Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 86-99). / Functional brain imaging technologies seek to expand our understanding of intact neural systems. Present day functional MRI (fMRI) measures the delayed hemodynamic response that is indirectly associated with neural activity. To study underlying molecular systems noninvasively but precisely, tools must be developed to modulate MRI contrast as a function of discrete molecular events within pathways of interest. In optical imaging modalities, genetically encoded sensors based on fluorescent proteins have provided an engineerable platform on which to optimize desirable device characteristics by exploiting the tools of molecular biology and protein biochemistry. Analogously, we seek to build genetically encodable sensors based on engineered metalloproteins whose effects on MRI contrast are regulated by specific biochemical interactions. In this work, we present two technological advancements toward realizing fMRI contrast sensors for molecular neuroimaging. First, a genetically encodable sensor for free calcium is described, consisting of a novel ferritin-based device that reversibly enhances NMR transverse relaxation times (T2) by Ca - dependent crosslinking. Second, we show that the T1 contrast effect of a recently proposed family of cytochrome P450-based MRI sensors can be significantly enhanced by substitution of the protein's native heme with a high spin manganese porphyrin. / by Victor S. Lelyveld. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 114-131). / The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common regulatory mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from normal cellular physiology. We describe a ligand-responsive protein-RNA interaction module that can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to sub- nanomolar affinities were identified. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for conserved sequence and structural motifs in TetR binding. We illustrate the utility of this chemically-inducible RNA-protein interaction to directly regulate translation in both a prokaryotic and eukaryotic organism. By genetically encoding TetR-binding RNA elements into the 5'-untranslated region (5'-UTR) of a given mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this modular system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from nonfunctional RNA-TetR interactions. We also demonstrate engineering this TetR-aptamer module to regulate subcellular mRNA localization. This is efficiently achieved by fusing TetR to proteins natively involved in localizing endogenous transcripts, and genetically encoding TetR-binding RNA aptamers into the target transcript. Using this platform, we achieve tetracycline-regulated enhancement of target transcript subcellular localization. We also systematically examine some rules for successfully forward engineering this RNA localization system. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and that can be used as a foundation for functionally and reversibly controlling multiple fates of RNA in cells. / by Brian J. Belmont. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. / "February 2008." Cataloged from PDF version of thesis. / Includes bibliographical references (p. 110-114). / Monoclonal antibodies have emerged as a promising class of therapeutics for the treatment of human disease, and in particular human cancer. While multiple mechanisms contribute to antibody efficacy, the engagement and activation of immune effector cells - mediated by the interaction of the conserved Fc regions of the antibody with the Fc gamma receptors (Fc[gamma]Rs) on immune cells - is critical to the efficacy of several. This thesis describes the engineering of antibody Fc domain interactions with Fc[gamma]Rs, using the' yeast S. cerevisiae. In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics. / by Stephen L. Sazinsky. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Title as it appears in MIT Degree awarded booklet, February 2013: Quantitative analysis of the Interleukin-2/15 receptor signaling in T lymphocytes. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 168-183). / IL-2 and IL-15 are common y-chain family cytokines critically involved in regulation of T cell differentiation and homeostasis. Both cytokines signal through a heterotrimeric surface receptor complex (IL-2/15R) composed of an [alpha], a [beta], and the common gamma ([gamma]c) chain. Signaling occurs through the shared [beta] and [gamma]c chains, and the a-chains function as high affinity ligand capture agents. Despite sharing signaling chains and pathways, IL-2 and IL-15 have nonredundant roles in T cell biology. In vitro, although IL-2 and IL-15 function as equivalent mitogens, they greatly differ in their activities as growth factors. T cells cultured with IL-2 are larger in size, show increased protein synthesis, and increased amino-acid uptake compared to T cells cultures in IL-15. Additionally, transient exposure of T cell to IL-15, but not IL-2, results in prolonged survival and proliferation after cytokine withdrawal. In vivo, IL-2 is critical for the initial clonal expansion and contraction of activated T cells, whereas IL-15 is critical for the differentiation, and maintenance of memory T cells. The mechanisms through which IL-2 and IL- 15 trigger distinct phenotypes while initiating identical signaling pathways remain elusive. Using mass spectrometry based proteomics, we found that IL-2 and IL-15 trigger a quantitatively and qualitatively identical phosphotyrosine signaling response in T cells. The distinct effects of IL-2 and IL-15 on T cells could not be explained through activation of qualitatively distinct signaling pathways. Instead, our results show that IL-2 and IL-15 mediate distinct T cell phenotypes through differential regulation of IL-2/15R signal strength and duration. The increased survival and proliferation of T cells in response to transient IL-15 exposure is mediated through its ability to trigger prolonged signaling through the IL-2/15R after cytokine withdrawal. Furthermore, we find that T cell proliferation in response to IL-2 and IL-15 stimulation requires a constant signal input from the IL-2/15R. Additionally, T cell proliferation and metabolism are controlled in a quantitatively distinct manner through IL-2/15R signal strength independent of the cytokine identity. Proliferation is controlled by signal strength in a digital manner, whereas cell size and metabolism are controlled in an analog fashion. The differences in cytokinereceptor binding affinity, receptor expression levels, physiological cytokine levels, and cytokinereceptor intracellular trafficking kinetics can result in the differential regulation of IL-2/15R signal strength and duration by IL-2 and IL-15. These results suggest that IL-2 and IL-15 can trigger distinct phenotypes by differentially regulating the strength and duration of IL-2/15R signal transduction, without having to activate distinct biochemical signaling pathways. These results provide important insights into the function of other shared cytokine and growth factor receptors, quantitative regulation of cell proliferation and metabolism through signal transduction, and improved design of cytokine based clinical immunomodulatory therapies for cancer and infectious diseases. / by Abhinav Arneja. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 199-213). / Upon exposure to environmental cues, protein modifications form a complex signaling network that dictates cellular response. In this thesis, we develop methods for using continuous logic-based models to aide our understanding of these signaling networks and facilitate data interpretation. We present a novel modeling framework called constrained fuzzy logic (cFL) that maintains a simple logic-based description of interactions with AND, OR, and NOT gates, but allows for intermediate species activities with mathematical functions relating input and output values (transfer functions). We first train a prior knowledge network (PKN) to data with cFL, which reveals what aspects of the dataset agree or disagree with prior knowledge. The cFL models are trained to a dataset describing signaling proteins in a hepatocellular carcinoma cell line after exposure to ligand cues in the presence or absence of small molecule inhibitors. We find that multiple models with differing topology and parameters explain the data equally well, and it is crucial to consider this non-identifiability during model training and subsequence analysis. Our trained models generate new biological understanding of network crosstalk as well as quantitative predictions of signaling protein activation. In our next applications of cFL, we explore the ability of models either constructed based solely on prior knowledge or trained to dedicated biochemical data to make predictions that answer the following questions: 1) What perturbations to species in the system are effective at accomplishing a clinical goal? and 2) In what environmental conditions are these perturbations effective? We find that we are able to make accurate predictions in both cases. Thus, we offer cFL as a flexible modeling methodology to assist data interpretation and hypothesis generation for choice of therapeutic targets. / by Melody K. Morris. / Ph.D.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nonetheless, there are several examples of variation between human and mouse erythropoiesis. We performed a comparative global gene expression study using data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Induction and repression of major transcriptional regulators of erythropoiesis, as well as major erythroid-important proteins, are largely conserved between the species. In contrast, at a global level we identified a significant extent of divergence between the species, both at comparable stages and in the transitions between stages, especially for the 500 most highly expressed genes during development. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of erythroid evolution. In developing a systematic framework to understand and study conservation and divergence between human and mouse erythropoiesis, we show how mouse models can fail to mimic specific human diseases and provide predictions for translating findings from mouse models to potential therapies for human disease. / by Novalia Pishesha. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 149-160). / Solar energy provides an unparalleled promise to generate enormous amounts of clean energy. As the solar industry grows rapidly with a focus on power generation, new, but equally important challenges are emerging, including how to store and transfer the generated solar energy. Light-driven water splitting to generate hydrogen has received increasing attention as a means of storing solar energy. However, in order to evolve hydrogen with no energy input beyond sunlight, it is important to develop a stable and efficient catalytic system for water oxidation, which is the more challenging half-reaction of photocatalytic water splitting. Over several billion years, cyanobacteria and plants have evolved highly organized photosynthetic systems for the efficient oxidation of water. Water oxidation by mimicking photosynthesis has been pursued since the early 1970s; however, the approaches have been primarily limited to the extraction and reconstitution of the existing natural pigments, photosystems, and photosynthetic organisms, which suffer from instability. Metal oxide catalysts, often coupled with pigments, are similar to the reaction centers in natural photosystems and have been shown to photochemically oxidize water. Unfortunately, various approaches involving molecular design of ligands, surface modification, and immobilization still show low catalytic efficiencies unless they are used under relatively harsh conditions (i.e., in highly alkaline or acidic solutions under ultraviolet radiation). The current work aims to demonstrate the impact of nano-scale assembly of organic and inorganic molecules on energy and charge transfers, and related redox reactions. Genetically modified M13 viruses are explored as biological scaffolds to guide the formation of metal oxide catalysts-pigments hybrid nanostructures that enable efficient transports of both energy and electrons for photochemical water oxidation. This dissertation deals with three aspects of the virus-templated nanostructures - photonic, photochemical, and electrochemical properties. First, organic pigments are arranged into a one-dimensional light-harvesting antenna on the M13 virus. Chemical grafting of zinc porphyrins to the M13 virus induces spectroscopic changes, including fluorescence quenching, the extensive band broadening and small red-shift of their absorption spectrum, and the shortened lifetime of the excited states. Based on these optical signatures a hypothetical model is suggested to explain the energy