Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 121-136). / Immune cells communicate with each other to mount an effective response to pathogens or maintain homeostasis. Communication and activation of the immune cell network can occur in part through secretion of or response to cytokines. Here, we couple experimental approaches with computational analysis to identify how cytokine communication impacts immune-rich cellular environments. The first part of this work focuses on on understanding intracellular signaling in response to TNF[alpha] -induced apoptosis in vivo. By applying a quantitative mechanistic modeling framework to phosphoproteomic data of inflammation in the context of a variety of genetic Ras mutations, we identify clear differences between signaling from N-Ras and K-Ras isoforms, and identify isoform specific contributions to intestinal apoptosis. The next part of this work focuses on understanding cytokine secretion from primary human immune cells especially as mediated by cell-cell communication. By coculturing pure immune cell types at known mixture ratios, and measuring multiplexed secretion, we gained insight into the regulation of individual cytokines in these mixtures as compared to expected values from corresponding measurements of monocultures. We find that monocultures are less predictive of cytokine secretion from physiological cell populations than controlled cocultures. In this work, we also elucidated a number of positive and negative synergies in communication between monocytes and CD4+ T cells. A particularly interesting result was finding a synergy between INF[gamma] and TNF[alpha] for production of CXCR3 chemokines. Overall, this combination of modeling and experimental work has made contributions to understanding regulation of the cytokine environment in multicellular environments, as well as contributions of specific cytokines of interest to cellular and tissue responses. / by Sarah B. Schrier. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 223-234). / The varied movements of the epithelium play vital roles in the development and renewal of complex tissues, from the separation of tissues in the early embryo, to homeostasis in the adult. Their movement is intricately connected to their proper functioning as selective barriers of the intestinal mucosae, as well re-epithelialization in the healing of wounds. Yet, considering their ubiquity and relevance, the basic origin of the collective motion of sheets has eluded a clear and quantitative interpretation in physical terms, prohibited by the lack of understanding of the relationship between motility, cell-cell contact, and their mediation by the mechanical properties of the substratum to which they adhere. Therefore, within this context, this thesis defines the prerequisites for both equilibrium and non-equilibrium coordinated cell motion. The timescales and lengthscales of the in vitro migration of an epithelial monolayer were calculated and compared under imposed constraints designed to mimic various states of in vivo epithelia. These constraints include assays that recreate the wound response of the epithelium such as what is seen in the cornea and epidermis, by unequivocally separating the influence of free space from cell damage in the induction of coordinated motion. The motion of the epithelium was further explored by the generation of gradients that reproduce asymmetry in the capacity for cells to migrate, divide, or undergo apoptosis, such as what is found along the crypt-villus axis of the intestine. Finally, as the epithelium adheres and migrates against the basal lamina, a substrate of uncertain in vivo mechanical properties, we explored the contribution of substrate viscoelasticity to the dynamics of coordinated migration. Parameterized this way, multiple modes of motility emerge, each distinct dynamically, phenotypically, and in their dependence on cell-cell contact. / by Michael Peter Murrell. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 151-162). / Multipotent stromal cells derived from bone marrow hold great potential for tissue engineering applications because of their ability to home to injury sites and to differentiate along mesodermal lineages to become osteocytes, chondrocytes, and adipocytes to aid in tissue repair and regeneration. One key challenge, however, is the scarcity of MSC numbers isolated from in vivo, suggesting a role for biomimetic scaffolds in the cells' ex vivo expansion before reintegration into target tissue. Toward this end, immobilized epidermal growth factor (tEGF) has recently been found to promote MSC survival and proliferation and is a prime candidate to be incorporated into scaffolds to control MSC behavior. To rationally and effectively design scaffolds to drive MSC responses of survival, proliferation, migration, and differentiation, we must first understand these responses and the underlying protein signaling pathways that mediate them. While our knowledge of MSC behavior is limited as a field, MSC migration is particularly less studied despite being critical for tissue and scaffold