Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 110-127). / For many cancers, dissemination of tumor cells to form metastases is not only a hallmark of the disease but an essential step to mortality. Migration and dissemination are complex, multistep processes, and study of their regulation has been challenging. Metastases need only be driven by a rare subpopulation of tumor cells, and a portion of dissemination is necessarily interaction with the cell's environment and thus cell extrinsic. Experimentally, there is additional uncertainty as exactly how to best assess migration outside of the complex in vivo environment. To develop a systems perspective of invasive disease, we first examine some of the experimental models used to study cell migration. We then apply this knowledge to examine regulation by proteases of endometrial cell invasion, and the pro-migratory effects of receptor crosstalk in breast carcinoma cells. Finally, extending from clear limitations in our knowledge of signaling regulation specifically within the invasive subpopulation of cells, we develop a model of ligand-mediated signaling for a receptor often expressed specifically during the process of dissemination. In total, this thesis extends systems biology techniques to the study of cell migration within the extracellular environment, with focus on that subpopulation of cells most directly implicated in the formation of metastatic disease. / by Aaron Samuel Meyer. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 145-156). / Post-translational modifications (PTMs) regulate cellular signaling networks by modifying activity, localization, turnover and other characteristics of proteins in the cell. For example, signaling in receptor tyrosine kinase (RTK) networks, such as those downstream of epidermal growth factor receptor (EGFR) and insulin receptor, is initiated by binding of cytokines or growth factors, and is generally propagated by phosphorylation of signaling molecules. The rate of discovery of PTM sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. The ten-fold increase in known phosphorylation sites over a five year time span can primarily be attributed to mass spectrometry (MS) measurement methods, which are capable of identifying and monitoring hundreds to thousands of phosphorylation sites across multiple biological samples. There is significant interest in the field in understanding these modifications, due to their important role in basic physiology as well as their implication in disease. In this thesis, we develop algorithms and tools to aid in analysis and organization of these immense datasets, which fundamentally seek to generate novel insights and testable hypotheses regarding the function and regulation of phosphorylation in RTK networks. We have developed a web-accessible analysis and repository resource for high-throughput quantitative measurements of post-translational modifications, called PTMScout. Additionally, we have developed a semi-automatic, high-throughput screen for unsupervised learning parameters based on their relative ability to partition datasets into functionally related and biologically meaningful clusters. We developed methods for comparing the variability and robustness of these clustering solutions and discovered that phosphopeptide co-clustering robustness can recapitulate known protein interaction networks, and extend them. Both of these tools take advantage of a new linear motif discovery algorithm, which we additionally used to find a putative regulatory sequence downstream of the highly tumorigenic EGFRvIII mutation that indicates casein kinase II (CK2) activity may be increased in glioblastoma. / by Kristen M. Naegle. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Mucosal barrier tissues are prominent targets for drugs against infection and chronic inflammatory disorders. One such mucosal barrier tissue, the endometrium, undergoes monthly cyclic remodeling via hormone-mediated growth, immune cell recruitment and proteolytic breakdown. Hormone response disruption has been associated with numerous endometrial pathologies, including endometriosis, adenomyosis, and infertility, which impacts upwards of 10% of women during their reproductive years. Currently, our understanding of endometrial biology is limited by the ability to replicate complex 3D physiology in vitro. Our ability to parse disease mechanisms and test efficacy of therapeutic interventions relies on development of reproducible models, adaptable to the limited numbers of cells available from patient biopsies. In this thesis, I address a critical gap in accessible tools to study and control endometrial biology in vitro and do so in a manner that can be translated to other epithelial-stromal mucosal tissues. Using the endometrium as an example mucosal barrier, I first establish design principles for the development of a synthetic, modular extracellular matrix (ECM) hydrogel suitable for 3D functional co-culture of epithelial and stromal cells. This 'one-size- fits-all' matrix features components that can be remodeled by cells and that responds dynamically to sequester local cell-secreted ECM characteristic of each cell type enabling long-term, hormonally responsive co-cultures. Next, I establish methods to expand and cryopreserve primary human endometrial epithelial cells, which maintain barrier and secretory function, further enabling studies using primary cells. Finally, we use data-driven network modeling of secreted proteins to understand how variation in cytokine signaling may alter hormone responsiveness and proteolytic remodeling in primary epithelial-stromal co-cultures. With the ability to create and parse more complex 3D tissue models using primary cells to recapitulate healthy and diseased states, we further enable basic understandings of disease pathologies and subsequent drug discovery efforts aimed at inflammation, wound healing and immune modulation. / by Christi Dionne Cook. / Ph. D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references . / Glycans are essential components of all living things because they function as key elements of cellular membranes and extracellular spaces by mediating cell-cell communication, transduction pathways, and cellular development, function, and survival. Because glycans are secondary gene products that depend on the availability of sub-cellular enzymes for synthesis, research on their structure, synthesis, and biological significance has lagged behind that of DNA and proteins due to both a lack of appreciation of their importance and the slow pace at which tools are being developed to study them. In this thesis, three projects focus on the development, application, and exploration of tools for glycan characterization. The first project resulted in the successful optimization of an analytical method to isolate and characterize O-linked glycans, on which relatively few research projects focus because of the limited availability of tools to isolate them and the need for specific analytical equipment to properly characterize them. Using this optimized method, the O-linked glycans of bovine mucin and fetuin were successfully profiled, and the analysis of the former provided the motivation for a second project focused on the significance of goblet cells and mucins in influenza infection. This project explored the potential benefits of a glycoprotein direct binding assay as a way to obtain quantitative information about lectin and influenza hemagglutinin specificities. Using mucins adsorbed to a polystyrene plate, it was possible to obtain quantitative binding constants for two commonly used lectins. The last project focused on the isolation and characterization of the cell surface N-linked glycans from chicken erythrocytes, turkey erythrocytes, and human tracheal epithelial (HTE) cells. Analysis of these cell types is warranted due to their importance as model systems to study influenza infection. The results of this project provide a context for future questions about the relevance of the erythrocyte model system for studying influenza binding specificities. All of these projects reiterate the importance of the study of glycobiology by showing how both the development and application of tools to study glycans can provide in new and interesting information about pathological processes related to human health and disease. / by Nia Beckley. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Mammalian noses can detect and distinguish an inestimable number of odors at minute concentrations. Four classes of G protein-coupled receptors (GPCRs) are responsible for this remarkable sensitivity: olfactory receptors (ORs), vomeronasal receptors (VNRs), trace amine-associate receptors, and formyl peptide receptors. Structural knowledge of these receptors is necessary to understand the molecular basis of smell. However, no structure exists for three main reasons. First, milligrams of protein are needed for crystallization screens, but most are expressed at low levels endogenously or in heterologous expression systems. Second, detergents capable of solubilizing and stabilizing these proteins in aqueous solution must be found. Third, the flexible nature of GPCRs can inhibit crystal lattice formation. Methods for overcoming each obstacle were developed. Milligrams of a VNR were expressed in HEK293 cells, and milligrams of 13 GPCRs were expressed in a cell-free system. All could be purified to >90%. The purified receptors had correct secondary structures, and could bind their ligands. The HEK293 and cell-free receptors had nearly identical structures and binding affinities, demonstrating that cell-free expression can be used for GPCR production and mutational studies. To demonstrate this, six variants of mOR103-15 with single amino acid substitutions were expressed. Ligand-binding measurements indicated which residues were involved in ligand recognition. The choice of detergent used in the cell-free system was critical, and significantly affected expression levels. A class of amphiphilic peptide detergents was designed and tested with the receptors. These detergents could be used to express milligrams of functional receptors. The peptide tail and head group properties did not significantly affect their function, suggesting that they may be a class of surfactants usable with multiple olfactory-related receptors, and even other membrane proteins. Lastly, the protein T4 Lysozyme (T4L) was fused in the 3rd intracellular loop of two receptors to increase potential crystal lattice contact points. Purified T4L variants had correct secondary structures, and could bind their ligands and initiate intracellular signaling. The methods described generated sufficient quantities of pure receptors for crystal screens. The large number of functionally expressed GPCRs indicates that these techniques can be applied to other olfactory-related receptors, and even other membrane proteins. / by Karolina Corin. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references. / An understanding of the factors involved in the human adaptation of influenza A viruses is critical for various aspects of influenza preparedness, including the development of appropriate surveillance measures, preventive strategies and effective treatments. A key step in influenza human adaptation is the acquisition of mutations in the viral coat glycoprotein, hemagglutinin (HA), which changes its binding specificity towards glycan receptors in the human upper respiratory epithelia (referred to as human receptors). In this thesis, determinants that mediate changes in HA-glycan receptor binding specificity are investigated, with focus on the molecular environments within and surrounding the glycan receptor binding site (RBS) of HA. The glycan receptor binding properties of HA from different influenza subtypes (H1N1, H2N2, H3N2 and H5N1) are studied using a combination of approaches including dose-dependent glycan binding, human tissue staining and structural modeling. Using these complementary analyses, it is shown in