jamie Berta Spangler. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 197-212). / Regulated cell-surface proteolysis underpins key processes of cellular growth and motility in both physiological and pathological contexts. However, comprehending how multiple proteolytic events cohesively integrate to yield context-dependent cellular behavior remains a challenge in the fields of both protease biology and systems biology in general. This work begins to address that challenge by quantitatively investigating the integrated effect of multiple diverse proteolytic events and their interaction with cell-signaling pathways from a computational network perspective, particularly focusing on A Disintegrin and Metalloproteinases (ADAMs). ADAMs have been studied for decades as the principal cell-surface "sheddases" responsible for cleaving growth factor ligands and receptor tyrosine kinase ectodomains from the cell surface. However, activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent sheddase function, and clinical trials targeting metalloproteinases in cancer have failed in part due to a poor understanding of the complex functions they mediate. This thesis outlines a conceptual framework for studying protease network biology (Chapter 1), describes novel experimental methods designed for such a framework (Chapters 2-3), and applies both to understand protease regulation in invasive disease (Chapter 4). Using combined measurement and computational modeling, we present a paradigm for monitoring and analyzing complex networks of protease activities that interface with signaling pathways to influence cellular migration in the invasive diseases of cancer and endometriosis. We find sheddase activity integrates with signaling pathways to direct cell migration, especially through concomitant proteolysis of both ligands and receptors. We find that indirect reduction of sheddase activity through kinase inhibition can lead to an accumulation of growth-factor receptors on the cell surface, consequently producing undesired compensatory signaling feedback. Thus, here we present a novel mechanism of rapid, protease-driven resistance to kinase inhibitors, and we subsequently demonstrate strategies for overcoming resistance through drug combinations. We develop a novel microfluidic platform to study protease activities in clinical samples, and apply the technology to study the peritoneal fluid from endometriosis patients. Results indicate joint dysregulation of sheddase activity with disease. Overall, this work provides a model for measuring, understanding, and targeting networks of proteases and the kinases with which they interact. / by Miles Aaron Miller. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 236-275). / Glycans are an important class of biological molecules that regulate a variety of physiological processes such as signal transduction, tissue development and microbial pathogenesis. However, due to the structural complexity of glycans and the unique intricacies of glycan-protein interactions, elucidating glycan structure-function relationships is challenging. Thus, uncovering the biological function of glycans requires an integrated approach, incorporating structural analysis of glycans, and glycan-proteins interactions with functional analysis. In this thesis, I develop new tools and implement integrated approaches to study glycans and glycan-binding proteins (GBPs). I apply these approaches to study glycans and GBPs in two areas: i) the role of hemagglutinin-glycan receptor specificity in human adaptation and pathogenesis of influenza and ii) the function of glycan regulation of cell-microenvironment interaction in cancer progression. Section 1: Influenza poses a significant public health threat and there is a constant looming threat of a pandemic. Pandemic viruses emerge when avian viruses acquire mutations that enable human adaptation, leading to infection of an antigenically naive host. Influenza Hemagglutinin (HA), and HA-glycan receptor interactions, play a central role in host tropism, transmissibility, and immune recognition. In section one, I develop and apply an integrated approach comprised of structural modeling, inter-amino acid network analysis, biochemical assays, and bioinformatics tools to study the hemagglutinin-glycan interaction and, in some cases, HA's antigenic properties. Using this approach, we i) identify the structural determinants required, and potential mutational paths, for H5N1 to quantitatively switch it's binding specificity to human glycans receptors, ii) identify the mutations that enable the 2013 outbreak H7N9 HA to improve binding to human glycan receptors in the upper respiratory tract, iii) uncover H3N2 strains that are currently circulating in birds and swine that possess features of a virus that could potentially re-emerge and cause a pandemic, and iv) characterize the glycan binding specificity of a novel 2011 Seal H3N8 HA. The approaches implemented here and the findings of these studies provide a framework for improved surveillance of influenza viruses circulating in non-human hosts that pose a pandemic threat. Section 2: Glycans are abundant on the cell surface, and at the cell-ECM interface where they mediate interactions between cells and their microenvironment. Despite this, the function of glycans in cancer progression remains largely understudied. Here, I develop an integrated approach to characterize the cell surface glycome, including N-linked, 0-linked glycans, and HSGAGs. This approach integrates glycogene expression data, analytical tools, and glycan binding protein reagents. I demonstrate that this platform enables rapid and efficient