infiltration. In this thesis, we quantitatively investigate the effects of tEGF and extracellular matrix (ECM) on MSC migration response and signaling. We take a systems level computational view to show a combined biomaterials and small molecule approach to control MSC migration. Cell migration is a delicately integrated biophysical process involving polarization and protrusions at the cell front, adhesion and translocation of the cell body through contractile forces, followed by disassembly of adhesion complexes at the cell rear to allow detachment and productive motility. This process is mediated by a multitude of crosstalking signaling pathways downstream of integrin and growth factor activation. Using a poly(methyl methacrylate)-grafted-poly(ethylene oxide) (PMMA-g-PEO) copolymer base, we modify the PEO sidechains with immobilized epidermal growth factor (tEGF) as a model system for biomimetic scaffolds. We systematically adsorb fibronectin, vitronectin, and collagen ECM proteins to alter surface adhesiveness and measure MSC migration responses of speed and directional persistence alongside intracellular activities of EGFR, ERK, Akt, and FAK phosphoproteins. While tEGF and ECM proteins differentially affected signaling and migration, univariate correlations between signals and responses were not informative, prompting the need for multivariate modeling to identify key patterns. Using decision tree "signal-response" modeling, we predicted that inhibiting ERK on collagen-adsorbed tEGF polymer surfaces would increase cell mean free path (MFP) by increasing directional persistence. We confirmed this experimentally, successfully demonstrating a two-layer approach-"coarse" biomaterials followed by small molecules "fine-tuning"-to precisely and differentially control MSC migration speed and persistence, setting the stage for combination therapies for bone tissue engineering. / by Shan Wu. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, September 2009. / "August 2009." Cataloged from PDF version of thesis. / Includes bibliographical references. / The primary motivation for this work is the manipulation of EGFR family signaling to influence regenerative responses of mesenchymal stem cells (MSC). Underlying the potential of regenerative medicine is the need to understand and control cell behavior. A 'cue, signal, response' paradigm has emerged as a framework for building predictive models for manipulation of cells to achieve desired responses. The HER receptor tyrosine kinase (RTK) family is an attractive target for manipulation of cues and signals, as its four members - epidermal growth factor receptor (EGFR or HERI), HER2, HER3 and HER4 - influence processes as diverse as development, wound healing, migration, and tissue homeostasis and family members are expressed by almost every cell type. All HER receptors require either homodimerization or heterodimerization with other family members for activation of signaling pathways, and the various dimer pairs are not equivalent in their ability to activate all the downstream pathways. Hence, signaling (and phenotypic) outcomes may be dictated not only by the number (or fraction) of each type of receptor ligated, but by the quantitative distribution of these receptors into various possible dimer pairs. The canonical physiological ligands for the HER family receptors are monomeric, allowing occupied receptors to freely homodimerize or heterodimerize. The premise of this work is that engineered bivalent ligands can drive specific dimerization events to enhance or inhibit signaling by various HER family receptors in a quantitative fashion that might be predicted on the basis of receptor expression. This work focuses on the design and implementation of engineered protein systems that are targeted to control homo and heterodimerization of HERI and HER3. One broad consequence of using homodimer ligands is to quantitatively force ligand-occupied HERI or HER3 to homodimerize and thus inhibit heterodimerization. Homodimerization may reinforce preferred signaling pathways (e.g, HERI-HERI vs HER1-HER2) - with implications for tissue regeneration and inhibit undesirable pathways (e.g. HER2-HER3) - with implications in cancer. Preliminary results suggest that whereas the monomeric HER3 ligand activates canonical signaling pathways expected from HER3-HER2 interactions, dimeric ligands inhibit signaling, presumably by forcing homodimerization of the kinase-inactive HER3 receptors. This thesis focuses on developing the design principles to use bivalent ligand dimers to control signaling, experimental testing of the hypothesis that signaling pathways can be controlled by such ligands and are quantitatively different than those for monovalent ligands, and demonstration of how such ligands influence proliferation of human marrow stromal cells, a cell type important for bone regeneration. In addition, the issue of practical implementation in a tissue engineering setting is addressed by implementing approaches to tether bivalent ligands to scaffolds in a manner that preserves signaling function. / by Luis M. Alvarez. / Ph.D.

