Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 99-107). / Microscopy has facilitated the discovery of many biological insights by optically magnifying small structures in cells and tissues. However, the resolution of optical microscopy is limited by the diffraction of light to ~200-300 nm, comparable or larger to the size of many subcellular structures. In this thesis, we describe a suite of tools based on a novel super-resolution microscopy approach called Expansion microscopy. Expansion microscopy (ExM) physically expands tissues so that the resolution of ordinary microscopes is increased -5 times by leveraging the swelling properties of polyelectrolyte hydrogels. Ordinary microscopes used with ExM are more accessible and faster than the specialized optical systems designed to image beyond the diffraction limit (e.g., STORM/PALM, STED, SIM), while yielding similar performance. Expanded tissues are also optically clear, allowing for unprecedented super-resolution imaging in thick tissues and facile reagent diffusion into the sample. We have since developed a variant of ExM, called protein retention ExM, in which proteins are directly anchored to the swellable gel using a commercially available cross-linking molecule. This strategy enables ExM of genetically encoded fluorescent proteins and commercial fluorescently labeled secondary antibodies. With these advancements, ExM can be carried out with purely commercial reagents and represents a simple extension of standard histological methods used to prepare samples for imaging. Furthermore, we have developed a variant of the ExM technology that enables RNA molecules to be directly linked to the ExM gel network via a small molecule linker and isotropic expansion. This technology, termed ExFISH, enables visualization of RNAs with nanoscale precision and single molecule resolution. We have demonstrated that the covalent anchoring of RNA also enables robust repeated washing and probe hybridization steps, opening the door to combinatorial multiplexing strategies. By leveraging these benefits, we have further developed in situ analysis tools which allow for highly multiplexed imaging of RNA identity and location with nanoscale precision in intact tissues. Taken together, these tools allow for spatially mapping molecular information onto cell types and tissue structures which could be invaluable for spatially complex biological processes such as brain function, cancer heterogeneity and organismal development. / by Fei Chen. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / 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. 119-124). / Cell size is often overlooked in the drive to define molecular mechanisms, but as a basic physical property it is an integrator of the cell's metabolic rate and indicator of cell fate. Development of the Suspended Microchannel Resonator (SMR), a microfluidic mass measurement system, enables femtogram cell mass resolution, and the resistive pulse (Coulter) technique provides high-speed electronic readout of cell volume. With these tools, we developed four methods to measure cell density, the ratio of mass to volume. We first measure the average density of cell populations using the SMR and a Coulter counter. We observe that cell density increases prior to bud formation at the G1/S transition of budding yeast, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we use the SMR to measure buoyant mass in high density media and monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator TOR, passage through START, and an intact actin cytoskeleton. These techniques are suitable for most non-adherent cells and subcellular particles to characterize cell growth in a variety of applications. We next develop two platforms to measure single-cell mass, volume, and density. These properties are calculated from two SMR buoyant mass measurements, each in different density fluids. These measurements are achieved by serially connecting two SMR structures through a microchannel with an intermediate T-junction, such that a cell is measured by each SMR in different density fluids. Similar measurements can also be made with one SMR by reversing the SMR fluid flow after a cell is measured-each cell re-enters the SMR in a higher density fluid for a second measurement. We find that the intrinsic cell-to-cell density variation is nearly 100-fold smaller than the mass or volume variation, and by simultaneously measuring density and mass, we identify distinct subpopulations of diseased and healthy cells that are indistinguishable by mass or volume alone. / by Andrea K. Bryan. