Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 143-159). / The ability to engineer complex genetic programs could have a huge impact on many industries, yielding organisms that can respond to their environment and perform functions relevant to manufacturing, agriculture, and medicine. However, such engineering efforts have proven difficult, in part because these programs often require precise levels of gene expression for proper function. It is especially tough to build programs that have robust activity, as any changes to the host cells can perturb the context of the genetic system and disrupt carefully tuned expression levels. Additionally, genetic programs often place high demands on host resources, which can adversely affect cell growth and further upset the intended function. In this thesis, we describe two regulatory systems in Escherichia coli that could serve to separate synthetic genetic programs from their host context, potentially leading to more robust activity. First, we build a 'resource allocator' by fragmenting T7 RNA polymerase variants into a conserved fragment and a set of variable fragments. The resource allocator limits the total number of polymerases that can be active in a genetic program, with the aim of protecting the host from being overburdened. This transcriptional budget can be allocated to different elements of the genetic program as necessary and further regulated using additional protein fragments. Second, we demonstrate a set of stabilized promoters that can maintain a level of gene expression independent of their genetic context. These promoters utilize a noncooperative incoherent feedforward loop to buffer differences in gene expression caused by changes in copy number. We demonstrate that stabilized promoters can be moved between plasmids and different locations on the genome with little change in expression. Further, they minimize the effects of other perturbations that can affect copy number, such as genome mutations and media composition. / by Thomas Hale Segall-Shapiro. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 151-171). / Hematopoiesis has long been used as a model system to study development and lineage decision-making. Within this branch of development, erythropoiesis is the process through which mature red blood cells arise from progenitor cells. In this context, GATA1 is widely considered to be the master transcription factor for erythropoiesis, controlling the expression of a vast majority of the genes involved in red blood cell maturation. GATA1 dysfunction has been shown to cause several human disorders, including anemias and thalassemias, and has been linked to the onset of various types of leukemia. GATA1 has been shown to function as both a gene activator and repressor, posing the question of how it distinguishes between various categories of genes and regulates them. In this thesis, we apply a combination of systems and molecular biology approaches in order to gain a better understanding of various regulatory mechanisms centered around GATA1. In the main study of this thesis, we uncover a new physical interaction between GATA1 and the cohesin complex, which has previously been involved in establishing three-dimensional chromatin architecture in the nucleus. We collected chromatin interaction data in a murine cell line model for erythropoiesis, and identified tens of thousands of DNA looping events in both progenitor and differentiated cells. Integration of these chromatin interaction maps with gene expression and transcription factor occupancy datasets revealed new principles underlying gene regulation, and suggests that GATA1 plays a major role in orchestrating the 3D organization of the differentiating erythroid cell. In a second study, we identify a new feedback mechanism which facilitates the replacement of GATA2, a transcription factor expressed at earlier stages of hematopoiesis, by GATA1. We show that Fbw7, an ubiquitin ligase protein trans-activated by GATA1, targets GATA2 for proteosomal degradation, thus reducing its halflife and leading to more efficient GATA factor switching. Finally, in a last section, we characterize the GATA1 -interacting transcription factors zfp281 and zfp148, and show that they play functionally redundant roles in erythroid development. Altogether, this thesis presents new insights into GATA1 various aspects of GATA1 biology, which will contribute to our understanding of mechanisms of gene regulation. / by Gabriela Pregernig. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2015.. / Cataloged from PDF version of thesis. "December 2014." / Includes bibliographical references (pages 93-108). / Adjuvants are immunomodulators and/or formulations/delivery vehicles which enhance immune responses to vaccines. The lack of progress in the development of an HIV humoral vaccine is due, in part, to the absence of available adjuvants which can be sufficiently potent with minimal adverse side effects. The main goal of this thesis was to develop nanoparticles as HIV vaccine adjuvants. Building upon previous work in the Irvine lab, we determined the potency of lipid-coated microparticles was due in part to the in situ generation of antigen-displaying liposomes. Synthetic liposomes were nearly as potent as lipid-coated microparticles, but with a 10-fold greater antigen conjugation efficiency. We subsequently optimized unilamellar liposomes as delivery vehicles for surface-displayed HIV antigens. For vaccines with a recombinant gpl20 monomer (part of the HIV envelope trimer), immunization at 0 and 6 weeks with 65 nm or 150 nm diameter liposomes with 7.5 pmol gpl20 was found to induce strong anti-gp120 titers which competed with the broadly-neutralizing antibody VRC01. The second HIV antigen used was a peptide derived from the membrane proximal external region (MPER) of the gp41 protein. High-titer IgG responses to MPER required the presentation of MPER on liposomes and the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced optimizing the MPER density to a mean distance of -10-15 nm between peptides on the liposomes surfaces. Lastly, we explored the adjuvant potential of cyclic dinucleotides (CDNs) with MPER liposome vaccines. Encapsulation of CDN in PEGylated liposomes enhanced its accumulation in draining lymph nodes (dLNs) 15-fold compared to unformulated cyclic dinucleotide. Liposomal CDN robustly induced type I interferon in dLNs, and promoted durable antibody titers comparable to a 30-fold larger dose of unformulated CDN without the systemic toxicity of the latter. This work defines several key properties of liposome formulations that promote durable, high-titer antibody responses against HIV antigens and demonstrates the humoral immunity efficacy of nanoparticulate delivery of cyclic dinucleotides, which is an approach broadly applicable to small molecule immunomodulators of interest for vaccines and immunotherapy. / by Melissa C. Hanson. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 123-130). / Experimenter control of target gene expression is a fundamental component of molecular biology research. In many systems, tools exist that allow generalizable control of gene expression at the transcriptional or post-transcriptional level. Plasmodium falciparum, the protozoan parasite responsible for the majority of death and sickness due to malaria, remains challenging to manipulate in the laboratory. No robust and generalizable tool for gene expression control has been developed in the parasite. To address this need, we engineered a new system for control of protein translation in eukarvotes, and applied it to P. falciparum. This system is based on the ligand-regulated interaction between an RNA aptamers and the TetR-repressor protein. Although such protein-RNA interactions are abundant in nature and are known to effectively mediate control of gene expression, our system is unique in its direct modulation by an exogenous chemical. By genetically encoding TetR-binding RNA aptamers in the 5' untranslated region (5'UTR) of an mRNA, translation of a downstream coding sequence is repressed by TetR in vivo and induced upon adding a non-toxic tetracycline analog. We first define the system's component molecular interactions in vitro, followed by optimization of the constituent parts for convenience and performance. We then further optimize the system and validate its performance in two model systems, the budding yeast Saccharomvces cerevisiae and cell-free rabbit reticulocyte extracts. We show the broad utility of the system in P. falciparum for controlling expression of reporter and endogenous proteins trafficked to a variety of subcellular compartments. Induction and repression are rapid and homogeneous across the cell population. Placing a drug resistance determinant tinder inducible control, we are able to modulate P. falciparum drug sensitivity, demonstrating the usefulness of the system for controlling relevant parasite biology. In the process of constructing and validating a novel tool for gene expression in P. falciparum. we built a new series of gene expression vectors for molecular biology work in the parasite. In addition to developing optimized protocols for plasmid construction, we built a standardized, sequence-defined family of plasmids for malaria research. In all, we present a generalizable, well-defined toolkit for genetic programming of P. falciparum. / by Stephen J. Goldfless. / Ph. D.

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.