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.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 108-111). / Molecular motors play an important role in driving some of the most complex and important tasks in biological systems, ranging from transcribing RNA from a DNA template (Polymerases) to muscle contraction (Myosin) and propelling bacteria (Flagellum). Key to the understanding of the fundamental principles and designs by which molecular motor function has been the kinesin family. Missing, however, is a clear understanding of the series of events that take place at the atomistic level when kinesin walks on a microtubule and generates force. Recent MD simulations have identified the force-generating mechanism in kinesin, the cover-neck bundle, and strongly suggest that the formation of the CNB by the N-terminal cover strand and the C-terminal neck linker of the motor head are responsible for force generation. In this thesis we present tools developed in the Lang Laboratory to further elucidate the stepping motion and force generation mechanism of kinesin using Drosophila kinesin as a model system. We demonstrate the function of a force clamp specifically designed for the laboratory and show traces of WT kinesin walking under constant load. We also purified and tested kinesin mutants running under a force load. We present two assays specifically designed to study the interaction between kinesin and the last 10-18 C-terminal residues of a-p tubulin, the E-hook. We were unable to observe kinesin - e-hook interactions, such as those suggested by the formation of tethers, when the e-hook was bound to the surface. In the case of e-hook in solution, our results indicate that 2G kinesin was still functional and its stall force approximately 3 pN just as for the case when no e-hook is present. We also propose ways that the work in this thesis can be expanded. The force clamp can be easily adapted to study novel kinesin mutants under constant load in 2D. In addition, the force clamp can be used to probe the kinesin - e-hook interactions by looking at kinesin walking over microtubules with cleaved e-hooks. The e-hook assays presented in this thesis can also be expanded to include higher concentrations of e-hook or be performed using labeled e-hook to assess single molecule interactions and concentrations. / by Ricardo González Rubio. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 232-252). / In this thesis, new tools have been developed for decoding structure-function relationships governing complex biopolymers that have emerged as key players in biology, biotechnology, and medicine. Specifically: (1.) The first part of this thesis addresses the structure-function relationship of synthetic biodegradable plastics that are at the forefront of nanotechnology for spatiotemporally-regulated targeting and sustained release of drugs to treat Cancer and other chronic diseases. A Voxel-based 3-D platform for accurately simulating all phases of polymeric nanoparticle erosion and drug release is introduced. Using the developed Voxel platform, the release of anti-inflammatory and anti-cancer drugs such as doxorubicin and dexamethasone from poly lactic-co-glycolic acid (PLGA) nanoparticles is precisely predicted. The Voxel platform emerges as a powerful and versatile tool for deducing the dynamics in interplay of polymer, drug, and water molecules, thus permitting the rational design of optimal nanotechnology treatments for cancer. (2.) The second part of this thesis is focused on development of tools to elucidate structure-function relationships of complex polysaccharides (glycans) that specifically interact with proteins to modulate a host of biological processes including growth, development, angiogenesis, cancer, anticoagulation, microbial pathogenesis, and viral infections. First, towards the fine structure determination of complex linear glycans (glycosaminoglycans or GAGs), enzymatic tools are developed for both depolymerizing GAGs at specific linkages and for effectively modulating their functional groups. Specifically, the development and integrated biochemical-structural characterization of the Chondroitinase ABC-II enzyme that depolymerizes dermatan sulfate and chondroitin sulfate GAGs (CSGAGs), and the 6-0- Sulfatase and N-Sulfamidase enzymes that de-sulfate functional groups on heparin and heparan sulfate GAGs (HSGAGs) are described. Second, the interaction of branched glycans with proteins is analyzed using the interplay of Influenza virus surface proteins (mainly Hemagglutinin and Neuraminidase) with host cell surface sialylated glycan receptors as a model system. For this purpose, the novel triple reassortant "Swine Flu" pandemic virus (or 2009 HINI virus) is studied. Finally, in order to overcome the challenges facing protein structure prediction in the "Twilight Zone" of low homology that is rampant in glycan-binding protein (lectin) families, a new tool is introduced for modeling the 3-D structure of proteins directly from sequence. Specifically, it is identified that protein core atomic interaction networks (PCAINs) are evolutionarily non-tinkered and fold-conserved, and this finding is utilized towards assignment of folds, structures, and potential glycan substrates to lectin sequences. It is further demonstrated that the developed tool is effective universally; thus emerging as a promising platform for generic protein sequence-to-structure and function mapping in a broad spectrum of biological applications. / by Venkataramanan Soundararajan. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 46-51). / This work investigated single-walled carbon nanotube (SWNT)/polymer-protein A complexes for optically reporting antibody concentration via a change in near infrared fluorescent emission after antibody binding. SWNT have potential as biosensors because of extraordinary sensitivity, lack of photobleaching, and optical activity in a near-infrared window. A SWNT sensor could provide label-free measurements of antibody concentration in a continuous fashion, which may aid selection of production strains. Protein A itself, dextran, poly vinyl alcohol, DNA sequences, and chitosan were used as polymers for wrapping SWNT. Nonspecific binding to solution-phase constructs was found to be a major problem with these approaches. Chitosan hydrogels encapsulating SWNT also show nonspecific responses. / by Peter Bojö. / M.Eng.