Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, June 2009. / "April 2009." Cataloged from PDF version of thesis. / Includes bibliographical references. / Tamoxifen resistance is the biggest problem in endocrine treatment against hormone receptor positive breast cancer patients. HER2 is a membrane receptor tyrosine kinase that is known to correlate with poor disease outcome and unresponsiveness to endocrine treatment. Although much work has been done over the past decades to elucidate pathways involved in HER2 receptor signaling, the map of network-wide signaling events that contributes to the resistance to Tamoxifen treatment has not been characterized, making it difficult to pin-point the downstream drug target to revert the Tamoxifen resistance. To gain a molecular understanding of the mechanisms by which cells gain drug resistance, we have employed a proteomic analysis by mass spectrometry to quantitatively analyze cellular tyrosine phosphorylation signaling events in breast cancer model systems and human tumor samples. As a result of research, we have identified the major differences in downstream signaling pathways between Tamoxifen sensitive and Tamoxifen resistant breast cancer cell line models. These findings were further analyzed in Tamoxifen sensitive, and Tamoxifen treated/recurred patient samples to study clinical relevance. Specifically, we determined that P13K/Akt, MEK/ERK, and Src/FAK/Abl pathways are major components of the Tamoxifen resistance. We further showed that they signaling components are possible drug targets to revert Tamoxifen resistance. This study revealed cell-context specific network-wide changes in signaling events in response to use of therapeutic drugs. This is, to our first knowledge, the first phosphoproteomic analysis of the signaling network in breast cancer to address Tamoxifen resistance. We believe that same approach is applicable to other drug resistance problems in various disease settings. / by Hideshiro Saito-Benz. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 191-209). / Mounting effective adaptive immune responses requires a large naive T-cell population with a wide diversity of target specificity. Naive CD8⁺ T-cells depend on T-Cell Receptor (TCR) and ye cytokine signals for their homeostatic survival and proliferation, but differences in sensitivity to these homeostatic signals among T-cell clones have been generally attributed to differences in TCR specificity. This thesis describes the novel identification and characterization of intrinsic heterogeneity in the TCR-independent abilities of CD8⁺ T-cells to respond to homeostatic ye cytokines, and survive in their absence. These differences were predictably marked by expression of CD5, a surrogate marker of TCR:spMHC binding avidity. In vitro, CD5I T-cells proliferate more robustly to saturating levels of the y, interleukin (IL) cytokines IL-7, IL-2 and IL-15, while CD5" cells have prolonged survival in the absence of dedicated homeostatic cues. IL-7 is the most critical cytokine for naive T-cell homeostasis, and a detailed analysis of IL-7 signaling revealed that IL-7 responsiveness is primarily determined by IL-7 receptor (IL-7R) expression, which is correlated with CD5 expression. While T-cells share common relationships between IL-7-induced signaling and responses, the signaling network encodes distinct signaling requirements for survival, proliferation and CD8a induction responses. As a result, all T-cells survive when treated with high doses of IL-7, but only cells with a critically high level of IL-7R expression can induce sufficient signaling to proliferate. IL-7 depletion also scales with IL-7R expression, and the 'overconsumption' of IL-7 by CD5hiIL-7Rh T-cells can compromise their prolonged survival. In vivo, lymphoreplete mice preserve the homeostatic diversity of CD5 expression by maintaining physiological IL-7 levels that promote neither preferential proliferation nor survival of CD5hiIL-7Rh' and CD5'"IL-7R'" T-cells. However, elevated IL-7 levels in lymphopenic mice or lymphoreplete mice administered with exogenous IL-7 yield preferential expansion of CD5hiIL-7Rh T-cell subsets, elevating the mean CD5 expression of the T-cell repertoire. This demonstration of functional intrinsic heterogeneities in IL-7R expression between CD8⁺ T-cells supports a previously under-appreciated role for IL-7 in maintaining not only the size but also the diversity of the T-cell repertoire. Furthermore, the exemplified potential for preferential expansion of more auto-reactive CD5"I T-cells subsets has important implications for the design of cytokine therapies. / by Megan Joan Palmer. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 115-123). / The identification and quantification of high-dimensional relationships is a major challenge in the analysis of both biological and chemical systems. To address this challenge, a variety of experimental and computational tools have been developed to generate multivariate samples from these systems. Information theory provides a general framework for the analysis of such data, but for many applications, the large sample sizes needed to reliably compute high-dimensional information theoretic statistics are not available. In this thesis we develop, validate, and apply a novel framework for approximating high-dimensional information theoretic statistics using associated terms of arbitrarily low order. For a variety of synthetic, biological, and chemical systems, we find that these low-order approximations provide good estimates of higher-order multivariate relationships, while dramatically reducing the number of samples needed to reach convergence. We apply the framework to the analysis of multiple biological systems, including a phospho-proteomic data set in which we identify a subset of phospho-peptides that is maximally informative of cellular response (migration and proliferation) across multiple conditions (varying EGF or heregulin stimulation, and HER2 expression). This subset is shown to produce statistical models with superior performance to those built with subsets of similar size. We also employ the framework to extract configurational entropies from molecular dynamics simulations of a series of small molecules, demonstrating improved convergence relative to existing methods. As these disparate applications highlight, our framework enables the use of general information theoretic phrasings even in systems where data quantities preclude direct estimation of the high-order statistics. Furthermore, because the framework provides a hierarchy of approximations of increasing order, as data collection and analysis techniques improve, the method extends to generate more accurate results, while maintaining the same underlying theory. / by Bracken Matheny King. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / In title on title-page "[beta]" appears as the lower-case Greek letter. Vita. Page 284 blank. Cataloged from PDF version of thesis. / Includes bibliographical references. / Inspired by the toxicity mechanism of cisplatin in testicular cancer, a series of bi-functional genotoxicants has been designed that supplement their DNA damaging properties with the ability to interact with tumor specific proteins. One such compound, 11[beta] -dichloro links an aniline mustard moiety to a steroid ligand for the androgen receptor (AR). In vitro and in vivo (cell cultures, mouse xenografts) studies highlighted the potent antitumor properties of the molecule, which can prevent growth of AR positive tumor xenografts in mice. However, 11 p-dichloro also proved highly effective against many cancer lines that do not express the AR, more so than other nitrogen mustards commonly used in chemotherapy. To understand better the AR independent mechanism, the toxicity of 116 -dichloro was investigated in Saccharomyces cerevisiae, by interrogating the complete yeast single-gene deletion mutant library. Surprisingly, the screen revealed that the mutants most sensitive to 11[beta] -dichloro are not the ones lacking genes involved in DNA repair, but rather mutants lacking genes involved in mitochondrial and ribosomal function. While some of the sensitive mutants are also sensitive to other DNA damaging agents, almost half of them are uniquely sensitive to 11[beta] -dichloro, suggesting a DNA-damage independent mechanism of action. Based on the yeast findings, we tested mechanistic hypotheses in HeLa cells (an AR negative human cancer cell line), and discovered that 11[beta] -dichloro induces a large amount of reactive oxygen species (ROS), accompanied by a significant depletion of the antioxidant pool and a perturbation of the mitochondrial inner membrane potential. We also discovered that cotreatment with antioxidants such as N-acetyl cysteine or vitamin E alleviates the toxicity of 11[beta]-dichloro, suggesting that ROS play an important role in the mechanism of toxicity. In terms of transcriptional responses, previous observations were confirmed that 11[beta] -dichloro upregulates expression of genes involved in the unfolded protein response (UPR) and sterol biosynthesis. Additional cellular responses consistent with these pathways were the striking increase in cytosolic calcium and significant changes in the size of the cells. Taken together, our results highlight the multifaceted toxicological profile of 11[beta] -dichloro, indicating that besides DNA damage, the generation of ROS and induction of UPR may be the additional pathways responsible for the potent anticancer effects of this agent. / by Bogdan I. Fedeleş. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The development of synthetic vectors enabling efficient intracellular delivery of macromolecular therapeutics such as nucleic acids and proteins could potentially catalyze the clinical translation of many gene and protein-based therapies. However, progress has been hindered by a lack of safe and effective materials and by insufficient insight into the relationship between key delivery properties and efficacy. Accordingly, working with a promising class of cationic, degradable gene delivery vectors, poly(-amino ester)s (PBAEs), we develop novel, hydrophobic PBAE terpolymers that display dramatically increased gene delivery potency and nanoparticle stability. We then develop a technique based on size-exclusion chromatography that enables the isolation of well-defined, monodisperse PBAE polymer fractions with greater transfection activities than the starting polymer. This technique also allows us to elucidate the dependence of gene delivery properties on polymer molecular weight (MW). Subsequently, we examine the cellular uptake and trafficking mechanisms of PBAE/DNA polyplexes, and demonstrate that polyplex internalization and transfection depend on a key endo/lysosomal cholesterol transport protein, Niemann-Pick C1 (Npcl). Finally, working with cationic lipids termed lipidoids, which have shown exceptional potency for the delivery of RNAi therapeutics, we develop these materials for intracellular delivery of proteins using a simple and novel approach in which nucleic acids serve as a handle for protein encapsulation and delivery. Preliminary in vivo experiments suggest the potential application of this approach toward lipidoid-mediated delivery of protein-based vaccines. Taken together, the work presented here advances the development of polymer and lipid materials for the safe and effective intracellular delivery of DNA and protein therapeutics. / by Ahmed Atef Eltoukhy. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. "September 2018." / Includes bibliographical references (pages 57-73). / A key requirement for understanding the workings of the brain is to fill in the explanatory gap between molecular phenomena and identifiable behavior at the organismal level. Magnetic resonance imaging (MRI) provides a unique tool for bridging this gap, as it allows for imaging tissue throughout whole organisms. Although functional MRI (fMRI) is already a workhorse technique in human neuroscience research, current fMRI methods give us limited information about brain mechanisms because they rely on blood flow changes that are only indirectly coupled to cellular and molecular events. To associate cellular or molecular specificity to MRI, there is a need for genetically targeted, analyte-specific sensors. Calcium is a molecule of great interest to biology since its fluctuations are highly correlated with neural activity. While much progress has been made in pursuit of genetically encoded calcium sensors none allow for deep tissue imaging of whole rodent brains. In this thesis we demonstrate that genetically encodable calcium sensors based on known MRI gene reporter ferritin show modest sensitivity. To achieve higher amplification we leverage the hemodynamic response, which is coupled to neuronal activity through a calcium-activated enzyme, neuronal nitric oxide synthase (nNOS). We show that chemical stimulation of ectopically expressed neuronal nitric oxide synthase (nNOS) elicits an artificial hemodynamic response detectable by MRI. To distinguish signaling from endogenous nNOS we use a two-prong strategy to engineer a suite of enzymes with altered inhibition constants compared to nNOS. We demonstrate that these engineered enzymes (NOSTICs) exhibit calcium-dependent catalytic activity. One such NOSTIC was then virally delivered to rodent brains and shown to express in certain cell populations. Hemodynamic responses from these cell populations were recorded following electrical stimulation using MRI. The imaging strategy demonstrated here thus offers a novel and potentially powerful approach for cell-targeted functional imaging of the brain. / by Souparno Ghosh. / 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. / 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.