Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / 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. / Antibody-dependent cell-mediated cytotoxicity (ADCC) is implicated in the efficacy, to some degree, of most anti-cancer monoclonal antibodies. This interaction is mediated through Fc gamma receptor (Fc[gamma]R) binding to the antibody Fc. Activating and inhibitory Fc[gamma]Rs are expressed on immune cells and bind to the Fc regions of IgGs, which either promote or hinder responses against the tumor cell. Since mouse models of cancer are currently the most established in this field, studying the effect of modulating individual murine Fc[gamma]Rs would provide insight into this type of therapeutic for humans. The ectodomains of murine Fc[gamma]Rs are highly homologous and therefore it has been difficult to engineer antibody Fc regions to specifically bind only one Fc[gamma]R. To address this challenge, we engineered the human tenth type III fibronectin (Fn3) domain scaffold to bind individual murine FcγRs. The Fn3 scaffold has the advantage of binding at epitopes on the Fc[gamma]R that are distinct from the Fc binding region. Fn3 clones have been isolated with specificity to each known murine Fc[gamma]R: Fc[gamma]RI, Fc[gamma]RIIB, Fc[gamma]RIII, and Fc[gamma]RIV. Measured KDs of Fn3 binding to Fc[gamma]R range on the order of 1-100nM, which fall within the range of normal Fc-Fc[gamma]R binding affinity or even higher affinity. Candidate Fn3 clones are fused to tumor antigen specific scFvs and a murine serum albumin (MSA) to maintain in vivo half-life. Each scFv- MSA-Fn3 construct was antigen specific and bound specifically to the Fc[gamma]R that it was designed to target. To confirm the biological activity of the Fn3 clones, phagocytosis assays with peritoneal macrophages were conducted. Pharmacokinetic studies have shown all scFv-MSA-Fn3 constructs to have approximate beta half-lives of 25 hours in C56BL/6 mice. Biodistribution of scFv-MSA-Fn3 constructs demonstrate preferential accumulation in antigen positive subcutaneous tumors. Multiple in vivo models were optimized to detect antitumor efficacy of our engineered constructs. In a subcutaneous tumor model with aggressive prophylactic dosing, our binders to the activating Fc[gamma]RI, Fc[gamma]RIII, and Fc[gamma]RIV demonstrate similar control to the mIgG2a antibody. These tools will allow us to conduct future studies on the immune response of triggering individual FcγR in models of cancer. The binding of human IgG1 to human Fc gamma receptors (hFcγR) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 (N297) of the Fc, with deglycosylation resulting in a complete loss of hFc[gamma]R binding. Thus, aglycosylated variants that can bind to hFc[gamma]Rs have the potential to allow therapeutic antibodies to be produced in virtually any expression system. Previously, we demonstrated that aglycosylated human IgG1 Fc variants are capable of engaging the human Fc gamma RII subset of the low-affinity hFc[gamma]Rs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFc[gamma]R engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with Fc gamma RIIIA, and that these variants can also bind the human Fc gamma RII subset. In this study, we also assess the biophysical properties and serum half-life of the aglycosylated IgG variants. Phagocytosis assays with monocytes and macrophages were performed to determine which constructs optimally drove tumor cell killing. A mathematical model of phagocytosis suggests that hFc[gamma]R dimers of hFc[gamma]RI were the main drivers of phagocytosis. / by Tiffany F. Chen. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 80-89). / Many major human oncogenes contribute to cancer in large part by activating the mitogenactivated protein kinases (MAPK) ERK1 and ERK2 (ERK). These kinases are critical in normal physiological processes from development to memory, and their activation in cancer drives growth and metastatic invasion. Understanding the effects of ERK signaling is especially important since vemurafenib, the first pharmaceutical directly targeting the MAPK pathway, has recently been approved to treat advanced melanoma. ERK controls cellular phenotypes by phosphorylating over two hundred known substrate proteins, however new ERK targets are reported frequently. We have used a chemical genetics approach to identify over one hundred novel substrates of ERK2. This approach utilizes an ERK2 kinase with "gatekeeper" mutation that allows it to bind bulky ATP analogs (AS-ERK2). AS-ERK2 can be used to label its direct substrates with thiophosphate in an in vitro kinase reaction. To achieve sufficient sensitivity we have improved on existing protocols for identification of thiophosphorylated peptides. Our improved protocol identified over one hundred novel ERK2 substrates in 3T3-L1 fibroblasts and in colon carcinoma cell lines. We investigated one novel ERK2 substrate, the transcriptional repressor ETV3, in detail and found that phosphorylation abrogates binding to DNA by ETV3, and that mutation of key phosphorylated residues to alanine blocks this effect. We also identified several thousand ETV3 targets across the genome. The wide range of genes targeted by ETV3 suggests that it may act as a key regulator of cell cycle and metabolism in some cell types. We have also identified ERK2 substrates in the DLD1 colon carcinoma cell line, including several mRNA splicing factors and members of the MLL family of histone 3 methyltransferases. We are using high-throughput sequencing and biochemical experiments to determine whether these phosphorylation sites control the function of MLL proteins. Taken together these investigations greatly expand our knowledge of the ERK signaling pathway and have revealed greater connectivity among biological processes than had been appreciated. / by Scott M. Carlson. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / 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 (pages 175-194). / Immune cells participate in dynamic cellular interactions that play a critical role in the defense against pathogens and the destruction of malignant cells. The vast heterogeneity of immune cells motivates the study of these interactions at the single-cell level. In this thesis, we present new tools to characterize how individual immune cells interact with each other and with diseased cells. We develop a nanowell-based platform to investigate how natural killer (NK) cells interrogate and attack diseased target cells. This platform enables integrated analysis of cytolytic activity, secretory activity, receptor expression, and dynamic parameters of interactions between thousands of individual NK cells and target cells. Using this platform, we show that NK cells operate independently when lysing a single target and that motility during contact is associated with the secretion of certain cytokines. Extending the platform, we investigate how contact with a target induces the shedding of CD16 from the surface of an NK cell. NK cells use CD16 to recognize antibody-coated target cells, and thus the loss of CD16 is of clinical interest. We show that the loss of CD16 is correlated to the length of time that the NK cell spends in contact with a target but that not all NK cells that shed CD16 exert common effector functions. In wells with multiple NK cells, shedding occurs in a more coordinated manner than would be expected by chance alone. Next, we compare the functional properties of NK cells with distinct repertoires of inhibitory receptors. Inhibitory receptors prevent NK cells from attacking healthy cells, and their expression can confer NK cells with increased functional activity in a process known as "licensing". We show that despite forming prolonged contacts with target cells, unlicensed NK cells are less likely than licensed NK cells to secrete cytokines. Finally, we present tools to study other modes of interaction between immune cells. Towards this end, we develop and apply fluorescent cellular barcoding strategies to efficiently analyze the secretory properties of individual immune cells from different populations. Altogether, this thesis contributes new tools for single-cell analysis and applies them to reveal new insights about intercellular interactions. / by Yvonne J. Yamanaka. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013 / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 143-158). / One of the most marvelous features of microbial life is its ability to thrive in such diverse and dynamic environments. My scientific interest lies in the variety of modes by which microbial life accomplishes this feat. In the first half of this thesis I present tools to leverage high throughput sequencing for the study of environmental genomes. In the second half of this thesis, I describe modes of environmental adaptation by bacteria via gene content or gene expression evolution. Associating genes' usage and evolution to adaptation in various environments is a cornerstone of microbiology. New technologies and approaches have revolutionized this pursuit, and I begin by describing the computational challenges I resolved in order to bring these technologies to bear on microbial genomics. In Chapter 1, I describe SHE-RA, an algorithm that increases the useable read length of ultra-high throughput sequencing technologies, thus extending their range of applications to include environmental sequencing. In Chapter 2, I design a new hybrid assembly approach for short reads and assemble 82 Vibrio genomes. Using the ecologically defined groups of this bacterial family, I investigate the genomic and metabolic correlates of habitat and differentiation, and evaluate a neutral model of gene content. In Chapter 3, I report the extent to which orthologous genes in bacteria exhibit the same transcriptional response to the same change in environment, and describe the features and functions of bacterial transcriptional networks that are conserved. I conclude this thesis with a summary of my tools and results, their use in other studies, and their relevance to future work. In particular, I discuss the future experiments and analytical strategies that I am eager to see applied to compelling open questions in microbial ecology and evolution. / by Sonia C. Timberlake. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. The diversity in lesions induced by these agents led to the evolution of several DNA repair pathways that suppress the mutagenic and toxic effects of DNA damage. Ironically, many cancer chemotherapeutics are themselves genotoxic due to their ability to target tumor cells that are more vulnerable to DNA damage. Therefore, knowledge about DNA damage and repair is relevant to cancer prevention, susceptibility and treatment. Despite its fundamental importance, measurement of DNA damage and repair in people is far from routine, primarily due to technological obstacles. Recently, we have developed the CometChip, a novel platform for measuring DNA damage that has its basis in the well-accepted comet assay. To advance the CometChip for broader utility in screens and population studies, we further improved the platform to provide greater sensitivity, reproducibility and throughput. We then characterized the conditions of the CometChip to analyze different DNA repair pathways. We showed that the CometChip is effective for evaluating repair kinetics and for detecting deficiencies of base excision repair, nucleotide excision repair, mismatch repair, and non-homologous end-joining. The ability to assess multiple repair pathways opens door to many applications. In terms of basic research, we applied the CometChip technology to perform cross-pathway analysis of two repair proteins, Xrccl and DNA-PKcs. Results confirm known activities as well as reveal new cross-pathway functions of these proteins. In terms of clinical relevance, we used the CometChip to evaluate small molecule inhibitors of DNA repair proteins, which are potential chemotherapeutics. In terms of public health, we exploited the CometChip to learn about differences in DNA repair among individuals. We discovered that there are subpopulations of people who share similar BER characteristics and there are shared factors between BER and NER among individuals. Taken together, the CometChip platform represents a significant technological advance. From integrated analysis of repair proteins, to evaluation of clinically relevant chemicals, to studies of inter-individual variations, the CometChip has demonstrated value in uncovering new information about DNA repair in humans. / by Jing Ge. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 125-144). / The volume of protein structure data has grown rapidly over the past 30 years, leaving a wake of facts that still require explanation. We endeavored to answer a few open questions on the structure-function relationship of intriguing mechanochemical protein systems. To this end this thesis work contains five studies that offer novel insights into molecular biomechanical systems that may guide future basic research or applications development. The first study concerns the biophysics of cadherin-mediated cell sorting observed in developing solid tissue. We investigated the evolutionary dynamics of the cadherin superfamily of cell-cell adhesion proteins to infer a structural basis for their paradoxical mixture of pairwise binding specificity and promiscuity. Our analysis predicts a small set of specificity-determining residues located within the protomer-protomer binding interface. The putative specificity-determinants form a design space with potential for engineering novel cell-cell adhesive interactions. The second study addresses the open question of how to automatically identify regions within a protein that engage in allosteric communication. To identify allostery we developed and tested two computational tools that operate on protein conformational dynamics data. These tools are useful for generating testable hypotheses about proteins with multiple functional sites for the design of non-competitive protein inhibitors. The third study asks, "What is the consequence of allosteric cooperation between the tandem binding sites in a class of proteins that bundle filamentous actin (F-actin)?" Through simulation we demonstrate that cooperative F-actin bundling tends to strengthen bundles by driving the formation of cross-links between neighboring filaments while depleting F-actin binding sites that are occupied but not cross-linked. We hence propose that allostery may be a natural feature of ABPs with tandem F-actin binding sites if nature indeed selects for sturdy F-actin bundles. The final two studies examine the impact of two structural perturbations to Factin on its mechanics. Using structure-based computer modeling we develop a simple explanation for the mechanism by which the structure of actin's polymorphic subdomain 2 mediates 4-fold changes in F-actin's flexibility. We further demonstrate that two calponin homology domains stabilize F-actin by binding in a configuration that tends to relax the stress concentration at actin-actin interfaces. / by Philip W. Bransford. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / Cataloged from PDF version of thesis. / Includes bibliographical references. / There is increasing recognition that aberrant protein modifications play an important role in the pathophysiology of inflammation and oxidative stress in cells. We recently discovered that N⁶-formylation of lysine is an abundant endogenous modification of histone and chromatin proteins. The high abundance of N⁶-formyllysine in histone proteins and its chemical similarity to the biologically important N⁶- acetyllysine has raised questions about its mechanisms of formation and biological consequences. Using novel ultrasensitive and specific liquid chromatography-coupled tandem mass spectrometry methods (LC-MS/MS) to quantify N⁶-formyllysine lesions in proteins, we aimed to investigate the sources as well as the fate of this abundant endogenous protein modification. We present evidence that endogenous formaldehyde is a major source of N⁶-formyllysine and that this adduct is widespread among proteins in all cellular compartments. We observed in vitro as well as in vivo that formaldehyde exposure leads to a dose-dependent increase in N⁶-formyllysine protein adducts, with the use of isotopically-labeled formaldehyde to dissect endogenous from exogenous formaldehyde as sources of the adduct. Further, other isotope labeling studies revealed that lysine demethylation in histone proteins is not a source of N⁶-formyllysine. With regard to N⁶-formyllysine persistence in cells, our investigation of histone deacetylases revealed that despite chemical similarity of N⁶-formyllysine to N⁶-acetyllysine, the former is refractory to removal by histone deacetylases, which suggests that they will persist throughout the life of individual histone proteins. If not repaired, lysine formylation could accumulate to significant levels. The resemblance of N⁶-formyllysine to N⁶-acetyllysine, together with recent studies that mapped its location on many conserved lysine acetylation and methylation sites along histone proteins, support the idea that this abundant protein modification could interfere with normal regulation of gene expression, potentially leading to an epigenetic mechanism of disruption of cell function. / by Bahar Edrissi. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Page 165 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 151-164). / Experiments in biology are often combinatorial in nature and require analysis of large multi-dimensional spaces, but the scales of these experiments are limited by logistical complexity, cost, and reagent consumption. By miniaturizing experiments across nanoliter-scale emulsions that can be processed at large scales, droplet microfluidic platforms are poised to attack these challenges. Here we describe a droplet microfluidic platform for combinatorial experiments that automates the assembly of reagent combinations, with order-of-magnitude improvements over conventional liquid handling. Moreover, our design is accessible, requiring only standard lab equipment such as micropipettes, and improves the chemical compatibility of droplet microfluidic platforms for small molecules. We applied our platform to two experimental problems: combinatorial drug screening and microbial ecology. First, we used our platform to enable screening of pairwise combinations of a panel of antibiotics and 4,000+ investigational and approved drugs to overcome intrinsic antibiotic resistance in the model Gram-negative bacterial pathogen E. coli. This screen processed 4+ million droplet-level assays by hand in just 10 days to discover more than 10 combinations of antibiotics and non-antibiotic drugs for further study. We then applied our platform to microbial ecology, where the interactions between microbes in communities can dictate functions important for both basic science and biotechnology. As a proof of concept, we used our platform to survey 960 pairwise interactions of microbes isolated from soil, and deconstruct higher-order interactions in a 4-strain community. Altogether, we expect that our platform can be used to efficiently attack combinatorial problems across molecular and cellular biology. / by Anthony Benjamin Kulesa. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 73-83). / Environmental bacteria display tremendous genetic diversity, but we are still learning how this diversity arises and relates to their wide range of habitats. Investigating how bacteria adapt helps us understand their contributions to environmental processes and informs forward engineering of bacteria for industrial applications. Experimental evolution is a powerful approach, with microbes especially, but it has mostly been applied to model organisms and metabolic functions. In the work here, we investigated the possibility, degree, and variability of adaptation of an environmental Vibrio strain by applying a little-used selection method appropriate to a relevant condition, salinity. We successfully isolated mutants with higher salt tolerance by selecting on salt gradient plates. Resequencing the genomes of the evolved strains revealed unprecedented hypermutation in three of nine parallel lineages. These mutator lines arose independently, and each of them accumulated more than 1500 single-base mutations. By comparison, there are only 302 single-base differences between the ancestor strain and another strain isolated in the wild. Hypermutation was associated with a deletion resulting from improper prophage excision. Members of this family of prophages are found in other proteobacteria, including well-studied human pathogens, from very different environments. Mutators are known to arise spontaneously in wild and clinical bacteria, but the extent of their adaptive contribution is unknown. We have preliminary evidence that this mechanism of evolution could be relevant in the environment, where horizontal gene transfer and mobile elements play known, significant roles in bacterial evolution. / by Sean Aidan Clarke. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / One of the primary goals of cancer chemotherapy is the design of antitumor agents that achieve selective targeting of tumor cells while minimizing toxicity to normal tissues. We have synthesized a series of DNA damaging agents that are designed to disrupt selectively the DNA repair and signaling pathways in tumor cells. My dissertation focuses on the mechanistic studies of the rationally designed genotoxicants E2-7aX and 2Pl, which target cancer cells that express the estrogen receptor (ER). The studies on the biological activity of E2-7ca in ovarian cancer cells reveal that the compound induces levels of cytotoxicity comparable to those of cisplatin, which is the current front-line therapeutic for ovarian cancer. E2-7ca induces crosslink formation in the ovarian cell line SKOV-3, which potentially leads to the S-phase arrest observed in these cells. Further, the arrest is persistent even after drug removal, indicating that the persistence might be one of the reasons for the enhanced toxicity of E2-7ca compared to the control compound chlorambucil. E2-7ca induces cell death in SKOV-3 cells through autophagy, as opposed to the classical cell death pathway of apoptosis. Mechanistic studies on the role of ER in E2-7a toxicity in SKOV-3 cells show that the absence of the ER (facilitated by the knockdown of ER protein through RNA interference in SKOV-3 cells) makes the cells less sensitive to the drug. In addition, the ER(-) population displays a lower level of drug- DNA adducts than its ER(+) counterpart, as detected by Accelerator Mass Spectrometry (AMS). The combination of these pieces of evidence strongly suggests that as per its intended mechanism of action, E2-7a. mediates its effects through the involvement of the ER, and appears to be a promising candidate as an antitumor agent in the clinical setting. In addition, the toxicity and DNA damage caused by the 2-phenylindole compounds 2PI and 2PI(OH) were studied in ovarian cancer cell lines; these studies underscore the differences in the mechanism of action of the two compounds. Both the compounds demonstrate promise as potent anti-tumor agents, and the selectivity of these compounds in different cellular contexts (ER (+)/(-) or p53 (+)/(-)), once established, could help strengthen the basis of their therapeutic efficacy. / by Sreeja Gopal. / Ph.D.