Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Recent advances in chemotherapeutic development have targeted vital mechanisms that ensure survival of cancer cells; these include the ability to evade immune surveillance, undergo metabolic adaptations and form a defense mechanism against oxidative stress. Cancer cells often possess higher endogenous levels of reactive oxidative species (ROS) compared to normal cells due to the cumulative effects from genomic instability, inflammation and oncogene activation, and they become increasingly reliant on the cell antioxidant network to prevent this elevated oxidative stress from becoming toxic. Thus, chemotherapeutics that inhibit the antioxidant network and thereby elevating ROS are thought to be promising candidates that can selectively eliminate tumor cells. Despite the promises of these molecules, chemotherapeutics modulating ROS levels have mostly fallen short of their projected impact. We believe that a thorough understanding of the quantity, location and duration of ROS generation needed to cause tumor cell toxicity, will be important for understanding the mechanism of current successful chemotherapeutics and designing future ROS-based drug candidates. In this thesis, we explored the use of genetically encoded sensors and generators of ROS, H₂O₂ in particular, to answer these important redox biology questions. We began by developing a deeper understanding of how to use these protein-based peroxide sensors in a quantitative manner. We created a technique quantifying intracellular peroxide levels by converting the fluorescence signal outputs from these sensors into more meaningful intracellular concentrations. This was accomplished via a combination of kinetic modeling, biochemical measurements and image analysis techniques. We also explored the cell to cell heterogeneity in sensor response to H₂O₂ stimulation, and found that the intracellular expression level of the sensor is correlated with the ratio-metric response of the probe. Further kinetic modeling analysis showed that the slow recycling step of activated sensor was responsible for the correlation. In the second part of the thesis, we used these sensors in combination with enzymatic generators that can produce H202 endogenously in a kinetically controlled manner. These tools allowed us to quantitatively determine that there are two toxicity thresholds, a total accumulation of H₂O₂ and intracellular concentration, that are needed for H₂O₂ -mediated cell death. We also applied these tools to investigate the mechanism of two ROS-based chemotherapeutics, phenethyl isocyanate (PEITC) and piperlongumine. We found that depletion of the glutathione antioxidant by these drugs was unimportant to the toxicity mechanism, and the amount of oxidative stress generated by these compounds was not enough to induce significant toxicity by itself. The final part of the thesis involves technology development for a next generation enzymatic ROS generator. We explored the use of P450-BM3, an enzyme that can generate superoxide and hydrogen peroxide through a reaction that requires only NADPH and oxygen. While this reaction in the wild type protein is slow, it can be engineered to have much higher catalytic rates. We demonstrated through various protein engineering approaches that we could create P450-BM3 proteins with enhanced generation of H₂O₂. We also were able to express correctly folded, active enzymes inside mammalian cells that utilize intracellular NADPH and oxygen to produce H₂O₂. / by Beijing Kara Huang. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / 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 (p. 162-176). / It is well established that extracellular pH (pHe) becomes acidic in several important physiological and pathological contexts, including the tumor and wound microenvironments. Although it is known that acidic pHe can have profound effects on cell adhesion and migration processes integral to tumor progression and wound healing, the molecular mechanisms underlying the cellular responses to acidic pHe are largely unknown. Transmembrane integrin receptors form a physical linkage between cells and the extracellular matrix, and are thus capable of modulating cell adhesion and migration in response to extracellular conditions. In this thesis, computational and experimental approaches are used to investigate the role of acidic extracellular pH in regulating activation and binding of integrin [alpha]v[beta]3, and to characterize the consequences for downstream subcellular- and cellular-scale processes. Molecular dynamics simulations demonstrate that opening of the integrin [alpha]v[beta]3 headpiece occurs more frequently in acidic pHe than in normal pHe, and that this increased headpiece opening can be partially attributed to protonation of ASP[beta]127 in acidic pHe. These computational data indicate that acidic pHe can promote activation of integrin [alpha]v[beta]3. This is consistent with flow cytometry and atomic force microscope-enabled molecular force spectroscopy experiments, which demonstrate that there are more activated [alpha]v[beta]3 receptors on live [alpha]v[beta]3 CHO-B2 cell surfaces at acidic pHe than at normal pHe 7.4. Put together, these