characterization of the N- and 0-linked glycome in a model cell system, representing metastatic versus non-metastatic cancer cells. Next, I apply this integrated approach to uncover new roles of glycans. I study the role that HSGAGs play in regulating cancer stem cell (CSC) activity in breast cancer. Here, we report that SULF1, an HSGAG modifying enzyme, is required for efficient tumor initiation, growth and metastasis of CSCs. Furthermore, we identify a putative mechanism by which SULF1 regulates interactions between CSCs and their microenvironment. The approaches implemented here and the finding of these studies Overall, this thesis provides important tools, approaches and insights to enable and improve the study of glycans and glycan binding proteins. Together the work here provides a framework for decoding structure-function relationship of glycans. / by Nathan Wilson Stebbins. / Ph. D.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 89-95). / Understanding the elastic and viscoelastic responses of biological soft tissues and engineered polymer simulants is of great interest to predicting and preventing penetrative injuries. Detailed understanding of the mechanical processes at work could aid in the development and evaluation of protective strategies such as armor and helmets, and repair strategies including robotic surgery or needle-based drug delivery. However, due to the mechanical complexity of so-called "soft tissues," including nonlinear viscoelastic behavior, surface adhesion, material failures and shock effects, the experimental characterization of various soft tissues is challenging and individual mechanical processes are often impossible to decouple without computational models and simulations. This thesis presents two finite element models designed to provide both replicate the results of indentation and impact experiments on synthetic polymers, aimed to decouple competing mechanical characteristics of contact based deformation. The first of these models describes the indentation on polydimethylsiloxane bilayer composites, with the aim of describing the relative effects of a adhesion and viscoelastic properties on the measured deformation response. That model expands on this objective via the analysis of the effects of surface adhesion commonly associated with highly compliant polymers and tissues. The second model attempts to replicate impact of a high velocity projectile on a relatively stiff material, polyurethane urea, and on a comparatively compliant polymer, gelatin hydrogel. These models provide means to simulate, predict and characterize material response, validated by comparison with available experiments. Such validated models can be used to simulate and design new materials as tissue simulants or as protective media that predictably dissipate concentrated mechanical impact. / by Kyle Geiser. / S.M.

Thesis (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.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 121-132). / During the process of metastasis, tumor cells must undergo changes that enable them to detach from a tumor, exit surrounding tissue during intravasation, enter into circulation and eventually stop at a distant site for extravasation. Here, we measure the physical changes in the deformability of tumor cells, indicated by the length of time required to pass through a microfluidic constriction in a suspended microchannel resonator (SMR), as related to different stages of the metastatic process-particularly, in an epithelial-mesenchymal transition (EMT) and existence in the circulation. We find that a mesenchymal population of murine tumor cells (MMTV-PyMT) that had undergone a spontaneous EMT at the primary tumor site were more deformable than the parental population of epithelial cells. In contrast, MMTV-PyMT and Ep5 murine breast carcinoma cells that had received signaling from platelets to undergo an epithelial-mesenchymal-like transition maintained the same deformability or became less deformable, respectively. In all cases, however, epithelial and mesenchymal tumor cells both take much longer to pass through a constriction than typical blood cells, as confirmed by examining various human cancer cell lines (H1975, SKBR-3, MDA-MB231, PC3-9). Using a syngeneic mouse tumor model, we find that cells that are able to exit a tumor and enter the circulation are not required to be particularly more deformable than the cells initially injected into the mouse. However, in a limited study of prostate cancer patients, various circulating tumor cells (CTCs) can pass through a constriction quickly because some are relatively small in size, while others are more deformable than typical tumor cell lines and more mechanically similar to blood cells. Nonetheless, due to the ambiguity in cell identity when a heterogeneous sample like blood is assessed by the SMR, there was a need to correlate each cell's precision biophysical measurement to its molecular expression. I thus developed a technique whereby cells can be sorted off-chip based on their passage time and/or buoyant mass characteristics, and collected into a 96- well plate. The proof-of-principle is demonstrated by sorting and collecting cells from cell linespiked blood samples as well as a metastatic prostate cancer patient blood sample, classifying them by their surface protein expression and relating them to distinct SMR signal trajectories. Taken together, our results provide impetus for further studies on the mechanical properties of CTCs as well as the future utilization of this platform for other types of biophysical-molecular characterizations. / by Josephine W. (Shaw) Bagnall. / Ph. D.