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.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 77-92). / Sequencing the human genome has spurred systematic work on understanding how gene expression and genomic integrity contribute to disease. To date, 3,519 genes have been identified as the underlying cause of specific single gene disorders. However, complex diseases still pose a daunting challenge that require both an understanding of cell function as well as how the genome interacts with its cellular environment. Sequencing technologies are now routinely applied to interrogate gene variants, gene expression patterns, chromosome accessibility, among other measurements to infer gene and cell function. We build upon past work to address the challenge of targeting sequencing effort to cells and genomic loci of interest to probe the molecular mechanisms behind disease. In this thesis, we demonstrate two novel targeted sequencing methods that can enable a greater understanding of cell function. (1) The development of targeted sequencing in pooled single cell RNA-seq libraries and (2) the development of a novel sequencing approach that allows for the quantification and identification of single stranded break (SSB) locations across the genome. First, we introduce a new targeted sequencing approach to identify rare cells of interest in pooled sequence libraries. Improved throughput in single cell sequencing has enabled the transcriptional profiling of thousands of cells at once. However, due to reliance on pooled library construction methods, it is now more difficult to focus on and analyze particular cells of interest, apart from analyzing the library in its entirety. We designed multiplex PCR primers to simultaneously enrich targeted cells from a complex DNA library pool of single cells. We show how molecular enrichment can be used to efficiently target rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC). Next, we demonstrate a new targeted sequencing approach, called NickSeq, to locate and quantify DNA SSBs with single nucleotide resolution. SSBs are the most common form of DNA damage at an estimated 10,000 per cell per day, but there is no available method to robustly determine the exact sites of damage. SSB accumulation correlates with disease, but it is unknown how the location and amount of damage relate to health outcomes. We intentionally create a unique mutational signature at the SSB that is a fingerprint for this specific type of DNA damage when the locus is sequenced. Taken as a whole, we introduce two novel strategies to further understand cell function through studying rare cells in single cell populations and analyzing DNA SSB damage in relation to cell health. This work demonstrates that targeted sequencing approaches have promise for understanding the molecular mechanisms behind aberrant cell function, a necessary step in the prevention and treatment of disease. / by Navpreet Singh Ranu. / Ph. D.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 85-88). / Resource limitations in bacterial cells can present significant hurdles that preclude correct synthetic circuit behavior. In a simple circuit with one constitutively expressed protein and one protein whose expression is inducible, it has been shown that inducing the expression of the second protein causes a significant decrease in the level of the first. In this thesis, we explore the possibility of reducing the effects of resource limitations by adding a high-gain negative feedback loop to one of the circuits. The loop includes an sRNA construct. We explore different implementations of this circuit and model them mechanistically. Furthermore, we begin physically implementing one of the circuit designs by testing intermediate constructs. Finally, we also explore the hypothesis that exogenous circuits on plasmids compete for a pool of resources that is spatially separated from the resources that the genome utilizes. Through our work, we show results that support the spatial separation hypothesis. / by John Elias Yazbek. / S.M.

Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. Includes CD-ROM with 9 videos in the .avi format and 2 video in the .mp4 format. / Includes bibliographical references. / Megakaryocytes (MKs), one of the largest and rarest hematopoietic stem cells in the bone marrow, have traditionally played a primary role in hemostasis as precursors to platelets, which are importantly, one of the most abundant cell types in the peripheral circulation. While platelets are studied for their various roles in inflammation, the role of MKs within the innate immune system has not been explored. In a series of comprehensive in vitro experiments, we have demonstrated that both cord blood-derived MKs and MKs from a megakaryoblastic lineage have innate immune cell functions, including: phagocytosis, formation of extracellular traps, and chemotaxis towards pathogenic stimuli. MKs were also observed to directionally release platelets towards pathogenic stimuli. In addition to their primary role as immune cells, MKs were also shown to contain extranuclear histones, which the MKs release along with budding platelets into the circulation. These small packages of histones can play a major role in inflammation and immunothrombosis by promoting inflammation and coagulation. By evaluating blood and tissue samples from patients diagnosed with sepsis, we demonstrated that there is an increased MK concentration both in the peripheral circulation, as well as in the lungs and kidneys. Platelets from patients with sepsis also appeared to have a specific phenotype, including increased DNA and histone staining. MK number in the circulation and end-organs, as well as platelet histone expression appeared to be correlated with both prognosis and type of infection. This newly recognized role of MKs as functional innate immune cells may have significant implications for the role of MKs in conditions such as sepsis and, pending a more profound mechanistic understanding, may further lead to the development of novel targets for the treatment of sepsis. / by Galit Hocsman Frydman. / Sc. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 125-137). / This thesis describes the identification and characterization of a novel 'layer' of intrinsic non-genetic functional heterogeneity within the seemingly homogeneous naive CD8⁺ T cell population in their survival and proliferation capacities. This heterogeneity is predictably marked by the surface level of CD5. NaYve CD8⁺ T cells that are also CD5hi have a greater intrinsic capacity to proliferate both in response to IL7 alone or identical stimulation of the TCR, pathway (same dose of PMA/ionomycin) as compared to CD51' cells. In contrast, CD510 cells survive better in conditions of cytokine deprivation. The selective proliferation in response to IL7 as well as the relative CD5 level is preserved even after several rounds of activation-induced proliferation and differentiation into memory-like cells in vitro, suggesting that the relative CD5 level could be used as a lineage marker to predict the proliferation capacity of CD8⁺ T cells. Microarray analysis of two naive TCR transgenic CD8⁺ T cells, from the same genetic background, but with marked differences in CD5 levels, namely OT-1 and F5 Rag-/- T cells, revealed consistent differences in their cell survival, proliferation and metabolic pathways. Analysis of upstream regulatory networks suggests that the E2A family of transcription factors and miR-181 are likely involved in setting the proliferation and survival capacities of naive T cells, possibly during thymic development. Our estimates of spMHC-induced signals in OT-1 (CD5hi) and F5 (CD50) cells, based on cytosolic Ca2⁺ influx measurements, suggest that the differences in their lymphophenia-induced proliferation, which were previously attributed to putative corresponding differences in their strength of interaction with self-peptide MHC, may also largely be a result of intrinsic differences in their proliferation capacities in response to 1L7. Further, the potential of exogenous IL7 therapy to skew the CD8⁺ T cell repertoire towards the CD5hi phenotype was demonstrated with in vivo studies in mice. Conversely, antibody-mediated depletion of IL7 has the opposite result. / by Vinay Subhash Mahajan. / Ph.D.