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.

Thesis (S.M. in Toxicology)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 50-54). / One and a half million new cancer cases are reported each year in the United States. Despite this overwhelming burden of disease, current preventative treatments and early detection techniques are inadequate. With cancers, as with many aggressive diseases, time is of the essence; earlier detection begets better patient prognoses. While contemporary technology offers physicians the ability to battle cancers via stage-one detection, few reliable biomarkers have been developed to assist in detection of upstream tumorigenesis or interpretation of early genomic assault. The objective of the work described here is to identify early biomarkers specific to tumorigenesis, which correlate with initial genomic assault and subsequent mutation in the gpt-delta (B6C3F1) mouse model. The test compound used in this work is aflatoxin B₁, a known human carcinogen produced by fungal spoilage of food materials. Aflatoxin B₁ was one of the carcinogens used in the "training set" of compounds that defined the B6C3F1 animal model. My work had three goals with regard to upgrading this model as a tool for contemporary toxicology. In the first part of the thesis, a kinetic profile was generated of AFB 1-DNA adduct formation and removal. In the second part of the work, adduct levels and specific DNA damage patterns of males and females were compared (males are generally more sensitive than females to this and other toxins). Third, nursing mother mice were treated with two chemo-preventive agents in an attempt to determine if chemoprevention of the dam would lead to protection of her children. This thesis documents generation of a 48-hour time-course assessing adductburden in four-day-old B6C3F1 neonates. These burdens are measured in adducts per mega-base of genomic DNA (based on a single 6mg/kg dose of AFB₁). As previous studies show that 6mg/kg at this age results in near 100% liver tumorigenesis, this timecourse provides significant intuition for the onset and persistence of DNA damage. The results showed that AFB₁-N7-Guanine adduct formation maximized at two hours post dosing and then decreased rapidly; its FAPY derivative proved to be much more stable with time. A slight excess adduct burden was observed in males from 2-48 hours post dosing. Systematic differences in gene expression were observed in nursing female mother mice that were either treated or not with R,S-sulforaphane or D3T (3H-1,2- dithiole-3-thione). While minimal gene expression changes were observed in pups nursed by a 2mg R,S-sulforaphane treated dam, those nursing from a 5mg R,S-sulforaphane treated mother experienced much greater effects. At 300 umol/kg doses of D3T to the mother, no statistically significant gene expression profile alterations were observed in the pups. The work described here did not identify conditions in which a chemopreventive pattern of gene expression in the mother could be transferred efficiently to her offspring. / by Jason T. Bouhenguel. / S.M.in Toxicology

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Title as it appears in MIT Commencement Exercises program, June 2013: Modulation of the ERK1/2 and p53 signaling pathways by nitric oxide during the response to cisplatin in melanoma cells. Vita. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 51-58). / Malignant melanoma causes the highest mortality rate in skin cancers. Although cisplatin has proved efficacious in the treatment of various solid tumors, melanoma seems particularly resistant to this chemotherapeutic drug. Reports show that melanoma patients whose tumors express nitric oxide (NO) synthase and/or nitrotyrosine are often faced with poor prognosis. Moreover, it has been shown that NO produced by melanoma cells sustains lower sensitivity to cisplatin toxicity in vitro. Because inflammatory products such as NO and reactive oxygen species (ROS) are associated with the genesis and evolution of cancer, we hypothesized that these oxidative species may regulate key components of the response of melanoma to cisplatin. Using a system for controlled delivery of NO to simulate the NO levels believed to occur during inflammation, we showed that human melanoma (A375) cells pre-exposed to submicromolar NO concentrations were protected from a subsequent challenge with cisplatin. This protection was strongly associated with increased activity of the MAP-kinase cascade leading to activation of ERK1/2, as well as with downstream modulation of the apoptotic factors Bax and Bcl-2, and the transcription factors p53 and MiTF. Although NO favored increased expression and phosphorylation of p53, it also increased the expression of the p53 inhibitor MDM2, which may have counteracted p53-induced apoptosis upon cisplatin treatment. Also, likely via ERK1/2 activation, NO favored phosphorylation of MiTF, which is associated with survival signals. Furthermore, NO displayed the remarkable ability to overcome the effect of U0126, a MEK inhibitor, and promoted continuous phosphorylation of ERK1/2 (and hence cell survival), in contrast to cells not exposed to NO. Results also demonstrated that cisplatin-induced apoptosis was substantially decreased by the antioxidant precursor N-acetylcysteine (NAC). Unlike exogenous NO, cisplatin-induced ROS were linked to lower activation of ERK1/2, which was reversed by NAC. During the treatment with cisplatin, NAC led to lower levels of p53, which may have partially contributed to increased cell survival. However, in contrast to NO, NAC did not significantly alter the effects of cisplatin upon MiTF and apoptotic proteins studied. Altogether, our findings illustrate the complexity of the regulation of signaling components by oxidative species of distinct natures. / by Chase Thaddeus Maceo Anderson. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The stability of the genome is constantly challenged by both endogenous and exogenous DNA damaging agents. DNA damage, if left unrepaired, can give rise to permanent genetic alterations that ultimately increase our risk of cancer and other diseases. To combat these threats, eukaryotic cells activate a DNA damage response (DDR) that coordinates a wide variety of cellular processes including cell cycle progression, DNA repair, or in the case of severe/irreparable damage, apoptotic cell death. In addition to its role in cancer prevention, the DDR is fundamental in cancer treatment as noted by the numerous DNA damage-based chemotherapies. Thus, understanding how cell fate is determined by consequences of DDR is important for basic biological science and for medical applications. Alkylating agents comprise a major class of DNA damaging chemotherapeutics. The 0⁶MeG DNA lesion is a highly mutagenic, carcinogenic, and cytotoxic lesion produced by SNl methylating agents. Additionally, persistent 0⁶MeG lesions induce apoptosis in an 0⁶MeG DNA methyltransferase (MGMT) repair- and mismatch repair (MMR)-dependent manner. Here, we examine the DNA damage response induced by the 0⁶MeG lesion at both the molecular and cellular level after treatment with the SN1 methylating agent N-methyl-N'-nitro-Nnitrosoguanidine (MNNG). A systems-level approach combining various experimental techniques was used to quantitatively monitor the temporal regulation of DDR network proteins and, in parallel, phenotypic responses (cell cycle arrest, DNA replication, and apoptosis) induced by 0⁶MeG lesions. Through this approach, we have shown that TK6 human lymphoblastoid cells undergo cell cycle delay through both the first and second cell cycle post treatment. Furthermore, we demonstrate that 0⁶MeG triggers an intra-S-phase arrest in the second S-phase that ultimately leads to cell cycle progression and survival in cells with low/repairable amounts of damage or apoptosis in cells with high/irreparable amounts of damage. Based on the signaling and phenotypic data acquired, we developed a conceptual model for MMR's role in triggering cell cycle arrest and cell death. In addition, exploration of the global transcriptional response provided an unbiased approach to further elucidate previously unrecognized biological processes involved in the response to MNNG-induced damage. Taken together, our results have enhanced our understanding of the cellular response to alkylation-induced damage and will contribute to the development of personalized chemotherapeutic treatments in the future. / by Ericka Marie Noonan. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 217-232). / The critical first step in the host infection by influenza A virus is the binding of the viral surface glycoprotein hemagglutinin (HA) to the sialylated glycan receptors terminated by N-acetyineuraminic acid (Neu5Ac) expressed on the host cell surface. Glycans terminating in Neu5Ac that is a2-6 and a2-3 linked to the penultimate galactose serve as receptors for human- and avian- adapted influenza A virus respectively. This thesis focuses on studying HA, glycan receptors and their interactions both in a biochemical and physiological context to understand the role of these interactions in influenza A virus pathogenesis. The first Specific Aim of this thesis deals with understanding the molecular determinants of glycan receptor-binding specificity and affinity of HA (or avidity in the context of the whole virus) and how these properties govern antigenic drift and efficiency of airborne transmission. This approach contributed to uncovering the relationship between receptor-binding affinity and efficiency of transmission of the 2009 H1N1 pandemic influenza A virus and also predicting the evolution of this virus into a more transmissible strain. The second Specific Aim of this thesis focuses on understanding the distribution of the glycan receptors for human-adapted HA (going beyond a2-3/aX2-6 linkages), in ferret (animal model for influenza research) and in human respiratory tracts. Based on this understanding, this part of the thesis contributed to developing new anti-viral strategies based on targeting the host glycan receptors (instead of the common strategies that directly target the viral proteins) Overall, this thesis has provided functional insights into the role of HA-glycan interaction in viral pathogenesis. As part of this research, various tools and methods were developed. Further, such an approach paves way for elucidating the functional significance of important protein-glycan interactions in other disease models. / by Akila Jayaraman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references. / Due