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references. / In recent years, the field of systems biology rapidly expanded in both basic and translational science. This method of investigation revolves around an iterative cycle of observing a system, making predictions about its behavior using a model, and testing these hypotheses with further experiments. Though computational approaches have achieved astonishing sophistication, these models are fundamentally limited in their predictive power by the quality of data they are given. Thus, the lack of tools to capture information-rich data has become a bottleneck for our ability to predict and perturb biological systems. This thesis focuses on developing tools to collect data that captures the complexity of signaling networks to deepen our understanding of the mechanistic processes occurring inside the cell. In particular, we present a method capable of measuring phosphorylation changes in the cell with 10-second resolution. One of the best-characterized proteins in biology is the Epidermal Growth Factor Receptor (EGFR), which has long been associated with diseases including cancer. Despite development of several EGFR inhibitors, the clinical efficacy of targeting this receptor has been minimal. This shortcoming is attributable primarily to the incredible complexity of the EGFR signaling network, which includes hundreds of proteins throughout the cell. In this thesis, we use EGFR as a model to demonstrate the utility of measuring phosphorylation dynamics with high temporal resolution. We present an extensive characterization of EGFR signaling behavior across a range of growth factor concentrations, exposing distinct regimes of network activation. Bioinformatic analysis uncovers unexpected relationships within the data that uncover previously obscured biological distinctions within the system. This information is used to generate and test specific mechanistic hypotheses using broad and targeted perturbations. We explore the relationship between phosphorylation and complex formation of receptors and adaptors, finding evidence for distinct recruitment mechanisms for Shc and Gab1. Inhibition of phosphatase activity in the system shows unexpected behaviors in the form of specific phosphatase activity against sites on EGFR and Gab1 and ligand-independent activation of ERK. Examination of the data suggests a connection with Src family kinases as contributors to EGFR signaling. Further exploration with targeted inhibition of Src and P13K create a quantitative mechanistic explanation for EGFR signaling. Lastly, inhibition of the network with clinically relevant tyrosines kinase inhibitors reveals temporally distinct effects of inhibitors in early signaling. Combination of broad kinase and phosphatase inhibition produces unusual results that raise further questions of EGFR signaling. Together, the tools presented here for studying early signaling events at the systems level will contribute to our understanding of complex biological systems. / by Raven Jon Reddy. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Glioblastoma (GBM) is the most common malignant form of brain cancer. Even with treatment including surgery, radiation, and temozolomide chemotherapy, the 1 year survival rate is only 35%. To identify specific mediators of GBM progression in a genetically engineered murine model of proneural GBM, we quantified signaling networks using mass spectrometry. We identified oncogenic signaling associated with the GBM model, such as increased phosphorylation of ERK1/2, P13K, and PDGFRA, relative to murine brain. Phosphorylation of CDK₁ Y₁₅, which causes G₂ /M cell cycle arrest, was measured to be the most differentially phosphorylated site, with a 14-fold increase in the tumors. We used syngeneic cell lines to investigate this checkpoint further and treated these cells with MK-₁₇₇₅, an inhibitor of Wee₁, the kinase responsible for phosphorylation of CDK₁ Y₁₅. MK-₁₇₇₅ treatment resulted in mitotic catastrophe of these cells, as measured by increased DNA damage, abnormal percentages of cells in cell cycle phases, and death by apoptosis. This response was abrogated by inhibiting CDK₁ with roscovitine, a CDK inhibitor, demonstrating the necessity of active CDK₁ for MK-₁₇₇₅ induced mitotic catastrophe. To assess the extensibility of targeting Wee₁ and the G₂/M checkpoint in GBM, we treated patientderived xenograft (PDX) cell lines with MK-₁₇₇₅. The response was more heterogeneous, but we measured decreased CDK₁ phosphorylation, increased DNA damage, and death by apoptosis. These results were validated in a flank GBM PDX model where treatment with MK-₁₇₇₅ increased mouse survival by 1.74-fold. We also quantified the signaling differences in our murine GBM model after treatment with sunitinib, an inhibitor of its driver receptor tyrosine kinase, PDGFRA. Treatment increased survival but lead to a morphological change causing a more invasive phenotype. Pro-migratory signaling was characterized by mass spectrometry, such as increased phosphorylation of Eno₁, ELMO₂, and tubulins. Invasion was further characterized in a lung cancer model where we identified signaling specific to different ligands that result in similar levels of invasion. We have demonstrated that unbiased, quantitative phosphotyrosine proteomics has the ability to reveal therapeutic targets in tumor models and signaling differences between treatments. / by Rebecca S. Lescarbeau. