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.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / DNA damaging agents produce a plethora of DNA lesions, which can lead to various outcomes. In order to understand the biological consequences of DNA lesions, and hence gain insights into the carcinogenic mechanisms of DNA damaging agents, individual lesions need to be evaluated for their genotoxic and mutagenic potentials in cells. In this work, we devised and validated a new strategy to study the in vivo consequences of DNA lesions using next-generation sequencing. By labeling different samples with unique oligonucleotide sequences as barcodes, multiple lesions can be simultaneously evaluated in multiple cell strains with different repair and replication capabilities. This high-throughput, multiplex approach greatly relieves the burden on researchers, and reduces the time and cost for a large-scale investigation. We applied this methodology to the investigation of ethenoguanine lesions, which are generated by oxidative stress and vinyl chloride exposure. N²,3-Ethenoguanine potently induces G to A mutations, the same type of mutation previously observed in vinyl chloride-associated tumors. We also found that N²,3-ethenoguanine cannot be repaired by AlkB, a DNA repair enzyme capable of repairing all other etheno lesions. Our observations suggest this lesion may have a functional role in vinyl chloride-induced or inflammation-driven carcinogenesis. The in vivo genotoxicity and mutagenicity of four 1,3-butadiene-induced adenine adducts were evaluated. Previous in vitro studies have shown that some of these lesions are highly mutagenic. Surprisingly, we found that none of them was significantly mutagenic in any of the conditions investigated. This observation suggests that there may be unknown mechanisms mitigating the mutagenic effect of the butadiene-induced lesions. Finally, we extended our methodology one step further by simultaneously analyzing the mutagenicity of 8-oxoguanine, a prevalent oxidative lesion, in all 16 adjacent-base sequence contexts. Our result shows that sequence context can significantly modulate 8-oxoguanine mutagenicity. The observed 8-oxoguanine mutational pattern clustered closely with COSMIC (Catalogue of Somatic Mutation in Cancer) Signature 18 of human cancers, providing support that this signature may result from oxidative damage. By applying the same type of analysis to other DNA lesions, researchers may identify the underlying processes that are responsible for the human cancer mutational signatures with unknown etiologies. / by Shiou-chi Chang. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 136-146). / From the fabrication of fine chemicals, to the increasing attainability of a non-petrochemical based energy infrastructure, catalysts play an important role in meeting the increasing energy and consumable demands of today without compromising the global health of tomorrow. Development of these catalysts relies on the fundamental understanding of the effects individual catalyst properties have on catalytic function. Unfortunately, control, and therefore deconvolution of individual parameter effects, can be quite challenging. Due to the nanoscale formfactor and wide range of available surface chemistries, biological catalyst fabrication affords one solution to this challenge. To this end, this work details the processing of M13 bacteriophage as a synthetic toolbox to modulate key catalyst parameters to elucidate the relationship between catalyst structure and performance. With respect to electrocatalysis, a biotemplating method for the development of tunable 3D nanofoams is detailed. Viral templates were rationally assembled into a variety of genetically programmable architectures and subsequently templated into a variety of material compositions. Subsequently, this synthetic method was employed to examine the effects of nanostructure on electro-catalytic activity. Next, nanoparticle driven heterogeneous catalysis was targeted. Nanoparticle-protein binding affinities were leveraged to explore the relationship between nanoparticles and their supports to identify a selective, base free alcohol oxidation catalyst. Finally, the surface proteins of the M13 virus were modified to mirror homogeneous copper-ligand chemistries. These viruses displayed binding pocket free copper complexation and catalytic efficacy in addition to recyclability and solvent robustness. Subsequently, the multiple functional handles of the viron were utilized to create catalytic ensembles of varying ratios. Single and dendrimeric TEMPO (4-Carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl) were chemically conjugated to the surface of several catalytically active phage clones further tailoring catalytic function. Taken together, these studies provide strong evidence of the utility of biologically fabricated materials for catalytic design. / by Jacqueline Ohmura. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 143-162). / Cell-cell communication is critically important to the function of the immune system, allowing a systems-level determination of the appropriate type of immune response to a perturbation. The immune system has at its disposal multiple types of responses, some beneficial and others harmful, all of which require coordination among immune cells and between the immune system and non-immune tissue cells. In this thesis, we have explored the use of multiple experimental and computational methods to understand how intercellular communication shapes the immune response in health and disease. Applications of this work are primarily focused on endometriosis, a disease characterized by the presence of endometrial glands and stroma located outside of the uterus. Disease initiation (cell survival) and progression (including neovascularization and neurogenesis) are thought to depend on interactions with the immune system, particularly macrophages. We have investigated these interactions on several levels, using both clinical samples and 3D in vitro culture models. The model systems used here include endometrial stromal and epithelial cells as well as peripheral blood monocytes with which to study dynamic processes within either the eutopic endometrium or the endometriotic lesion environment. / by Abby Shuman Hill. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2017. / "January 2017." Cataloged from PDF version of thesis. / Includes bibliographical references. / Metastasis, which accounts for 90% of cancer deaths, critically depends on the ability of cancer cells to migrate through the dense extracellular matrix (ECM) surrounding the solid tumor. Recent advances in cancer biology have revealed that non-cancerous cells and biophysical forces in tumor microenvironment can influence metastasis. Specifically, macrophage, one of the most abundant tumor-associated stromal cell types, has been shown to assist cancer cell invasion. However, exactly how macrophages affect the different aspects (e.g. speed and persistence) of cancer cell migration, especially in 3D ECM that mimics the in vivo tumor microenvironment, remains largely unknown. In addition to macrophages, elevated interstitial flow (the flow of tissue fluid) within the tumor tissue has been shown to influence cancer cell and fibroblast migration. Nevertheless, the effects of this tumor-associated biophysical force on macrophages are still unknown. In this thesis, we first explored how macrophages control the subtle details (speed vs. persistence) of cancer cell migration. Using a microfluidic migration assay, we found that macrophage-released TNFa and TGF1 enhance cancer cell migration speed and persistence in 3D ECM in an MMP-dependent fashion via two distinct pathways. Specifically, macrophagereleased TGF1 enhances cancer cell migration speed via the induction of MTl-MMP expression in cancer cells. In contrast, macrophage-released TNFa and TGFp1 synergistically enhance cancer cell migration persistence via the induction of NF-KB-mediated MMP1 expression. Therefore, these results suggest that macrophages control different aspects of cancer cell migration in 3D differently, and both TNFa and TGFp1 released by macrophages need to be simultaneously inhibited to effectively limit macrophage-assisted cancer cell metastasis. In a separate study, we investigated how tumor-associated interstitial flow (IF) affects macrophage migration and protein expression. We discovered that IF promotes macrophage migration in 3D ECM via the flow-induced activation of FAK and Akt. Interestingly, IF also directs the preferential migration of macrophages against the direction of flow (upstream). Moreover, we show that IF polarizes macrophages toward a pro-metastatic M2 phenotype via integrin/Src-dependent STAT3/6 activation. Since IF emanates from tumor core to stromal tissue surrounding the tumor, these results suggest that IF can promote metastasis by not only recruiting macrophages from stroma into tumor, but also enhancing the M2 polarization of macrophages in the tumor microenvironment. Keywords: Tumor Microenvironment, Macrophages, Interstitial Flow, Migration, and Polarization. / by Ran Li. / Ph. D.