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.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 59-60). / Similar to biological human ankle, today's commercially available powered ankle-foot prostheses can vary impedance and deliver net positive ankle work. These commercially available prostheses are intrinsically controlled. Users cannot intuitively change ankle controller's behavior to perform movements that are not part of the repetitive walking gait cycle. For example, when transition from level ground walking to descending stairs, user cannot intuitively initiate or control the amount of ankle angle deflexion for a more normative stair descent gait pattern. This paper presents a hybrid controller that adds myoelectric control functionality to an existing intrinsic controller. The system employs input from both mechanical sensors on the ankle as well as myoelectric signals from gastrocnemius muscle of the user. This control scheme lets the user to modulate the gain of command ankle torque upon push off during level ground walking and stair ascent. It also allows the user to interrupt level ground walking control cycle and initiate ankle plantar flexion during stair descent. As a preliminary study, ankle characteristics such as ankle angle and torque were measured and compared to biological ankle characteristics. Results show that the proposed hybrid controller can maintain existing controller's biomimetic characteristics. In addition, it can also recognize to a qualitative extent the intended command torque for ankle push off and user's desire to switch between control modalities for different terrains. The study shows that it is possible and desirable to use neural signals as control signals for prosthetic leg controllers. Keyword: Myoelectric control, powered prosthesis, proportional torque control / by Jing Wang. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Vita. Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / The inability of articular cartilage to repair itself after acute injury has been implicated in the development of osteoarthritis. The objective of this work was to develop methods for delivering growth factors to cartilage and to test the ability of a self-assembling peptide scaffold, (KLDL)3, with or without growth factors to augment repair. Delivery methods included growth factor adsorption, scaffold-tethering, and modification of growth factor structure. (KLDL)3 was modified to deliver IGF-1 and TGF-[beta]1 to chondrocytes and bone marrow- derived stromal cells (BMSCs), respectively, by adsorption and by biotin-streptavidin tethering. This study showed that while TGF-[beta]1 can be effectively delivered by adsorption, IGF-1 can not. Additionally, while tethering these factors provided longterm sequestration, tethering did not stimulate proteoglycan production in vitro. A full-thickness, critically sized, rabbit cartilage defect model was used to test the ability of (KLDL)3 with or without chondrogenic factors (TGF-[beta]1, dexamethasone, and IGF-1) and BMSCs to stimulate cartilage regeneration in vivo. (KLDL)3 alone showed the greatest repair after 12 weeks with significantly higher Safranin-O, collagen II immunostaining, and cumulative histology scores compared to untreated contralateral controls. Ongoing studies include the evaluation of (KLDL)3 in a clinically relevant sized equine defect co-treated with micro-fracture and subjected to strenuous exercise. A fusion protein was created by adding a heparin-binding domain to IGF-1 (HBIGF- 1), converting IGF-1 from a short-acting growth factor to one that can be retained and locally delivered in articular cartilage in vivo. It was shown that HB-IGF-1 is retained in cartilage through binding to negatively charged glycosaminoglycan chains, with chondroitin sulfate the most prevalent type in cartilage. HB-IGF-1 was shown to bind adult human cartilage and to be preferentially delivered and retained in rat articular cartilage after intra-articular injection. In contrast, unmodified IGF-1 was not detectable after intra-articular injection. These results suggest that modification of growth factors with heparin-binding domains may be a clinically relevant strategy for local delivery to cartilage. Taken together, these results show that (KLDL)3 self-assembling peptide hydrogels are customizable for growth factor delivery and can promote cartilage repair in vivo. In addition, the fusion protein HB-IGF-1 is preferentially retained in cartilage tissue compared to un-modified IGF-1. / by Rachel E. Miller. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Inflammation is a major risk factor for many types of cancer, and the physiological processes involved in inflammation can contribute to many aspects of cancer development. Inflammation entails reprogramming of cell behaviors that resemble cancer, such as increased proliferation and signals for survival and migration, and it also entails production of reactive chemical species, which can damage DNA to promote genetic instability, another hallmark of cancer. While much research has been dedicated to studying the relationships between inflammation and cancer, it has been difficult to distinguish the relative contributions of modified cell behavior and de novo mutagenesis to the development of cancer. Furthermore, few studies have addressed the role(s) inflammation plays in cancer initiation versus promotion. Here, we utilized a transgenic mouse for detecting mutations in a variety of models of inflammation to parse the mechanisms by which inflammation contributes to mutations and cancer. The RaDR mouse, developed in the Engelward lab, contains a ubiquitously expressed transgene that enables detection of sequence rearrangement mutations following aberrant homologous recombination (HR). These mice also contain the Gpt-[delta] transgene for detecting point mutations and deletions, enabling unprecedented breadth and depth of possible mutation analyses in a single tissue. Our studies began by querying whether elements that regulate inflammation protect against mutagenesis in RaDR animals. We then studied RaDR mutagenesis in several models of intestinal inflammation and cancer. Together, these experiments showed that inflammation does not significantly induce de novo sequence rearrangement mutations, but it greatly increases the overall burden of mutant cells in a tissue as a result of heightened proliferation and clonal expansion. We also used the RaDR mouse model to expand upon studies of DNA repair pathway balance. DNA damage is addressed by a network of pathways, each designed to identify and repair specific types of lesions. One of the most important repair pathways for DNA damage caused by inflammation is the Base Excision Repair (BER) pathway, and we have previously found that BER intermediates can increase the frequency of mutagenic HR. Here, we expand upon that information, showing that acceleration of the BER pathway by increased expression of an initiating enzyme does not increase sequence rearrangement mutations, provided the downstream pathway can be resolved efficiently. Together, the studies described herein demonstrate that inflammation is unlikely to initiate cancer via sequence rearrangement mutations, but inflammation is a strong promoter of cancer in part through increased clonal expansion of mutant cells. / by Jennifer Elizabeth Kay. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Tissue engineering is a potentially valuable tool for clinical treatment of diseases where host tissues or organs need to be replaced. Progression of engineering metabolically complex organs and tissues has been severely limited by the lack of established, functional vasculature. The thesis work described herein focused on methods of establishing and studying specific endothelial cell types in vitro for potential applications in establishing functional microvascular architecture. To achieve these objectives, a model system of primary liver sinusoidal endothelial cells (LSEC) was initially studied due to the high metabolic requirements of the liver, as well as the unique phenotype that they possess. We were able to demonstrate that free fatty acids were able to rescue LSEC in culture, promote proliferation, and maintain their differentiated phenotype. Our work with lipid supplementation in serum-free conditions provides flexibility in engineering liver tissue with a functional vasculature comprised with relevant endothelial types encountered in vivo. Following up our work with LSEC, we explored the human dermal microvascular endothelial cell (HDMVEC) system to understand the signaling mechanisms involved in sprouting angiogenesis. Engineered tissues that are implanted will require integration with host vasculature. We established a method to collect large signaling data sets from a physiologically relevant in vitro culture system of HDMVEC that permitted angiogenic sprouting. We were able to find statistically significant data regarding how angiostatic cues like Platelet Factor 4 can modulate angiogenesis signaling pathways. Our results from working with both types of endothelial cell systems provide insight into potential methods for establishing specialized microvasculature for engineered tissues, both in propagation of differentiated endothelial cells in vitro and promotion of tissue/organ survival following their implantation. / by Ta-Chun Hang. / Ph.D.