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2016. / Cataloged from PDF version of thesis. "September 2015." / Includes bibliographical references (pages 60-66). / The genome holds the blueprint of life and heredity. In the case of the mammalian genome it is comprised of billions of DNA bases grouped into elements that we are beginning to understand (ie genes) and others we know little about (ie noncoding DNA). Forward and reverse genetics in cells and animal models is key to discovering causal mechanisms relating molecular and genetic events to phenotypes. Therefore, the ability to sequence and edit DNA is fundamental to understanding of the role of genetic elements in normal biology and disease. Recently developed genome editing technologies are now making it possible to modify the genome in its endogenous context, opening up exciting possibilities for understanding its function. The RNA-guided endonuclease Cas9 from microbial type II CRISPR (clustered regularly interspaced short palindromic repeat) systems has been harnessed to facilitate facile genetic manipulations in a variety of cell types and organisms. Cas9 can be easily reprogrammed using RNA guides to generate targeted DNA double strand breaks, which can stimulate genome editing. A unique advantage of the Cas9 system is that Cas9 can be combined with multiple guide RNAs to achieve efficient multiplexed genome editing in mammalian cells, which opens up the possibility of interrogating multigenic biological processes. In this thesis, we utilize the Cas9 technology to facilitate genome editing experiments in vivo in mice. First, we create a Cas9 knockin mouse and demonstrate its utility for in vivo and ex vivo genome editing experiments. Then, we leverage the Cas9 knockin mouse and viral-mediated delivery of guide RNA to model lung adenocarcinoma to obtain pathology consistent with traditional transgenic mouse models and human patients. Finally, we utilize pronuclear injection of Cas9 mRNA and guide RNA to generate mice harboring a mutation in the autism-associated gene CHD8 and investigate the underlying behavioral and molecular phenotype. These applications broadly demonstrate the potential of Cas9 for interrogating genetic elements in vivo towards understand their role in normal biological processes and disease. / by Randall Jeffrey Platt. / Ph. D.

Thesis: M. Eng, Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 61-65). / Amitotic cells with large, hollow bell-shaped nuclei, or metakaryotic stem cells, are the post-embryonic stem cells of the fetal organs from about the fourth week post conception through physical maturity. These metakaryotic stem cells, after acquiring necessary genetic, and possibly other events, are also the stem cells of precancerous, cancerous and metastatic lesions of carcinogenesis. Furthermore, our lab has discovered that metakaryotic stem cells, both in fetal development and tumor growth, use a peculiar mode of DNA synthesis and segregation that involves inter alia expression of large amounts of RNA polymerase beta during DNA synthesis. It was hypothesized that an inhibitor of DNA polymerase beta would be toxic to metakaryotic stem cells at concentrations lower than necessary to kill eukaryotic non stem cells. The polymerase beta small-molecule inhibitor chosen for this study was pamoic acid, a napthoic acid derivative. With it we determined the relative sensitivity of metakaryotes and eukaryotic cells in the human colorectal cancer cell culture, HT- 29mes, that expresses characteristics expected of colorectal cancer metastases. We conclude that, at 300 [mu]M and above pamoic acid does not selectively kill metakaryotes in cell culture below that concentration which kills eukaryotes. Rather, pamoic acid acts in a similar fashion as X-rays: eukaryotic non-stem cells are killed at lower doses than those that kill metakaryotic stem cells. Treatment of pamoic acid with these concentrations causes concomitant declines in colony-formation potential for both metakaryotes and eukaryotes alike. At lower overall survival levels, surviving colonies appear to have arisen from metakaryotic cells, not eukaryotic cells as evidenced by the presence of visible metakaryotic cells in most colonies and the ability of such colonies to support continuous growth upon passaging. We conclude with the possibility that this specific polymerase beta inhibitor is not an effective metakaryocide in culture, insofar as they are not selectively toxic for these stem cells in the HT-29mes colorectal cancer cell line. / by Tushar Vinod Kamath. / M. Eng

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The epithelial cell-extracellular matrix interface primarily comprises of complex glycans and glycoconjugates. The widespread distribution of these glycans on the epithelial cell surface makes them ideal targets for interaction with microbial pathogens. In this thesis, a framework of integrated approaches was developed to characterize the structure-function relationships of host cell surface glycans and examine their role in mediating hostpathogen interactions. The first part of the thesis involves a study of the effect of secreted bacterial sphingomyelinases on the epithelial cell surface proteoglycan (a large glycan- protein conjugate), syndecan-1 and on epithelial tight junctions. The findings presented in this work suggest mechanisms by which sphingomyelinases could enhance bacterial virulence by regulating epithelial cell function. The second part of the thesis investigates the glycan binding requirements that govern the human adaptation and transmission of influenza A viruses by characterizing the molecular interactions between sialylated glycan-receptors and viral hemagglutinin (HA). The study puts forth the concept that the topology or shape (going beyond the chemical c2-3 versus a2-6 sialic acid linkage) adopted by the sialylated glycans is the critical determinant for efficient human adaptation of these viruses. In conclusion, this thesis provides insights into the molecular mechanisms of host-pathogen interactions and enables development of improved strategies for targeted antimicrobial therapies. / by Aarthi Chandrasekaran. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 107-119). / Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, M13 bacteriophage libraries are engineered as a biochemical platform for de novo semisynthetic enzymes, functionally modular and widely stable. Carbonic anhydrase-inspired hydrolytic activity via Zn²+ coördination is first demonstrated. The phage clone identified hydrolyzes a range of carboxylic esters, is active from 25°C to 80°C, and displays greater catalytic efficacy in DMSO than in water. Reduction-oxidation activity is subsequently developed via heme and copper cofactors. Heme-phage complexes oxidize multiple peroxidase substrates in a pH-dependent manner. The same phage clone also binds copper(II) and oxidizes a catechol derivative, di-tert-butylcatechol, using atmospheric oxygen as a terminal oxidant. This clone could be purified from control phage via Cu-NTA columns, enabling future library selections for phage that coördinate Cu²+ ions. The M13 semisynthetic enzyme platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work could tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality. / by John P. Casey, Jr. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 104-111). / Studying biophysical properties of cells can provide insight into the metabolic mechanisms and regulation of cell cycle processes. Though size is considered to be a fundamental property of cell state, its measurement on a single-cell basis with high-resolution has been elusive primarily due to enormous experimental barriers. This thesis discusses the use of a cantilever based suspended microchannel resonator (SMR) to measure mass, and resistive pulse based Coulter counter to measure volume. First, we discuss the implementation of several engineering principles that have enabled the SMR to measure size with a high precision and temporal resolution. As a result, growth rates can now be estimated at a single-cell basis with unprecedented precision of ~170 fg.hr-¹. Second, we employ the SMR to investigate the coordination between the fundamental processes of cell growth and cell division cycle. Contrary to the reigning 60-yr old hypothesis of a deterministic size-control of the cell cycle, it is observed that cells display significant size variability at the Start checkpoint of the cell cycle. Furthermore, the measurements find only a weak size-control on the time spent in G1. Remarkably, it is observed that the cell's initial growth rate is a significantly better predictor of G1 duration than its initial size. Third, we develop a method to enable continuous, long-term volume measurement. Based on a commercial Coulter counter device, it provides a complementary technique for high-throughput measurement and continuous sampling of cell volume, as well volumetric growth rate on a population-scale. / by Amneet Gulati. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 243-264). / Hematopoietic stem cell or bone marrow transplantation is a curative treatment for multiple hematologic malignancies. However, the myeloablative conditioning regimens preceding cell delivery have rendered the rapid and sustained hematopoietic recovery after transplantation an outstanding challenge. Successful long-term engraftment of hematopoietic stem cells is dependent largely on the surrounding stroma components or hematopoietic niche. Cell types within this niche that support hematopoietic recovery include two adherent cell types, mesenchymal stromal cells (MSCs) and vascular endothelial cells (VECs). The niche also contains many biophysical and mechanical cues including cell contractility against other cells or the matrix, pulsatile fluid flow, differences in localized niche stiffness, and occupation of fluid volume by macromolecules. This thesis aims to understand how VECs and MSCs respond to these cues ex vivo, and how these cues can be used to engineer VEC and MSC phenotypes that can predictably support hematopoietic recovery in vivo. VEC-mediated angiogenesis and angiocrine signaling are known to support hematopoietic recovery in vivo. In this thesis, we first explored how the biophysical cue of macromolecular crowding (MMC) and the mechanical cue of strain can regulate angiogenesis. The addition of synthetic MMC to in vitro cultures replicates the endogenous occupation of fluid space due to macromolecules. We explored how MMC affects the basement membrane formation of VECs, and determined that MMC can increase the deposition, areal spread, and alignment of basement membrane proteins. Even with the addition of biochemical signals from pericytes, this biophysical cue of MMC played a dominant role in the organization of the basement membrane. Pericytes that surround blood vessels and the basement membrane have been shown to exert contractile forces, which results in a hoop strain in the blood vessel wall. We translated this strain to in vitro VEC cultures by applying static, uniaxial strain to confluent VEC monolayers using a polydimethyl siloxane (PDMS) substrata, which allowed us to decouple the mechanical cue of pericytes from their chemical signaling. The application of 10% engineering strain was sufficient to induce cell-cycle re-entry in a quiescent monolayer. We then went on to demonstrate in a quasi-3D assay that straining the VECs also produced angiogeniclike sprouts. Together, these results show that biophysical and mechanical cues of the hematopoietic niche alone are sufficient to direct VEC-derived extracellular matrix formation and to induce angiogenic sprouting. Thus, future models of in vitro angiogenesis must include these cues to more comprehensively and accurately replicate the in vivo hematopoietic niche. Paracrine signaling from MSCs is crucial in regulating the self-renewal capacity and differentiation of hematopoietic stem and progenitor cells (HSPCs) that re-populate the bone marrow compartment in vivo. Thus, we then explored if and how to modulate MSC paracrine signaling or the MSC secretome. Like VECs, MSCs are known to respond to microenvironment cues such as substratum stiffness. We developed tissue-culture compatible PDMS-based substrata with tunable viscoelastic properties to assay potential mechanosensitivity. We characterized the bulk and surface properties of this substrata to verify that we could tune stiffness across three orders of magnitude without altering material surface biochemistry. When we expanded the MSCs on compliant substrata (elastic modulus ~I kPa), we found that we could increase the expression of osteopontin as well the expression of at least a dozen other secreted proteins without altering cell capacity for terminal differentiation. We observed changes in the MSC secretome that were significantly correlated to the viscoelastic properties (shear storage and loss moduli G' and G", respectively, and the ratio of G"/G' as tan [delta]) of the substratum material. These results suggested that we could mechanically modulate the MSC secretome using the viscoelastic properties of the extracellular substrata. Finally, we went on to explore how these mechanically modulated changes in MSC phenotype could regulate hematopoiesis in vitro and support hematopoietic recovery in vivo. To do so, we used statistical regression modeling (partial least squares regression or PLSR) to identify the components of the MSC secretome that were significantly correlated with improved radiation rescue and hematopoietic recovery in mouse models of hematopoietic failure. We then characterized the expression of these key secretome components in our mechanoprimed MSCs. The mechanoprimed MSCs expressed equal or higher concentrations of these proteins as a diameter-defined subpopulation of MSCs we previously identified to be therapeutically effective. Using the regression parameters from PLSR and the new expression data from our mechanoprimed MSCs, we then predicted how our mechanoprimed MSCs would elicit radiation recovery of the bone marrow compartment in vivo. From these computational predictions, we found that our mechanoprimed MSCs could potentially improve survival proportion in this in vivo model of hematopoietic failure. Thus, we tested mechanoprimed MSCs by expanding them in co-culture with HSPCs to determine if the MSCs could regulate hematopoiesis in vitro. We found that mechanoprimed MSCs could maximize the proliferation or expansion of HSPCs when co-cultured on top of our most compliant PDMS substrata (~I kPa). When grown on stiffer PDMS substrata (100 kPa), those MSCs could prime differentiation of the HSPCs down myeloid lineages, which include red blood cells. Together, these results demonstrate that these mechanoprimed MSCs can be used to modulate the ex vivo expansion and differentiation of HSPCs. Lastly, we tested these mechanoprimed MSCs in our sub-lethally irradiated mouse models of hematopoietic failure. Our mechanoprimed MSCs significantly increased the survival of the mice. Interestingly, this increased survival and improved hematopoietic recovery outperformed the survival predicted from our regression model. We also observed recovery of red blood cells, white blood cells, and platelets in mice treated with mechanoprimed MSCs, suggesting complete recovery of all hematopoietic lineages. In summary, we have explored how biophysical and mechanical cues can modulate VEC and MSC phenotype in vitro. In the case of VECs, the results presented in this thesis further the development of more accurate in vitro models of angiogenesis. Accurate in vitro models of angiogenesis are necessary to elucidate the mechanisms by which VECs regulate hematopoietic recovery in vivo. We also characterized the components of the MSC secretome correlated with improving hematopoietic recovery and demonstrated that we could engineer the expression of these same MSC secretome components using substratum viscoelastic properties. Lastly, we validated that these mechanically modulated MSCs led to improved survival outcome in vivo. The work presented in this thesis furthers our understanding of how biophysical and mechanical cues regulate hematopoietic niche components that participate in indirect repair of the bone marrow. We also demonstrated how these same cues can be applied in vitro to improve cell-based therapies for hematopoietic recovery in vivo. / by Frances D. Liu. / Ph. D.