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 155-165). / Communities of microorganisms are found nearly ubiquitously on earth. They survive and proliferate through interactions within and between microbial species, which are mediated by the exchange of small signaling modules. Understanding how they regulate the interactions is both crucial and challenging, with applications including industrial biotechnology, human health and environmental sustainability. In microbial ecology, researchers have been trying to culture pure and mixed species in different conditions to elucidate the rules behind the interactions. However, the studies have been complicated by multiple variables at both the genotype and phenotype levels. To address these challenges, I demonstrate a synthetic ecological system as a proof of principle to observe microbial population level behaviors. Using a formalized design process and engineering principles, I design and construct a synthetic multi-module ecological system for population homeostasis. The synthetic ecological system consists of four functionally distinct modules - quorum sensing, high threshold killing, low threshold killing, and intermediate rescuing modules. The system is able to maintain the yeast population within a programmable range in liquid culture. However, when the same system is studied in solid medium, heterogeneity in growth rate and population size is observed. To further study the heterogeneity issue in solid medium, I develop a cell deposition platform to evaluate sub-population level or even single-cell level behavior. With a commercial Nano eNabler machine, cells with pre-defined patterns are deposited on agarose surface. This technique can be used to study microbial communities in a spatially distributed fashion. / by Jingjing Sun. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / CD-ROM contains copy of thesis in .pdf format and files in .mov format. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 76-84). / Cytotoxic lymphocytes (CTLs) traffic through tissues in search of antigen and mount protective immune responses against viral infections and cancer. While molecular mechanisms of CTL antiviral effector functions have been established in vitro, they have been defined in the absence of physiological dynamics and migration. Furthermore, longterm dynamics of single cells have been inaccessible in vivo, where brief imaging durations have been achieved (-30-60 min). Presently, several key aspects of CTL dynamics and function remain unknown: whether individual CTLs migrating within tissues kill multiple targets, if CTLs exhibit spatiotemporal coordination of effector functions, or if migrating CTLs effect these functions in different compartments. Thus, a mechanistic understanding of multidimensional CTL function might directly inform therapeutic strategies. In this thesis, we first developed an approach for long-term high-speed optical imaging of cellular dynamics for continuous periods of 24 hours. HIV-specific CTLs were visualized as they encountered CD4+ target cells within a three-dimensional extracellular matrix tissue model supporting migration of both CTLs and targets. Using this approach, we found that high-avidity CTLs engaged, arrested, and killed the first target encountered with near-perfect efficiency. These CTLs remained in contact with dead targets for hours, accumulating TCR signals and upregulating antiviral cytokine and chemokine secretion for >12 hours, but were refractory to killing additional targets. By contrast, lower-avidity CTLs exhibited poor efficiency and target migration directly impeded CTL killing. Thus, high-avidity CTLs coordinate multiple antiviral functions in four dimensions (3D space and time): effectively destroying the first detected infected cell during an initial "commitment phase", but rapidly transitioning to a prolonged "secretory phase." In vivo, coordination of lytic and non-lytic effector functions will direct the local inflammatory milieu and recruit additional effectors to the tissue. We conclude that the efficiency of antigen recognition by individual migrating CTLs is a critical, but previously undefined, parameter of CTL function. Furthermore, TCR avidity and initial CTL efficiency are prerequisites for sustained antiviral polyfunctionality; together these parameters define a highly effective, multidimensional CTL response, which may inform the design of increasingly effective vaccines. / by Maria Hottelet Foley. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 96-100). / The link between abnormal microRNA expression and cancer has been widely reported. However, little is known about the relationships between temporal microRNA expression and changes in cell behavior. To better understand how microRNA expression is involved in cell responses it is necessary to know what time dependent changes happen in response to cellular stimuli. Here, we demonstrate that, in the hepatocellular carcinoma (HCC) cell line Huh7, microRNA expression changes resulting from treatments with different combinations of the cytokines IFN-[gamma] and TRAIL follow a time-dependent pattern that correlates with cell death. An initial stimulus with IFN-y, followed by a second stimulus with TRAIL is most effective at inducing cell death. By applying other combinations of these two cytokines, we induce different levels of cell death after 48 and 72 hours of the initial treatment. MicroRNA expression data from high throughput sequencing analysis was used to construct data-driven multivariate models. Expression profiles associated to different cytokine treatments were identified using principal component analysis (PCA) and, cell death was defined as a function of microRNA expression using partial least square regression (PLSR). Differential expression analysis was performed to identify relevant microRNAs from the conditions most highly associated to cell death. Global microRNA expression one hour after the second cytokine treatment is most predictive of cell death. Several microRNAs were identified as strong predictors of cell death, including let-7c, miR- 181a and miR-92b, and others. Gene ontology analysis of the targets of these, and other highly predictive microRNAs, suggests that there is an enrichment of apoptosis related targets for the microRNAs that are up-regulated upon cytokine treatment. These studies illustrate that the expression dynamics of microRNAs provide important insights into the role of microRNAs in cell decisions processes, bringing us closer to designing new strategies for diagnosis and treatment of HCC. / by Nancy Guillen. / Ph. D.