atomistic- and molecular-level data suggest a novel mechanism of outside-in integrin activation regulation by acidic extracellular pH. Next, the consequences of acid-induced integrin activation for subcellular- and cellular-scale processes are investigated. Kymography experiments show that [alpha]v[beta]3 CHO-B2 cell membrane protrusion lifetime is increased and protrusion velocity is decreased for cells in pHe 6.5, compared to cells in pHe 7.4. Furthermore, [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 form more actin-integrin adhesion complexes than cells in pHe 7.4, and acidic extracellular pH results in increased cell area and decreased cell circularity. Cell migration measurements demonstrate that [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 migrate slower than cells in pHe 7.4, and that the fibronectin ligand density required for peak migration speed is lower for cells in pHe 6.5. Together, these data show that acidic pHe affects subcellular- and cellular-scale processes in a manner that is consistent with increased integrin activation in this condition. Finally, the migration behavior of [alpha]v[beta]3 CHO-B2 cells, bovine retinal microvascular endothelial cells, and NIH-3T3 fibroblasts in an extracellular pH gradient is investigated. Results demonstrate that NIH-3T3 fibroblasts do not exhibit directional preferences in the pHe gradient, but that [alpha]v[beta]3 CHO-B2 cells and bovine retinal microvascular endothelial cells migrate preferentially toward the acidic end of the gradient. These data suggest that acidic extracellular pH may serve as a cue that directs migration of angiogenic endothelial cells to poorly vascularized regions of tumors and wounds. Overall, this thesis research results in multiscale, in-depth understanding of extracellular pH as a critical regulator of cell function, with associated implications for tumor growth, wound healing, and the role of proton pumps in cell migration. / by Ranjani Krishnan Paradise. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 125-133). / Using molecular imaging in vivo, biomolecular and cellular phenomena can be investigated within their relevant physiological context, addressing a central challenge for 21st century biomedicine and basic research. To advance neuroscience in particular, molecular-level measurements across the brain inside the intact organism are required. However, existing imaging strategies and available probes have been limited by serious constraints. Magnetic resonance imaging (MRI) provides deeper tissue penetration depth than optical imaging and better spatial resolution and greater versatility in sensor design than radioactive probes. The most important drawback for MRI probes has been the need for high concentrations in the micromolar to millimolar range, leading to analyte sequestration, complications for noninvasive brain delivery, and toxicity. Efforts to address the sensitivity problem, such as nuclear hyperpolarization, introduce their own technical constraints and so far lack generality. Here, we introduce a conceptually novel molecular imaging technique based on artificially induced physiological perturbations, enabling molecular MRI with nanomolar sensitivity. In this imaging strategy, we take advantage of blood as an abundant endogenous source of contrast compatible with multiple imaging modalities including MRI and optical imaging to decouple the concentration requirement for molecular sensing from the concentration requirement for imaging contrast. Highly potent vasoactive peptides are engineered to respond to specific biomolecular phenomena of interest at nanomolar concentrations by inducing dilation of the microvasculature, increased local bloodflow, and consequently, large changes in T₂*-weighted MRI contrast. This principle is exploited to design activatable probes for protease activity based on the calcitonin gene-related peptide (CGRP) and validate them for brain imaging in live rats; to use CGRP as a genetic reporter for cell tracking; and to create fusions of a vasoactive peptide from flies to previously characterized antibodies capable of crossing the blood-brain barrier (BBB), suggesting the possibility of minimally invasive brain delivery of such probes. We demonstrate the feasibility of highly sensitive molecular MRI with vasoactive probes at concentrations compatible with in situ expression of probes and delivery across the BBB, and show that vasoactive peptides are a versatile platform for MRI probe design which promises unprecedented in vivo molecular insights for biomedicine and neuroscience. / by Adrian L. Slusarczyk. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Animal noses have evolved the ability to rapidly detect a seemingly infinite array of odors at minute concentrations. The basis of this sensitivity are the olfactory (smell) receptors - a large, highly related class of sensory G-protein coupled receptors that function together combinatorially to allow discrimination between a wide range of volatile and soluble molecules. However, the structural and functional mechanisms of these amazing receptors are not currently known. In order to begin to investigate the molecular mechanism(s) of olfaction, I have developed a mammalian expression system for the large-scale production and purification of functional olfactory receptor (OR) proteins in milligram quantities. Expressed OR genes were fabricated from scratch using PCR-based gene synthesis, which facilitated codon optimization and attachment of different affinity tags for purification. Established methods for the production and purification of rhodopsin were adapted to olfactory receptors through extensive optimization (including a full-spectrum screening of over 45 detergents). Key to the efficient extraction and solubilization of olfactory receptors tested is the use of novel zwitter-ionic fos-choline detergents. Following initial experiments on the inducible expression of a human olfactory receptor (hOR17-4) in adherent HEK293S cell cultures, the system was successfully scaled up using a suspension bioreactor. Large-scale culture allowed the purification of >10 milligrams of hOR17-4 monomer at >90%, which was suitable for subsequent X-ray crystallization screening trials. The purified protein was also characterized using several spectroscopic methods and shown to possess the correct secondary structure and several predicted post-translational modifications. To assay the functionality of purified (nonmembrane- bound) hOR17-4, we successfully developed an in vitro assay method using surface plasmon resonance (SPR) to demonstrate that the receptor retains functional selectivity in binding specific odorant ligands in a concentration-dependent manner. The application of these techniques to other olfactory receptors already shows promise and could lead to a generalized method for obtaining large quantities of any olfactory receptor in a rapid and simple manner. Such methods could prove extremely useful in elucidating the structural and functional mechanism(s) of olfactory receptors and in their integration into OR-based biosensor devices. / by Brian Lee Cook. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 110-127). / For many cancers, dissemination of tumor cells to form metastases is not only a hallmark of the disease but an essential step to mortality. Migration and dissemination are complex, multistep processes, and study of their regulation has been challenging. Metastases need only be driven by a rare subpopulation of tumor cells, and a portion of dissemination is necessarily interaction with the cell's environment and thus cell extrinsic. Experimentally, there is additional uncertainty as exactly how to best assess migration outside of the complex in vivo environment. To develop a systems perspective of invasive disease, we first examine some of the experimental models used to study cell migration. We then apply this knowledge to examine regulation by proteases of endometrial cell invasion, and the pro-migratory effects of receptor crosstalk in breast carcinoma cells. Finally, extending from clear limitations in our knowledge of signaling regulation specifically within the invasive subpopulation of cells, we develop a model of ligand-mediated signaling for a receptor often expressed specifically during the process of dissemination. In total, this thesis extends systems biology techniques to the study of cell migration within the extracellular environment, with focus on that subpopulation of cells most directly implicated in the formation of metastatic disease. / by Aaron Samuel Meyer. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 145-156). / Post-translational modifications (PTMs) regulate cellular signaling networks by modifying activity, localization, turnover and other characteristics of proteins in the cell. For example, signaling in receptor tyrosine kinase (RTK) networks, such as those downstream of epidermal growth factor receptor (EGFR) and insulin receptor, is initiated by binding of cytokines or growth factors, and is generally propagated by phosphorylation of signaling molecules. The rate of discovery of PTM sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. The ten-fold increase in known phosphorylation sites over a five year time span can primarily be attributed to mass spectrometry (MS) measurement methods, which are capable of identifying and monitoring hundreds to thousands of phosphorylation sites across multiple biological samples. There is significant interest in the field in understanding these modifications, due to their important role in basic physiology as well as their implication in disease. In this thesis, we develop algorithms and tools to aid in analysis and organization of these immense datasets, which fundamentally seek to generate novel insights and testable hypotheses regarding the function and regulation of phosphorylation in RTK networks. We have developed a web-accessible analysis and repository resource for high-throughput quantitative measurements of post-translational modifications, called PTMScout. Additionally, we have developed a semi-automatic, high-throughput screen for unsupervised learning parameters based on their relative ability to partition datasets into functionally related and biologically meaningful clusters. We developed methods for comparing the variability and robustness of these clustering solutions and discovered that phosphopeptide co-clustering robustness can recapitulate known protein interaction networks, and extend them. Both of these tools take advantage of a new linear motif discovery algorithm, which we additionally used to find a putative regulatory sequence downstream of the highly tumorigenic EGFRvIII mutation that indicates casein kinase II (CK2) activity may be increased in glioblastoma. / by Kristen M. Naegle. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Mucosal barrier tissues are prominent targets for drugs against infection and chronic inflammatory disorders. One such mucosal barrier tissue, the endometrium, undergoes monthly cyclic remodeling via hormone-mediated growth, immune cell recruitment and proteolytic breakdown. Hormone response disruption has been associated with numerous endometrial pathologies, including endometriosis, adenomyosis, and infertility, which impacts upwards of 10% of women during their reproductive years. Currently, our understanding of endometrial biology is limited by the ability to replicate complex 3D physiology in vitro. Our ability to parse disease mechanisms and test efficacy of therapeutic interventions relies on development of reproducible models, adaptable to the limited numbers of cells available from patient biopsies. In this thesis, I address a critical gap in accessible tools to study and control endometrial biology in vitro and do so in a manner that can be translated to other epithelial-stromal mucosal tissues. Using the endometrium as an example mucosal barrier, I first establish design principles for the development of a synthetic, modular extracellular matrix (ECM) hydrogel suitable for 3D functional co-culture of epithelial and stromal cells. This 'one-size- fits-all' matrix features components that can be remodeled by cells and that responds dynamically to sequester local cell-secreted ECM characteristic of each cell type enabling long-term, hormonally responsive co-cultures. Next, I establish methods to expand and cryopreserve primary human endometrial epithelial cells, which maintain barrier and secretory function, further enabling studies using primary cells. Finally, we use data-driven network modeling of secreted proteins to understand how variation in cytokine signaling may alter hormone responsiveness and proteolytic remodeling in primary epithelial-stromal co-cultures. With the ability to create and parse more complex 3D tissue models using primary cells to recapitulate healthy and diseased states, we further enable basic understandings of disease pathologies and subsequent drug discovery efforts aimed at inflammation, wound healing and immune modulation. / by Christi Dionne Cook. / Ph. D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references . / Glycans are essential components of all living things because they function as key elements of cellular membranes and extracellular spaces by mediating cell-cell communication, transduction pathways, and cellular development, function, and survival. Because glycans are secondary gene products that depend on the availability of sub-cellular enzymes for synthesis, research on their structure, synthesis, and biological significance has lagged behind that of DNA and proteins due to both a lack of appreciation of their importance and the slow pace at which tools are being developed to study them. In this thesis, three projects focus on the development, application, and exploration of tools for glycan characterization. The first project resulted in the successful optimization of an analytical method to isolate and characterize O-linked glycans, on which relatively few research projects focus because of the limited availability of tools to isolate them and the need for specific analytical equipment to properly characterize them. Using this optimized method, the O-linked glycans of bovine mucin and fetuin were successfully profiled, and the analysis of the former provided the motivation for a second project focused on the significance of goblet cells and mucins in influenza infection. This project explored the potential benefits of a glycoprotein direct binding assay as a way to obtain quantitative information about lectin and influenza hemagglutinin specificities. Using mucins adsorbed to a polystyrene plate, it was possible to obtain quantitative binding constants for two commonly used lectins. The last project focused on the isolation and characterization of the cell surface N-linked glycans from chicken erythrocytes, turkey erythrocytes, and human tracheal epithelial (HTE) cells. Analysis of these cell types is warranted due to their importance as model systems to study influenza infection. The results of this project provide a context for future questions about the relevance of the erythrocyte model system for studying influenza binding specificities. All of these projects reiterate the importance of the study of glycobiology by showing how both the development and application of tools to study glycans can provide in new and interesting information about pathological processes related to human health and disease. / by Nia Beckley. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Mammalian noses can detect and distinguish an inestimable number of odors at minute concentrations. Four classes of G protein-coupled receptors (GPCRs) are responsible for this remarkable sensitivity: olfactory receptors (ORs), vomeronasal receptors (VNRs), trace amine-associate receptors, and formyl peptide receptors. Structural knowledge of these receptors is necessary to understand the molecular basis of smell. However, no structure exists for three main reasons. First, milligrams of protein are needed for crystallization screens, but most are expressed at low levels endogenously or in heterologous expression systems. Second, detergents capable of solubilizing and stabilizing these proteins in aqueous solution must be found. Third, the flexible nature of GPCRs can inhibit crystal lattice formation. Methods for overcoming each obstacle were developed. Milligrams of a VNR were expressed in HEK293 cells, and milligrams of 13 GPCRs were expressed in a cell-free system. All could be purified to >90%. The purified receptors had correct secondary structures, and could bind their ligands. The HEK293 and cell-free receptors had nearly identical structures and binding affinities, demonstrating that cell-free expression can be used for GPCR production and mutational studies. To demonstrate this, six variants of mOR103-15 with single amino acid substitutions were expressed. Ligand-binding measurements indicated which residues were involved in ligand recognition. The choice of detergent used in the cell-free system was critical, and significantly affected expression levels. A class of amphiphilic peptide detergents was designed and tested with the receptors. These detergents could be used to express milligrams of functional receptors. The peptide tail and head group properties did not significantly affect their function, suggesting that they may be a class of surfactants usable with multiple olfactory-related receptors, and even other membrane proteins. Lastly, the protein T4 Lysozyme (T4L) was fused in the 3rd intracellular loop of two receptors to increase potential crystal lattice contact points. Purified T4L variants had correct secondary structures, and could bind their ligands and initiate intracellular signaling. The methods described generated sufficient quantities of pure receptors for crystal screens. The large number of functionally expressed GPCRs indicates that these techniques can be applied to other olfactory-related receptors, and even other membrane proteins. / by Karolina Corin. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references. / An understanding of the factors involved in the human adaptation of influenza A viruses is critical for various aspects of influenza preparedness, including the development of appropriate surveillance measures, preventive strategies and effective treatments. A key step in influenza human adaptation is the acquisition of mutations in the viral coat glycoprotein, hemagglutinin (HA), which changes its binding specificity towards glycan receptors in the human upper respiratory epithelia (referred to as human receptors). In this thesis, determinants that mediate changes in HA-glycan receptor binding specificity are investigated, with focus on the molecular environments within and surrounding the glycan receptor binding site (RBS) of HA. The glycan receptor binding properties of HA from different influenza subtypes (H1N1, H2N2, H3N2 and H5N1) are studied using a combination of approaches including dose-dependent glycan binding, human tissue staining and structural modeling. Using these complementary analyses, it is shown in this thesis that the molecular interactions between amino acids in and proximal to the RBS (referred to as amino acid interaction networks), including those between the RBS and glycosylation at sites proximal to the RBS, and interactions between the RBS and the glycan receptor together govern the high affinity binding of HA to human receptors. The thesis is divided into three sections. First, the evolution of glycan receptor binding specificity of recent human-adapted H3 strains such as A/Fujian/411/02 and A/Panama/2007/99 is investigated, with implications on vaccine production in chicken eggs. Second, the determinants of glycan receptor binding affinity of potentially pandemic avian viruses is studied in the context of the recently circulating H2 A/Chicken/Pennsylvania/2004 and the highly pathogenic H5 A/Vietnam/1203/2004. Here it is shown that mutations which cause human adaptation of H2 do not increase human receptor binding affinity in H5, and the importance of amino acid interaction networks is implicated. Third, determinants that govern the high affinity human receptor binding of pandemic influenza HAs is investigated using the prototypic 1918 H1N1 HA as a model system. The roles of amino acid interaction networks and the molecular interactions between the RBS and glycosylation at sites proximal to the RBS in contributing to the high affinity human receptor binding of 1918 H1N HA are investigated. The approaches presented in this thesis to systematically investigate molecular interactions between HA and glycan receptors that impinge on quantitative HA-glycan receptor binding affinity offer a new angle towards studying determinants of human receptor binding specificity and affinity of influenza A virus HAs. / by Xiaoying Koh. / Ph.D.