transfer occurring in the supramolecular porphyrin structures templated on the virus. Second, through further genetic engineering of M13 viruses, iridium oxide hydrosol clusters (catalysts) are co-assembled with zinc porphyrins. When illuminated with visible light, this system evolves about 100 oxygen molecules per surface iridium molecule per minute in a prolonged manner. In addition, porous polymer microgels are used as an immobilization matrix to improve the structural durability of the assembled nanostructures and enable the recycling of the materials. The system also maintains a substantial level of its catalytic performance after repeated uses, producing about 1,200 oxygen molecules per molecule of catalyst during 4 cycles. These results suggest that the multiscale assembly of functional components, which can improve energy transfer and structural stability, should be a promising route for significant improvement of photocatalytic water oxidation. Lastly, electrochemical properties of the virus-templated iridium oxide nanowires are examined as an electrochromic film on a transparent conductive electrode. The prepared nanowire film has a highly open porous morphology that facilitates ion transport, and the redox responses of the nanowires are limited by the electron mobility of the nanowire film. These results demonstrate that a bio-templating approach provides a versatile platform for designing complex nanostructures that can facilitate the transport of electrochemical molecules in a broad range of photoelectrochemical devices. / by Yoon Sung Nam. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, June 2016. / Cataloged from PDF version of thesis. "May 2016." / Includes bibliographical references (pages 113-134). / When engineers control gene expression, cells can be re-programmed to create living therapeutics or materials by initiating expression of biosynthetic pathways in response to specific signals. In this thesis, two new genetic tools were developed to aid the construction of genetic circuits and facilitate their delivery to bacteria isolated from diverse environments. First, antisense transcription was explored as a new tool for tuning gene expression in Escherichia coli. Antisense transcription was found to reliably repress gene expression and was applied tune simple genetic circuits. Second, an integrative conjugative element from Bacillus subtilis, ICEBsJ, was engineered to deliver exogenous DNA to diverse strains of undomesticated Gram-positive bacteria. Engineered ICEBsI conjugation was demonstrated in twenty different bacterial strains, spanning sixteen species and five genera. To demonstrate ICE's utility in creating new probiotics, the element was used to deliver functional nitrogen fixation pathways (nif clusters) to bacteria isolated from agricultural soils. Collectively, the tools presented here in provide a platform for programing bacteria from diverse environments for advanced applications. / by Jennifer Ann Noelani Brophy. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / 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 58-63). / Amid the myriad recent developments in synthetic biology, progress has been fastest in the areas with the most versatile tools for understanding and engineering biological systems. RNA synthetic biology and synthetic minimal cells are areas where design is limited by the availability of tools to observe, program, and manipulate the systems in question. In this work I present expanded toolsets to achieve these goals. The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular, programmable protein architecture for targeting unmodified RNA sequences. I report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which I call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. I validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. I further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems. Genetic circuits are a fundamental tool in synthetic biology; an open question is how to maximize the modularity of their design, to facilitate their integrity, scalability, and flexibility. Liposome encapsulation enables chemical reactions to proceed in well-isolated environments. I here adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. I demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) so that they contain multiple-part genetic cascades, that these cascades can be controlled by external as well as inter-liposomal communication without cross-talk, and that these cascades can also be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability. / by Daniel Alberto Martin Alarcon. / Ph. D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 46-52). / Preterm birth impacts 15 million babies every year, leading to morbidity, mortality, significant health care costs, and lifelong consequences. The causes of preterm birth are unknown, resulting in ineffective treatment, but it is correlated with ascension of vaginal bacteria through the cervix, which is normally protected by a dense mucus plug during pregnancy. This mucus plug, consisting of a tight meshwork of glycoproteins called mucins, should prevent pathogens from accessing the sterile uterine environment. Cervical mucus from women at high risk and low risk for preterm birth was collected and compared. The aim of this study was to discover differences that will lead to clues about why preterm birth occurs, and ultimately what can be done about it in terms of prevention and intervention. Using rheological techniques and a translocation assay, we found that cervical mucus from women at high risk is more translucent and more elastic under both elongational and shear stress, than cervical mucus in normal pregnancies. These properties more closely resemble mucus during ovulation, when spermatozoa can most easily penetrate the barrier, than mucus in normal pregnancy. Furthermore, high risk mucus is more permeable to beads of comparable size to viruses, suggesting the barrier is weakened and foreign particles may harmfully traverse it to cause intrauterine infection. The techniques in this paper have not been previously used to study cervical mucus in the context of preterm labor, but their results may have important implications. If these mucus properties in women indeed permit increased bacterial infection through the cervix, then they can be used to stratify patients, allowing for more personalized prenatal care to lower the rate of preterm birth. / by Grace Yao. / M.Eng.