Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 118-125). / The adaptive immune system is an extraordinarily diverse inventory comprised of highly specialized cells, the differentiation of which requires numerous lineage specifications at various developmental stages. The precise control of immune cell differentiation and the delicate balance of their population composition are crucial for effective protection against infectious environmental agents, without triggering autoimmune responses or allergies. It is therefore important to understand at the molecular level in individual cells how lineage commitment is regulated. I explored the heterogeneous gene expression during the lineage specification of single T helper cells, by quantitatively measuring mRNA and protein levels. I have discovered a paradigm of cell lineage specification governed by the signaling interplay between extracellular cues and intracellular transcriptional factors, where the strength of extracellular signaling dominates over the intracellular signaling components. In the presence of extracellular cues, T helper cells stochastically acquire any intermediate Thl/Th2 states. The states of T helper cells can be gradually tuned by depriving availability of extracellular cytokines, which are produced stochastically by a small subpopulation of cells. When extracellular cues are removed, the weak intracellular signaling network reveals its effect, leading to classic mutual exclusion of antagonistic transcriptional factors. / by Miaoqing Fang. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / 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. / The human gastrointestinal tract is home to a dense and dynamic microbial community. Recent advancements in sequencing technology have revealed numerous relationships between the composition of these communities and human and health and disease. In some cases, researchers have shown causal relationships between the presence or absence of particular microorganisms and disease. These findings offer promise for using microorganisms or microbial communities to modulate health and disease, but to date, we lack tools and mechanistic insight needed for rational engineering of these communities. Understanding how microorganisms enter, colonize, grow, and disperse to new hosts present key considerations for rational engineering of the human gastrointestinal tract. In this thesis, I use experimental studies of the human and murine gastrointestinal tract to address these considerations. In the first study, I examined endospores and other resistant cell types in the gastrointestinal communities of unrelated humans to identify the ecological role of these states in the distribution of bacterial populations in healthy people. I used this information to infer shared roles for these organisms in successional states in the human gut, and identify host- and diet-derived metabolites as environmental signals mediating the growth and colonization of these organisms. In the second study, I examined the potential for using targeted manipulations of diet to favor selective growth and colonization by an introduced bacterium in the murine gastrointestinal tract. I showed that resource exclusivity of this bacterium permits its selective expansion in this environment, and negatively impacts the growth of other commensals. Central to the goal of rational engineering of the gut microbiota, these studies reveal ecological considerations that may promote or inhibit colonization by introduced commensals in this complex ecosystem. This work invites provides a conceptual framework for integrating systems microbial ecology with engineering design to manipulate the composition of the gastrointestinal microbiota. / by Sean M. Kearney. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The high incidence of traumatic brain injury due to adverse impact events ranging from head collisions to ballistic attacks has prompted significant interest in synthetic polymer gels capable of mimicking key mechanical properties of brain tissue. These so-called brain tissue simulants are valuable tools for developing protective strategies because they can serve as test media to evaluate new helmets or optimize robotic surgery techniques. However, the so-called "soft matter" employed to date for ballistic applications, such as ballistic gelatin and clay, are crude mechanical representations of brain tissue. Therefore, there remains a need for a class of tissue simulant materials that more accurately replicates the mechanical behavior of brain tissue under impact loading, specifically in terms of deformation resistance and impact energy dissipation. This thesis focuses on design and synthesis of hierarchically structured gels, and mechanical characterization of these compliant gels for comparison with mammalian brain tissue. In particular, we use impact indentation to explore how the impact energy dissipation response varies as a function of species for brain tissue, or as a function of molecular composition and structure for synthetic gels. We find that a bilayered polydimethylsiloxane (PDMS) composite system enables the decoupling of the material's deformation resistance and energy dissipation characteristics, and can be tuned to fully match porcine brain tissue. However, given that the top PDMS layer is highly adhesive, we investigate whether adhesion plays a significant role in modulating the energy dissipation response, which has important implications in the utility of the tissue simulant material for ballistic applications. With a separate bilayered PDMS composite system, we decouple surface adhesion from bulk viscoelasticity, and quantify their individual contributions to impact energy dissipation. Through these experimental studies, in addition to a finite element computational analysis, we establish fundamental design principles and provide new insights regarding mechanisms that govern the extent of deformation and energy dissipation in compliant polymeric materials. Finally, we extend the capabilities of our impact indentation technique by demonstrating a novel analytical approach to extract viscoelastic moduli and relaxation time constants directly from the measured impact deformation response, thus significantly broadening the utility of impact indentation. With conventional characterization techniques such as shear rheology, several challenges arise when the material of interest has stiffness on the order of 1 kPa or lower, as is the case with brain tissue, largely due to difficulties detecting initial contact with the compliant sample surface. In contrast, impact indentation does not require contact detection a priori, and thus can potentially be utilized as a more accurate tool to characterize the viscoelastic properties of a wider range of soft matter for diverse biomedical or engineering applications, not limited to brain tissue simulants. This semi-analytical approach enables future studies to extract viscoelastic properties of brain tissue and tissue simulant polymers with increased accuracy and spatial resolution, in the context of traumatic brain injury, protection, and recovery. / by Bo Qing. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 107-124). / The extensive genomic diversity of complex systems, such as the human gut microbiome and the evolution of human cancer, has been revealed with advances in DNA sequencing. But we are still at an early stage in understanding this genomic diversity to expand our knowledge in biology and for biomedical applications. Taking the diverse human gut microbiome as an example, little is known about the rapid exchange of antibiotic resistance genes and virulence factors as part of the mobile gene flow between the microbes in the gut. Understanding such heterogeneous systems often involves studying the nature and behavior of the individual cells that constitute the system and their interactions. However, it is technically challenging to probe the genomic material of cells, the smallest unit of life and amplify single genomes for sequencing. Current single-cell technologies require complex instrumentation and the data quality is often confounded by biased genome coverage and chimera artifacts. We address these challenges with a new single-cell technology paradigm to make high-quality low-input genomic research accessible to scientists. We developed hydrogel-based virtual microfluidics as a simple and robust platform for the compartmentalization of nucleic acid amplification reactions. We applied whole genome amplification (WGA) to purified DNA molecules, cultured bacterial cells, human gut microbiome samples, and human cell lines in the virtual microfluidics system. We demonstrated whole-genome sequencing of single-cell WGA products with excellent coverage uniformity and markedly reduced chimerism compared with traditional methods. Additionally, we applied single-cell sequencing to identify horizontally transferred genes between the microbes in the gut and revealed human population activities' selective pressure in shaping the mobile gene pools. Altogether, we expect virtual microfluidics will find application as a low-cost digital assay platform and as a high-throughput platform for single-cell sample preparation. This work offers a significant improvement in making high-quality low-input genomic research accessible to scientists in microbiology and oncology. / by Liyi Xu. / Ph. D.

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.