to its common dysregulation in epithelial-based cancers and the extensive characterization of its role in tumor growth, epidermal growth factor receptor (EGFR) has long been an attractive target for monoclonal antibodies. Intense research has culminated in the approval of two antibody-based drugs against EGFR for cancer treatment, with numerous others in clinical trials. However, therapeutic efficacy of these drugs has been disappointingly low due to autocrine signaling, receptor mutation, and transport limitations, necessitating novel antibody designs and mechanisms of action. Recently, it was reported that treatment with combinations of antibodies can induce receptor clustering, leading to synergistic receptor downregulation and anti-tumor activity. The aim of this thesis is to elucidate the details of this phenomenon and to exploit this mechanism to design more effective therapeutic antibodies targeting EGFR. We first illuminate several key aspects of combination antibody-induced clustering. By screening a panel of pairwise combinations, we show that the most potently downregulating pairs consist of two non-competitive antibodies that target EGFR extracellular domain 3. We further find the mechanism underlying downregulation to be consistent with recycling inhibition. Lastly, in contrast to the agonism associated with ligand-induced downregulation, we demonstrate that combination mAb-induced downregulation does not activate EGFR or its downstream effectors and it leads to synergistic reduction in migration and proliferation of cells that secrete autocrine ligand. To enhance antibody binding and induced receptor clustering, we design multispecific antibodybased constructs that engage up to four distinct epitopes on EGFR. We engineer two classes of constructs: one consisting of a full EGFR-specific antibody fused to the variable domain of a second anti-EGFR antibody and the other consisting of a full EGFR-specific antibody fused to one or more EGFR-targeted tenth type three domains of human fibronectin. Both classes of constructs induce robust receptor clustering and downregulation in the absence of signal activation. In vitro downregulation correlates well with in vivo inhibition of tumor growth in several mouse xenograft tumor models and mutational analysis demonstrates that the efficacy of our fusions is attributable to both signaling effects and antibody-dependent cell-mediated cytotoxicity. Our multi-epitopic strategy may be readily applied to other receptor systems to form the basis for a new category of antibody-based therapeutics. / by Jamie Berta Spangler. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 197-212). / Regulated cell-surface proteolysis underpins key processes of cellular growth and motility in both physiological and pathological contexts. However, comprehending how multiple proteolytic events cohesively integrate to yield context-dependent cellular behavior remains a challenge in the fields of both protease biology and systems biology in general. This work begins to address that challenge by quantitatively investigating the integrated effect of multiple diverse proteolytic events and their interaction with cell-signaling pathways from a computational network perspective, particularly focusing on A Disintegrin and Metalloproteinases (ADAMs). ADAMs have been studied for decades as the principal cell-surface "sheddases" responsible for cleaving growth factor ligands and receptor tyrosine kinase ectodomains from the cell surface. However, activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent sheddase function, and clinical trials targeting metalloproteinases in cancer have failed in part due to a poor understanding of the complex functions they mediate. This thesis outlines a conceptual framework for studying protease network biology (Chapter 1), describes novel experimental methods designed for such a framework (Chapters 2-3), and applies both to understand protease regulation in invasive disease (Chapter 4). Using combined measurement and computational modeling, we present a paradigm for monitoring and analyzing complex networks of protease activities that interface with signaling pathways to influence cellular migration in the invasive diseases of cancer and endometriosis. We find sheddase activity integrates with signaling pathways to direct cell migration, especially through concomitant proteolysis of both ligands and receptors. We find that indirect reduction of sheddase activity through kinase inhibition can lead to an accumulation of growth-factor receptors on the cell surface, consequently producing undesired compensatory signaling feedback. Thus, here we present a novel mechanism of rapid, protease-driven resistance to kinase inhibitors, and we subsequently demonstrate strategies for overcoming resistance through drug combinations. We develop a novel microfluidic platform to study protease activities in clinical samples, and apply the technology to study the peritoneal fluid from endometriosis patients. Results indicate joint dysregulation of sheddase activity with disease. Overall, this work provides a model for measuring, understanding, and targeting networks of proteases and the kinases with which they interact. / by Miles Aaron Miller. / Ph.D.