this thesis that the molecular interactions between amino acids in and proximal to the RBS (referred to as amino acid interaction networks), including those between the RBS and glycosylation at sites proximal to the RBS, and interactions between the RBS and the glycan receptor together govern the high affinity binding of HA to human receptors. The thesis is divided into three sections. First, the evolution of glycan receptor binding specificity of recent human-adapted H3 strains such as A/Fujian/411/02 and A/Panama/2007/99 is investigated, with implications on vaccine production in chicken eggs. Second, the determinants of glycan receptor binding affinity of potentially pandemic avian viruses is studied in the context of the recently circulating H2 A/Chicken/Pennsylvania/2004 and the highly pathogenic H5 A/Vietnam/1203/2004. Here it is shown that mutations which cause human adaptation of H2 do not increase human receptor binding affinity in H5, and the importance of amino acid interaction networks is implicated. Third, determinants that govern the high affinity human receptor binding of pandemic influenza HAs is investigated using the prototypic 1918 H1N1 HA as a model system. The roles of amino acid interaction networks and the molecular interactions between the RBS and glycosylation at sites proximal to the RBS in contributing to the high affinity human receptor binding of 1918 H1N HA are investigated. The approaches presented in this thesis to systematically investigate molecular interactions between HA and glycan receptors that impinge on quantitative HA-glycan receptor binding affinity offer a new angle towards studying determinants of human receptor binding specificity and affinity of influenza A virus HAs. / by Xiaoying Koh. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 125-147). / Neurodegenerative diseases are a costly burden, both economically and in terms of human suffering. A common feature of neurodegenerative diseases is that they stem from problems with protein folding, but the underlying biology that leads to neuron death is not well understood. Due to this lack of mechanistic information there are currently no therapeutics that treat underlying mechanisms that lead to cell loss. This thesis explores the link between complications in protein folding and cell death. In the first part of this thesis, I combined modeling of the proteotoxicity of polyglutamine (as exemplified in Huntington's Disease) in Saccharomyces cerevisiae with microfluidics and automated microscopy. From these studies, I have found that glutamine-rich proteins suppress the toxicity of poly-glutamine expanded Huntingtin by physically interacting and sequestering the protein at the IPOD (insoluble protein deposit) quality control compartment. These studies have provided new insight into possible therapeutic strategies and how the proteomes of different cell types may protect or sensitize sells to specific proteotoxic stresses. In the second part of this thesis, I quantitatively and systematically studied the toxicity of a-synuclein, which is implicated in the synucleinopathy family of diseases, including Parkinson's Disease. To systematically study the effect of toxic levels of a-synuclein expression on cellular homeostasis, I constructed a library of fluorescent reporters and utilized automated, high-throughput microscopy to image changes in reporter localization and abundance in response to a-synuclein toxicity. The results from this study have illuminated a number of pathways that were not previously studied for a-synuclein toxicity and have tied together disparate findings from many other studies. Additionally, I have shown that our experimental strategy is generalizable and can be applied other yeast models of neurodegenerative toxicity, such as poly-glutamine and AO 1-42. In summary, the quantitative studies presented in this thesis have expanded our understanding of the mechanisms underlying a variety of toxicities related to neurodegeneration. The biological insights gained from these studies have helped illuminate new areas of inquiry that may be used to combat these diseases. / by William R. Hesse. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / One of the major goals of systems biology is understanding how a cell changes from a healthy state to a diseased state. Entire fields of cell biology have been built around studying how changes in the type and abundance of specific biomolecules affect disease status. However, one major knowledge gap in systems biology is the quantification of protein synthesis rate at any given time (i.e the Translatome). At this time, measurements of protein synthesis rates are limited to methods that use mRNA abundance as a proxy; however, there are regulatory steps on the level of translation that can confound correlation between mRNA abundance and protein synthesis rates. Here, I improve upon a proteomics based method for measuring newly translated proteins, biorthogonal non-canonical amino acid tagging (BONCAT), and adapt it for robust quantitative multiplexing analysis. In the BONCAT method, cells are pulsed with azidohomoalanine (Aha), a methionine analog that contains an azide functional group, such that proteins synthesized for the duration of the pulse incorporate Aha. By coupling pulsed stable isotope labeling of amino acids in cell culture (pSILAC) and Aha metabolic labeling of newly synthesized proteins with strain-promoted azide-alkyne cycloaddition and tandem mass tag (TMT) labeling, I am able to quantitatively interrogate the translatome in a multiplexed manner with high sensitivity and high temporal resolution. The multiplexed BONCAT protocol was applied to observe changes in temporal protein synthesis during the unfolded protein response (UPR) and epidermal growth factor (EGF) stimulation. Eliciting the UPR by blocking N-glycosylation results in a global downregulation of protein translation, but upregulation of several key protein-folding chaperones. Furthermore, protein translation machinery (ribosomal