Thesis (S.M. in Molecular and Systems Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Vita. / Includes bibliographical references (leaves 60-62). / The purpose of this work is to probe the dTDP-L-rhamnose pathway in an effort to develop small molecule inhibitors that could act as therapeutics for Mycobacterium tuberculosis. The necessity for newer, more effective treatments for tuberculosis is growing, as the bacteria evolve resistance to traditional treatments. In an effort to develop more effective and perhaps more abbreviated courses of treatment, a plan was developed to investigate a pathway involved in cell wall biosynthesis as a promising target: the dTDP-L-rhamnose pathway. This pathway plays an essential role in linking the peptidoglycan and arabinogalactan portions of the mycolic acid-arabinogalactan-peptidoglycan complex, a significant part of the mycobacterial cell wall. The mounting level of biochemical understanding of this pathway and its importance in bacterial cell wall biosynthesis indicates that it is not only a relevant target but also an accessible one. Of the four enzymes crucial to this biosynthetic pathway, one was chosen as the primary focus: dTDP-D-glucose-4,6- dehydratase (RmlB). There are 3 steps in the reaction mechanism of RmlB: oxidation of the C4 position of dTDP-D-glucose to form a 4-keto structure, dehydration of the C6 position via the elimination of water and a subsequent reduction to result in a 6-deoxy product. Crystal structures of this particular enzyme, dTDP-D-glucose 4,6-dehydratase (RmlB), complexed with single substrates or substrate analogs have provided a foundation for these studies, enabling the rational design of a small library of potential inhibitors. Twelve mechanism-based inhibitors of RmlB are proposed. These compounds reflect the current understanding of the mechanism and mimic the sugar portion of the sugar-nucleotide substrate at various steps throughout the reaction mechanism. Each of the proposed inhibitors is designed to inhibit one of the specific steps of the mechanism. While the intention of this project is to synthesize each compound in this library from commercially available starting materials in 15 steps or less, the primary goal of this particular dissertation is to synthesize 3 of the 12 proposed inhibitors from the commercially available starting material 1,5-anhydro-D-glucitol. The long term goal of this work is to produce these compounds in significant amounts in order to test their efficacy in an animal model of mycobacterial infection. / by Neena Sujata Kadaba. / S.M.in Molecular and Systems Toxicology

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 139-142). / The development of single-cell RNA-sequencing has led to a new degree of resolution in the characterization of complex, heterogeneous biological systems. However, existing methods are often limited in their ability to link these whole-transcriptome profiles with complimentary measurements of single-cell phenotype. In this thesis we present a microfluidic toolset which allows us to link a panel of single-cell phenotypic measurements - including lineage history, cell cycle stage, cell size, and growth rate - with their corresponding transcriptional profiles. Using a microfluidic platform that employs an array of hydrodynamic traps to capture and culture single cells for multiple generations we measured single-cell growth kinetics, lineage hierarchies and cell cycle stage. By subsequently releasing individual cells from this device for downstream scRNA-seq we were able to generate whole-transcriptome profiles of primary, activated murine CD8+ T cell and lymphocytic leukemia cell line lineages. For both cell types we found distinct transcriptional patterns associated with single-cell lineage relationships as well as cell cycle progression. In order to link single-cell size and growth rate measurements with gene expression, we have also developed a system that relies on an array of suspended microchannel resonators (SMR) - high resolution single-cell buoyant mass sensors - in combination with an automated method of isolating single cells to conduct scRNA-seq downstream. Using this platform, we were able to collect linked transcriptional and biophysical measurements for a murine leukemia cell line, primary murine CD8+ T cells, and a patient-derived glioblastoma multiforme (GBM) cell line. For all cell models measured, we found that single-cell buoyant mass showed a strong correlation with the expression of cell cycle genes. Furthermore, we found that single-cell growth rate and buoyant mass measurements can be used to characterize the degree to which GBM cells respond to drug treatment as well as determine transcriptional signatures associated with response and resistance. Taken together, we believe these single-cell phenotypic measurements will offer complementary contextual information to further resolve the heterogeneity of single-cell transcriptional data. As such, we expect these platforms to be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology. / by Robert J. Kimmerling. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 112-119). / Malaria accounts for over 500,000 deaths each year. While malaria is caused by multiple distinct parasites of the genus Plasmodium, P. falciparum is responsible for the majority of morbidity and mortality due to the disease. Despite this fact, molecular tools for genetic experimentation in the parasite remain underdeveloped. In particular, the ability to inducibly control gene expression and edit the genome in a site-specific manner present significant challenges. In addition, the building of genetic constructs poses challenges due to the required vector size and the high A+T richness of the P. falciparum genetic regulatory elements. This work begins by presenting the first vector family for use in the parasite made up of modular parts and encompassing all selectable markers and replication technologies in current use. It also discusses the development of a 2A like viral peptide tag for use in expression of multiple differentially localizing proteins from a single expression cassette. Based on this work, we were then able to construct vectors to reconstitute the T7 RNA polymerase expression system in P. falciparum, functionally creating the first system for directed expression of non-coding RNA in the parasite. We were then able to use this expression system to adapt a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system in the parasite and use it to achieve genome editing at high efficiency at multiple loci. The data imply an adaptable system to readily edit the genome of the parasite and holds promise for the ability to create gene knockouts, perform allelic replacements, and add regulatory elements into the parasite significantly faster than has been previously demonstrated. This also represents the first illustration of the functionality of a CRISPR/Cas9 system in any non-bacterial pathogenic organism. In addition, we were able to introduce the lac repression system in order to regulate the T7 RNA polymerase dependent production of RNA and have created the first inducible expression system for RNA in any apicomplexan parasite. This work provides several new molecular tools and frameworks to aid in the study of, and fight against, malaria. / by Jeffrey C. Wagner. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 192-205). / Human obesity is a world-wide health crisis that promotes insulin resistance and type 2 diabetes. Obesity increases intracellular free fatty acid concentrations in peripheral tissues, particularly the liver, which disrupts molecular mechanisms that maintain normal glycemia in response to fasting and feeding. The progression towards outright pathology in response to obesity is a highly complex process that involves coordinated dysregulation of a variety of molecular processes across multiple regulatory levels. The goal of this thesis was to apply a quantitative, multi-omic systems biology approach to the study of obesity-induce hepatic insulin resistance. We fed male C57BL/6J mice high-fat diets (HFD) to induce obesity and insulin resistance. In the first presented study, our group collected datasets to profile the hepatic epigenomes, transcriptomes, proteomes, and metabolomes of chow diet (CD) control and HFD-fed mice. I extended and applied an established computational modeling algorithm, namely the prize-collecting Steiner forest (PCSF), to simultaneously integrate these molecular data with protein-protein and protein-metabolite interactions into a tractable network model of hepatic dysregulation. This model uncovered a variety of dysregulated pathways and processes, some of which are not well-established aspects of insulin resistance. We further tested and validated some of these model predictions, finding that HFD induces serious architectural defects in the liver and enhances hepatocyte apoptosis. In the next study, we focused more specifically on hepatic transcription. We fed mice short and long-term HFDs and treated them with the type 2 diabetes drug metformin. Compared to non-treated CD controls, diet exerted the strongest effect on transcription, progressively inducing changes as HFD duration increased. We additionally stimulated mice with insulin and collected temporal transcriptomic profiles. We found that long-term HFD almost completely blunted normal insulin-induced transcriptional changes, but also found a small set of genes that are specifically insulin-responsive in HFD livers. We further characterized one of these genes and provided evidence supporting the notion that aspects of hepatic insulin signaling are intact during insulin resistance. In another study, we collected transcriptomic and epigenomic data from mice fed a calorie-restricted (CR) diet. Interestingly, we found a small set of genes altered in the same direction by both CR and HFD. We then used chromatin accessibility experiments to infer regulators associated with these gene expression changes and found roles for PPAR[alpha] and RXR[alpha]. We performed ChIP-Seq experiments for these factors and treated mice and primary hepatocytes with a PPAR[alpha] activator, uncovering a role for PPARα in the regulation of anaerobic glycolysis. We also validated novel predicted target genes of PPAR[alpha] involved in glucose metabolism. Finally, we profiled hepatic miRNAs in CD and HFD livers, finding that HFD progressively alters their expression levels. We implemented an enrichment procedure and a network modeling approach to analyze these data. We integrated additional mRNA and epigenetic data to infer miRNAs that may play regulatory roles during insulin resistance. In total, this thesis presents a unique comprehensive approach to the study of diet-induced hepatic insulin resistance that revealed new insights into pathology. / by Anthony Robert Soltis. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Page 193 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 185-192). / Rational drug design of small molecules has led to the development of robust therapeutics that are currently used in the clinic. However, key challenges remain in designing drugs against infectious disease targets that are susceptible to mutation. To achieve full clinical efficacy against rapidly-mutating targets, new methods must be developed for designing drugs. In this thesis, we utilize a three pronged approach for rational ligand design by developing methods, analyzing existing experimental data, and designing novel therapeutics. On-target mutations in infectious diseases often render inhibitors ineffective and are one of the key clinical failures of current therapies. We use HIV protease as a model system to understand mutation resistance. HIV protease substrates are unaffected or only moderately affected by resistance mutations that greatly decrease inhibitor binding. This idea has led to the design of broadly binding inhibitors using substrate mimicry. This is achieved by constraining inhibitors to bind within the consensus substrate volume, which we term the "substrate envelope". However, while the substrate envelope has been relatively successful, some inhibitors that are designed based on this model are sensitive to mutants. We performed a detailed biophysical binding energy decomposition of a flat and susceptible binder pair and found that the susceptible inhibitor forms stronger interactions with key residues. These residues are entirely characterized by examining known resistant mutants to approved HIV protease inhibitors. To generalize our findings, we cross-validate on a set of ten HIV protease inhibitors with previously measured sensitivity. We find that interaction energy successfully classifies susceptible and flat inhibitors. Based on these results, we extend the current design paradigm. We develop a methodology to minimize extraneous contacts with the active site and express it as an appropriate cost function, which is then minimized. We then implement this design scheme for HIV protease, yielding both flat and susceptible binders. Next, we apply rational drug design principles to other infectious disease targets. We first focus on the tuberculosis specific CIpP1P2 peptidase to optimize the antibiotic, acyl depsipeptides (ADEPs). We use component analysis to understand the biophysics of ADEP binding to the active site. We then design a series of analogs resulting in a two-fold affinity improvement along with enhanced peptidase activity. We also develop new methods to improve an anti-fungal for the treatment of Candida albicans. We use molecular docking to predict a binding mode for the lead compound and then account for receptor plasticity by performing molecular dynamics simulations. We use this improved receptor model to design novel analogs that are predicted to bind better than the parent compound. Lastly, we focus on disease diagnosis by developing a novel paradigm for MRI contrast agent design. We first integrate the governing thermodynamics and relevant parameters that influence imaging efficacy to develop an integrated workflow for contrast agent design. We then apply our methodology to the DOTA system and successfully explain differential activity of designed analogs. Put together, we demonstrate the power of rational design in various relevant biological contexts. Overall, this thesis presents new techniques, analysis, and applications of rational design to address unmet clinical problems. Work from this thesis accelerates the field of computational drug design, which has implications in many uncured diseases and diagnostics. / by Raja R. Srinivas. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Biotherapeutics have revolutionized medicine with their ability to achieve unprecedented molecular recognition and mediate complex biological responses. The intracellular delivery of biotherapeutics is an unmet scientific challenge and medical need. A wide variety of different treatment modalities depend on not only on the ability to achieve intracellular delivery, but to do so in a targeted manner. An independently-targeted, two-molecule system was developed to accomplish intracellular delivery in a uniquely specific manner. Immunotoxins were designed based on the plant toxin gelonin and targeted towards the canonical cancer-specific antigens: epidermal growth factor receptor and carcinoembryonic antigen. Using quantitative internalization flow cytometry matched with controlled exposure cytotoxicity, the number of internalized gelonin immunotoxins required to induce apoptosis in a single cell was found to be ~5x10⁶ molecules. This threshold to cytotoxicity was conserved across all gelonin constructs regardless of antigen target, binding scaffold, affinity, or cell line. Next, cholesterol-dependent cytolysins were targeted to the same antigens by genetic fusion to engineered fibronectin domains. When combined in vitro, targeted gelonin and cytolysin had synergistic cytotoxic effects and the presence of cytolysin reduced the intracellular barrier to cytotoxicity to < 10⁴ immunotoxin molecules. In vivo, these molecules induced nonspecific, dose-limiting toxicities at varying levels and were cleared from the plasma at rates consistent with their molecular weight. Dosed individually, neither compound was capable of controlling tumor xenografts, but when combined in a delayed dosing scheme they inhibited tumor growth and induced apoptosis throughout xenografts as confirmed by histology. Mathematical modeling was informed by in vivo experiments and provided insight in dosing and tumor exposure overlap. These results emphasize the necessity of a targeted intracellular delivery system and support the merit of the described approach. Additional research into the safety and efficacy of these molecules as well as the design of new constructs will certainly improve the clinical relevance of this technique. / by Christopher M. Pirie. / Ph.D.