Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / "December 2012." Cataloged from PDF version of thesis. / Includes bibliographical references. / Multiple signaling pathways are employed by endothelial cells to differentially respond to distinct hemodynamic environments and acquire functional phenotypes, including regulation of inflammation, angiogenesis, blood coagulation, and the vascular tone. In order to understand how these pathways interact, this thesis applies a systems biology approach through a two-step process. First, we constructed an integrated mathematical model for shear-stress-induced nitric oxide (NO) production to assemble the current understanding of this signaling system. Second, we conducted experiments to define how shear stress dynamically modulates the expression of components of the endothelial glycocalyx, a mechanosensor that regulates shear-stressdependent NO production. Nitric oxide produced by vascular endothelial cells is an anti-inflammatory mediator and a potent vasodilator. In order to understand the rich diversity of responses observed experimentally in endothelial cells exposed to shear stress, we assembled four quantitative molecular pathways previously defined for shear-stress-induced NO production. In these pathways, endothelial nitric oxide synthase (eNOS) is activated (a) via calcium release, (b) via phosphorylation reactions, and (c) via enhanced protein expression. To these pathways we added (d) an additional pathway describing the actual NO production from the interactions of eNOS with its various protein partners. These pathways were then combined and simulated. The integrated model is able to describe the experimentally observed change in NO production with time following the application of fluid shear stress, and to predict the specific effects to the system following interventional pharmacological or genetic changes. Importantly, this model reflects the up-to-date understanding of the NO system and provides a platform to aggregate information in an additive way. The endothelial glycocalyx is a glycosaminoglycan layer located on the apical surface of vascular endothelial cells. Previous studies have documented a strong correlation between the glycocalyx expression, local hemodynamic environment, and atheroprotection. Based on these observations, we hypothesized that the expression of components of the endothelial glycocalyx is differentially regulated by distinct hemodynamic environments. In order to test this hypothesis, human endothelial cells were exposed to shear stress waveforms characteristic of atherosclerosis-resistant or atherosclerosis-susceptible regions of the human carotid, and the expression of several components of the glycocalyx was then assessed. Interestingly, we found that heparan sulfate expression is higher and evenly distributed on the apical surface of endothelial cells exposed to the atheroprotective waveform, and is irregularly present in cells exposed to the atheroprone waveform. Furthermore, the expression of a heparan sulfate proteoglycan, syndecan-1, is also differentially regulated by the two waveforms, and its suppression mutes the atheroprotective-flow-induced cell surface expression of heparan sulfate. Collectively, these data links distinct hemodynamic environments to the differential expression of critical components of the endothelial glycocalyx. Taken together, these projects present in this doctoral thesis increase our understanding of endothelial mechano-activated pathways, and have demonstrated how we could use systems biology approach to unravel complex biological problems. / by Andrew Jia-An Koo. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / 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. / In this thesis, we developed two synthetic biology frameworks to facilitate the construction of useful genetic circuits, with a focus on circuits that sense miRNAs. miRNAs are an attractive biomarker for sensing since they regulate virtually all biological pathways in plants and animals, and because miRNA sensors can be easily designed by incorporating sequences complementary to the miRNA into a genetic circuit. Therefore, circuits that sense endogenous miRNAs can dynamically respond to cellular signaling or classify between cell types. However, the development of genetic circuits, and especially multi-input miRNA sensors, has traditionally been iterative, costly, and time-consuming. To this end, we have developed a framework to measure miRNA activity and generate accurate predictions for sensors with multiple miRNA inputs. We started by building the largest library of miRNA sensors to date (620 sensors) and used the library to measure miRNA activity in several cell lines. We then constructed multi-input sensors and determined design rules for predicting their function, namely that miRNAs repress targets synergistically in opposite UTRs and antagonistically within the same UTR. In our second framework, we developed a 'one-pot' method for high-information transfection and analysis that allows researchers to quickly determine performance of many tuned circuit variants in a single well. We used our one-pot method to quickly characterize a variety of genetic elements and to optimize the design of a miRNA sensor with inverted logic, a circuit topology we found difficult to design using traditional methods. / by Jeremy Jonathan Gam. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Joint injuries are common and often result in damage to cartilage, which has a limited ability to repair itself. Tissue engineering is a promising approach for improving cartilage healing in which biomaterials and chemical factors are supplied to direct cells to create a new tissue. The objective of this thesis was to optimize cartilage-like extracellular matrix production by investigating the effects of Dexamethasone (Dex) and HB-IGF- I (heparin-binding insulin-like growth factor-1) on cells encapsulated in the self-assembling peptide RAD and agarose hydrogels. Dex is a synthetic corticosteroid that has been shown to improve cartilage-like tissue production by bone marrow stromal cells (BMSCs), but the mechanisms underlying BMSC response to Dex are not understood. The hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine BMSCs in RAD and agarose hydrogels was tested. The effects of Dex were dependent on the hydrogel material and the species/age of the BMSCs. Importantly, Dex reduced aggrecanase-mediated degradation of matrix in both agarose and RAD hydrogels and for both young bovine and adult human BMSCs. HB-IGF-1, a fusion protein of the heparin binding domain of HB-EGF and IGF-1, can be retained in cartilage matrix and stimulate proteoglycan synthesis with a single dose, whereas unmodified IGF-1 easily diffuses out of cartilage tissue. The RAD peptide was used as a scaffold for retaining growth factor to stimulate encapsulated chondrocytes and adjacent cartilage tissue. RAD was modified by adsorption of HB-IGF-1 before and after RAD assembly, as well as adsorption of heparan sulfate (HS) and IGF-1. The RAD material retained HS adsorbed pre-assembly and HB-IGF-1 delivered in both adsorption methods. Adsorbed HB-IGF-1 and IGF-1 led to increased aggrecan content regardless of the method of adsorption. A trend was found for increased proteoglycan synthesis in adjacent explants as well. RAD self-assembling hydrogels are a promising material for culturing BMSCs undergoing chondrogenesis, retaining, and delivering HB-IGF-1. Dex decreases aggrecanase activity of differentiating BMSCs and adsorbed HB-IGF-1 appears to enhance aggrecan production by encapsulating chondrocytes and adjacent tissue. These findings show potential for improving cartilage repair in vivo. / by Emily Marie Florine. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2008. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 126-138). / Technologies that provide information about the concentrations or activities of specific molecules in living subjects have the potential to greatly advance science and medicine. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task because of its ability to image deep inside tissues at relatively high spatial and temporal resolution. However, the range of molecular phenomena currently accessible to MRI is limited by a lack of suitable molecular sensors. Most efforts to create such sensors have focused on synthetic contrast agents, whose complicated structures make them difficult to engineer, synthesize and deliver to target tissues. If MRI sensors could instead be made of proteins, a number of these difficulties could be mitigated. Here, we describe two platforms for the development of protein-based molecular sensors for MRI. The first is based on the heme domain of the bacterial cytochrome P450-BM3, which produces changes in TI contrast in MRI in response to small molecule binding. We developed a high-throughput assay that allowed us to evolve this protein into a sensor of the neurotransmitter dopamine (DA). We then used it to image DA release from cultured cells and cocaine-induced changes in DA transport in the brains of living rats. The second platform is based on the human iron storage protein ferritin (Ft), which enhances T2 contrast in MRI upon self-aggregation. We developed a system to express self-assembled Ft nanoparticles incorporating multiple surface functionalities, and used it to create a sensor for protein kinase A activity. Our results provide a proof of concept for two novel platforms for protein-based MRI sensor development, and highlight some key advantages of this approach over the synthetic methods used previously. / by Mikhail G. Shapiro. / Ph.D.

Thesis (M. Eng. in Biomedical Engineering)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 37-40). / Spinal Cord injury (SCI) leads to paralysis, decrease in quality of life and high lifetime medical costs. Direct nerve Functional Electrical Stimulation (FES) induces muscles to contract by electrically stimulating nerves, which shows promise for clinical applications in restoring muscle function in SC. However, Functional Electrical Stimulation is limited by the lack of graded response in muscle contraction and by high fatigability due to the reversal of recruitment order of motor units. Previous work showed that ion-selective membranes can be used to modulate Ca 2 ions in situ, decreasing the current threshold for nerve stimulation and eliciting a more graded muscle contraction response. This work developed polyimide-based cuff ion-selective electrodes to enable the future application of this technique in vivo. The developed electrodes were flexible, elastic and conductive. In vitro tests of the electrodes by stimulation of frog sciatic nerve reproduced the decrease in stimulation current threshold, which had been observed in planar glass-based electrodes, in the flexible polyimide-based electrodes. Additionally, cuffing the stimulated nerves with ion-selective electrodes was more effective at decreasing current threshold than planar stimulation. This work also analyzed data on twitch width, contraction time and relaxation time to infer effects of ion-selective electrodes on recruitment order. Stimulation with the ion-selective electrodes had higher twitch width, contraction time and relaxation time than traditional electrical stimulation at all force levels. The difference was particularly high at low force levels, indicating an effect of Calcium ion depletion on recruitment order. / by Ragheb Mohamad Fawaz El Khaja. / M.Eng.in Biomedical Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 189-211). / While a link between chronic inflammation and cancer has been established, the mechanisms of genotoxicity in inflammatory environments remain poorly understood. We hypothesized that inflammation may provide cues that allow cells to survive in the face of significant DNA damage as well as cues that foster cell proliferation. This study sought to elucidate changes that influence hepatocyte decision processes under conditions of chronic inflammation by using a set of extracellular signals, both soluble and matrix-related, which can be varied systematically to create a diverse range of intracellular signaling states and phenotypic outcomes. We developed an easily translatable model system that can maintain primary mouse hepatocytes in a differentiated state and study the independent extracellular cues which regulate hepatocyte behavior during chronic inflammation. This model system allowed systematic variation of oxygen concentration as well as matrix composition and stiffness. Collagen, polyacrylamide, and RADA gels were used to create extracellular environments resembling the various stages of normal and fibrotic liver. Through careful control of medium depth and incubator oxygen levels, we determined that oxygen tension and extracellular matrix affected hepatocyte differentiation independently, and that both high oxygen and a compliant matrix environment are necessary for prolonged maintenance of primary mouse hepatocytes in vitro. Optimization of a quantitative imaging solution enabled the capture of rare events occurring in individual cells within a larger cell population. Using TNF-alpha, Fas ligand and, IL-6 cytokines in conjunction with oxygen and matrix cues, we investigated how extracellular stimuli influence cell fate in a pseudo-inflammatory environment and demonstrated that partial execution of apoptosis is a possible mechanism for genotoxicity in hepatocytes during chronic inflammation. The results of this study improve our understanding of how cues in the extracellular environment combine to influence the behavior primary mouse hepatocytes. The system we developed can be used as a platform for a multitude of in vitro applications including studies regarding drug toxicity and inflammation. / by Lorenna Dianne Buck. / Ph. D.

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.