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.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 118-128). / Sequencing of microbial genomes has enabled new understanding of human health and disease. Certain microbes can support human health through the microbiota, helping to train our immune system or supply essential nutrients. In other cases, microbes may be pathogenic, overwhelming the immune system and causing infection. Low-cost and accessible DNA sequencing has allowed us to learn important information about microbial systems - we can identify what microbes are members of our microbiota and how they change with disease, as well as how pathogenic microbes evolve and acquire resistance to antibiotics. While the cost of sequencing has decreased and allowed for widespread use, studies are now limited by sample acquisition and preparation. In particular, microbial sample preparation has challenges at the limits of sensitivity (low signal to noise ratio) and at the limits of scale (large sample size). In this thesis, I developed methods to address both of these challenges and applied the techniques to study questions in basic biology and in clinical medicine. First, I developed a procedure to sample and sequence the lung microbiome in mouse models, where high background of mammalian DNA in lung samples poses a serious challenge for sequencing preparation. Along with my collaborator, I used this procedure to investigate the microbiome in a murine model of lung cancer. Second, I developed a platform for high-throughput sequencing preparation of bacteria at the scale of thousands of samples, with a 100-fold less cost per sample. I prepared and sequenced 3000 antibiotic-resistance bacteria from a clinical trial studying the role of decolonization procedures. This work provides new insights about microbes in the context of health and disease, and the methods developed here can make samples newly accessible for sequencing at the limits of scale or sensitivity. / MIT Presidential Fellowship MIT Hugh Hampton Young Fellowship NSF Graduate Research Fellowship / by Georgia Kerasia Lagoudas. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. "February 2016." / Includes bibliographical references (pages 139-151). / Cellular biophysical properties both reflect and influence cell state. These parameters represent the consequences of the interactions of multiple molecular events, and thus may reveal information otherwise obscured when measuring individual pathways in isolation. Previous work has demonstrated how precise measurements of certain of these properties, such as mass, volume, density and deformability using a suspended microchannel resonator (SMR) can help characterize cellular behavior and physiological role. Here, we expand upon this previous work to demonstrate the necessity of measuring multiple parameters simultaneously to fully determine cellular responses to environmental perturbations, and describe a situation in which changes to density and size promote survival under conditions of limited nutrient availability. We first investigate the relationship between cell density, volume, buoyant mass, and passage time through a narrow constriction under a variety of environmental stresses. Osmotic stress significantly affects density and volume, as previously shown. In contrast to density and volume, the effect of an osmotic challenge on passage time is relatively small. Deformability, determined by comparing passage times for cells with similar volume, exhibits a strong dependence on osmolarity, indicating that passage time alone does not always provide a meaningful proxy for deformability. Finally, we find that protein synthesis inhibition, cell cycle arrest, protein kinase inhibition, and cytoskeletal disruption result in unexpected relationships between deformability, density, and volume. Taken together, our results suggest that measuring multiple biophysical parameters can detect unique characteristics that more specifically reflect cellular behaviors. We next examine how cellular biophysical changes occurring immediately after growth factor depletion in lymphocytes promote adaptation to reduced nutrient uptake. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density prior to autophagy initiation, observed in both FL5.12 cells depleted of IL-3 and primary CD8+ T cells depleted of IL-2 and differentiating towards memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress. / by Vivian Hecht. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Page 168 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 155-167). / Insulin resistance is a condition that underlies type 2 diabetes and various cardiovascular diseases. It is highly associated with obesity, making it a pressing medical problem in face of the obesity epidemic. The obesity association also makes adipose tissue the target of interest for ongoing research. Previous work on adipose insulin resistance has largely been focused on deciphering the signaling defects and abnormal adipokine secretion profiles. There is increasing awareness that transcriptional control is a source of dysregulation as well as an avenue of therapeutic intervention for insulin resistance. However, knowledge of transcriptional regulation and dysregulation of adipose insulin resistance remains fragmentary. Here, we present a genome-wide perspective on transcriptional regulation of adipocyte biology and adipose insulin resistance. We made use of the latest high-throughput sequencing technology to interrogate different aspects of transcriptional regulation, namely, histone modifications, protein-DNA interactions, and chromatin accessibility in adipocytes. In combination with the transcriptional outcomes measured by microarray and RNA-sequencing, we (1) characterized a largely unknown histone modification, H3K56 acetylation, in human adipocytes, and (2) set up four diverse in vitro insulin resistance models in mouse adipocytes and analyzed them in parallel with mouse adipose tissues from diet-induced obese mice. In both cases, through computational analysis of the experimentally identified cis-regulatory regions, we identified existing and novel trans-regulators responsible for adipose transcriptional regulation. Furthermore, by comprehensive pathway analysis of the in vitro models and mouse models, we identified aspects of in vivo adipose insulin resistance that are captured by the different in vitro models. Taken together, our studies present a systems view on adipose transcriptional regulation, which provides a wealth of novel resources for gaining insights into adipose biology and insulin resistance. / by Kin Yui Alice Lo. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 149-165). / The liver regulates a myriad of vital functions including bile acid synthesis, hepatobiliary circulation, cholesterol homeostasis, drug metabolism, etc. This thesis focuses on the use of a 3D in vitro model of liver to study the effects of compounds and culture conditions on hepatobiliary transport and bile acid synthesis. In order to achieve this goal, protocols were developed to perform hepatic transport studies of bile acids in perfused 3D primary rat hepatocyte cultures. An established 2D sandwich culture model was used as a foundation for evaluation of variations in protocol parameters including culture medium composition and assay incubation times. In 2D sandwich cultures, dexamethasone (DEX) was essential for the formation of canalicular networks, whereas epidermal growth factor (EGF) disrupted the formation of these networks. Strikingly, EGF promotes cellular re-polarization and canalicular network formation in perfused 3D cultures, in contrast to its effect on 2D cultures. Perfused 3D cultures were found to have greater bile acid transport capabilities and closer to in vivo expression of certain liver transporters than 2D sandwich cultures on day 7. Perfused 3D cultures also had greater bile acid synthesis on day 7 than 2D cultures, however this was inhibited by EGF in both cultures. A numerical model was also developed for the reduction of experimental measurements necessary to determine pharmacokinetic parameters of hepatic transport, thus allowing for quantitative comparison of the effects of different culture conditions and culture platforms on transporter activity. The result of this thesis is the adaptation of a system for the study of hepatic transport and bile acid synthesis to 3D cultures. This system has also been previously shown to maintain other liver functions such as drug metabolism, and the work of this thesis thus allows for the concerted study of all these functions and potentially others. / by Jose Ricardo Llamas Vidales. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 134-146). / High throughput screening methods such as yeast surface display (YSD) are frequently able to isolate high affinity antibodies against clinical targets; however, the success of these candidates depends on selecting for both on-target binding and desirable biophysical characteristics. Development liabilities, including antibody aggregation and nonspecificity, can lead to problems during production and poor pharmacokinetics (PK). The exact structural and sequence determinants causing this poor developability are unknown, which leads to inexact methods to correct otherwise promising clinical candidates. In this thesis we outline the development of high throughput methods to interrogate developability of candidate antibodies on the surface of yeast and apply these methods to both determine the causes of nonspecificity and create new libraries with improved biophysical properties. We first analyzed methods for early stage assessment of monoclonal antibodies, finding a polyspecificity reagent (PSR) binding assay on the surface of yeast which can accurately predict antibody clearance rates in mice. While robust, this assay relies on production of a poorly defined mixture of protein components, and thus, we next looked at potential alternatives to a multicomponent PSR reagent. We found that chaperone proteins may work as well-defined, easily producible reagents with similar broad predictive power to predict downstream antibody behavior. Next, we applied these assays to assess core determinants of nonspecificity. We first analyzed a case study of two antibodies with identical target antigens but vastly different performance on preclinical assessments of biophysical characteristics. Through this matched case, we found differences in clearance rates can be driven wholly by variable-region mediated effects independent of neonatal Fc receptor (FcRn) binding. Focused on the antibody variable region, we next utilized our nonspecificity assay as a sorting tool to look at a naive repertoire library. We found significant nonspecificity in the VH6 class of antibodies, driven by a poorly behaved complementarity determining region (CDR) H2 sequence. Subsequently, we applied a similar sorting technique to two synthetic library designs to identify a set of motifs that can drive nonspecificity. These included motifs containing tryptophan, valine, glycine and arginine located in CDR H3. We then applied these discoveries to the design of a new, semi-synthetic single chain variable fragment (scFv) library and demonstrated its ability to isolate high affinity, highly specific candidate clones against a panel of antigens. Finally, we explored the use of an alternate yeast display system capable of easily switching between scFv-Fc display and secretion, which may aid in the rapid development and testing of candidate antibodies. Taken in whole, the work in this thesis aids the clinical development of antibodies. We have presented both methods to assess nonspecificity at an early stage in the development process as well as a set of motifs to be eliminated in future library designs. With these combined findings, we hope to increase the utilization of in vitro screening methods such as yeast display for the isolation of clinical candidate antibodies with favorable biophysical characteristics. / by Ryan L. Kelly. / Ph. D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Title as it appears in MIT Commencement Exercises program, June 2013: Signaling network changes in high fat diet-induced insulin resistance Cataloged from PDF version of thesis. / Includes bibliographical references (p. 73-80). / The goal of my research is to understand the mechanism by which high fat diets mediate insulin sensitivity and the role SirT3 plays in high fat diet-induced insulin resistance. Insulin resistance is defined as the inability of cells and tissues to respond properly to ordinary amounts of insulin and is a precursor to many metabolic diseases such as diabetes and cardiovascular disease. Obesity, brought on in large part by caloric excess from high fat diet feeding, is a major contributor to insulin resistance. The recent drastic increase in the prevalence of obesity makes it imperative that steps are taken to more effectively treat and cure obesity-linked diseases such as diabetes. To identify optimal therapeutic targets, it is crucial to first gain a mechanistic understanding of obesity-induced insulin resistance, and understand how specific changes in the signaling network affect insulin sensitivity. Previous work has demonstrated that levels of SirT3, a mitochondrial protein deacetylase, are diet dependent. Additionally, SirT3 expression levels have been shown to mediate insulin and glucose tolerance in animals in a diet-dependent manner. Perturbations in SirT3 levels also alter the levels of phosphorylation on several canonical insulin signaling proteins. In my research, I further investigated the link between SirT3, diet and insulin resistance from a signaling network perspective. Using mouse liver as a model system, I analyzed liver tissue from mice fed a normal diet (insulin sensitive) or mice fed a high fat diet, thus inducing insulin resistance. Quantification of phenotypic and network events in response to insulin and utilization of computational techniques revealed activated pathways and nodes mediating insulin response, some of which had not been previously associated with the canonical insulin signaling network. I extended the study to analyze the role SirT3 plays in diet-mediated insulin sensitivity by perturbing the level of SirT3 in mice on both normal chow and high fat diets. The results of this research are useful for designing more efficacious therapies to treat insulin resistance-induced diseases. / by Nina Louise Lee. / S.M.