proteins, initiation factors, and elongation factors) are downregulated to a much greater extent. In contrast to the UPR stress response, pro-growth EGF stimulation resulted in the upregulation of protein translation machinery. EGF stimulation also resulted in waves of temporally distinct protein synthesis, beginning with immediate and delayed early genes and followed by late response genes that determine cell fate. By sampling protein synthesis at both 30 minute and 15 minute intervals, I was able to further elucidate the order of protein synthesis with high temporal resolution. Comparison of protein translation with RNA sequencing and ribosome footprinting revealed tight correlations between RNA, ribosome occupancy, and protein synthesis. This comparison also allowed the distinction between protein synthesis driven by an increase in transcription versus that driven by an increase in translation. Interestingly, temporal delays between ribosome occupancy and protein synthesis were observed in many genes. These genes also demonstrated a unique codon bias compared to the average codon usage of the genome. An analysis of codon frequency revealed changes in global codon usage over time following EGF stimulation. Changes in chemical modifications of tRNA isoacceptors were also observed which may play a role in regulating protein translation. Finally, our multiplexed BONCAT method was leveraged to compare the translation response between MEK inhibitor resistant (MelJuso) and sensitive (MM415) melanoma cell lines. Using partial least squares regression (PLSR) and gene set enrichment analysis (GSEA), upregulation of melanoma lineage-dependent transcription factor MITF and MITF targets was observed in MM415s after binimetinib treatment, with no such response in the MelJuso cells. Using a small molecule inhibitor against MITF, we found that MITF inhibitions results in a protective effect against binimetinib in MM415 cells. However, the MM415 cells were resistant to siRNA-mediated knockdown of MITF. Further work needs to be done to characterize the role of MITF in the context of binimetinib sensitivity. / by Daniel Abram Rothenberg. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Therapeutic resistance continues to be a persistent challenge in medical oncology. In clinical settings, resistance can occur at the beginning of treatment, or may be acquired after an initial clinical response to the therapy. Several mechanisms of drug resistance have been described in cancer, including alterations in the drug transport and metabolism process, mutations in drug-target, activation of bypass signaling pathways, inhibition of cell-death pathways, and induction of an epithelial to mesenchymal transition (EMT) in response to cytotoxic or targeted therapies. In this study, I have investigated the molecular mechanisms underlying ZEB 1-induced EMT and established a new computational framework that uses inter-animal heterogeneity to identify drivers responsible for variable phenotypic responses across different animals. EMT describes a cell-state switching process wherein epithelial cells lose their tight cell-cell junction contacts, and acquire the ability to migrate and invade the surrounding stroma to enter into blood circulation. Given the widespread role of EMT in drug resistance, it is imperative to identify therapeutic strategies to inhibit this transition. To identify druggable targets to block EMT progression, and therefore overcome EMT-mediated therapeutic resistance, I studied the effects of ZEB 1 expression on cellular signaling networks. By quantifying changes in tyrosine phosphorylation at different time points during ZEB 1-induced EMT, I found that Src family kinases (SFKs) were activated within 24 hours of ZEB 1 expression. Inhibition of SFKs blocked not only ZEB 1-induced EMT, but also EMT initiated by TGFp- and EGF signaling pathways in both breast and NSCLC cell-lines. SFK inhibition also prevented EGFR inhibitor-induced EMT and drug resistance in NSCLC cells both in vitro and in vivo. Mechanistically, SFK activation stabilized ZEBI by promoting ERK1/2-mediated phosphorylation on three serine residues, S583, S646, and S679. Consequently, MEK inhibition phenocopied the effects of blocking SFK activity with regards to decreasing stability of ZEB 1 and inhibiting ZEB 1-induced EMT. These results provide a new therapeutic application of SFK inhibitors as a potential anti-EMT therapy, to enhance the susceptibility of cancer cells to chemo- or targeted therapies. In the second part of this thesis, I have described a computational framework that leverages inter-animal heterogeneity to identify molecular mechanisms underlying variable phenotypic responses across different animals. Substantial inter-animal variability in phenotypes within the same treatment group, limits our ability to draw conclusions or gain meaningful insights about a biological process by simply averaging the data. To identify molecular drivers for heterogeneous phenotypic responses, I have established a method where each animal is considered as an individual entity whose phenotypic response is dependent on the state of its underlying signaling networks. As a proof of concept, I have used this method to successfully predict the resistance mechanisms of CDK4/6 inhibitor, palbocilib in two GBM PDX and one MPNST PDX models. The GBM6 model activated EGFR signaling upon treatment with palbociclib whereas the GBM22 and MPNST3 models activated SFKs and PDGFRa signaling in resistant tumors. Across all three PDX tumor models, treatment with combination therapies, consisting of palbociclib and an inhibitor targeting the activated bypass signaling pathway, substantially prolonged survival of mice. Thus, these results suggest that inter-animal variability can be used as a tool to predict drivers for a specific phenotypic response across different treatment conditions. / by Vibhuti Agrawal. / 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-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.