This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / The epitranscriptome -- the naturally occurring system of chemical modifications on ribonucleic acid (RNA) -- / is an emerging frontier of research into how changes in the cellular environment are coupled with global rates of protein synthesis. Here we report the development of new analytical and computational approaches to study mechanisms of epitranscriptomic regulation and function in the context of (1) phenotypic antibiotic resistance in bacteria, and (2) proteostasis in eukaryotes. While at least 11 major classes of RNA have been identified to date, this work focuses on transfer RNA (tRNA), the most diversely modified species of RNA that plays a central role in the initiation, elongation and termination of translation. To provide context for investigating the epitranscriptomic regulation of microbial adaptation, we first use multivariate statistical modelling to integrate time-resolved, systems-level analyses of mycobacterial persistence using an in vitro model of tuberculosis infection. / Combining biochemical characterization of cellular pH and redox state, metabolic phenotyping, time-course metabolomics, whole-genome transcriptomics, and quantitative proteomics, we demonstrate that starved Mycobacterium bovis BCG (BCG) adapts to starvation by entering a ketotic state that results from coordinated metabolic shifts towards lipolysis and fatty acid [beta]-oxidation. We also show that management of toxic ketone body intermediates appears to be mediated by cytochrome P450 (CYP)-linked ketolysis and carbon cycling through CO₂ fixation, as evidenced by elevated endogenous reactive oxygen species production during starvation and the sensitivity of starved persisters to well-known CYP poisons. Using this model of mycobacterial pathogenesis, we next describe how BCG responds to nutrient deprivation by reprogramming the tRNA epitranscriptome to mediate selective translation of codon-biased stress response genes. / We discuss how insights from preliminary experiments with a new in-house method, Absolute QUAntification RNA-Seq (AQUA RNA-Seq), will deepen our mechanistic understanding of this alternative genetic code, and also describe a strategy for chemotherapeutic intervention to reverse phenotypic drug resistance. Finally, we detail the development of a new high-throughput platform to identify and quantify the role of the epitranscriptome in translational fidelity in Saccharomyces cerevisiae. Our results indicate that loss of certain tRNA-modifying enzymes induces the aggregation of stress response proteins with amino acid misincorporations that map to specific codon sites. / The research conducted under this thesis (1) advances our fundamental understanding of how genes are regulated at the level of translation, (2) establishes the role of the epitranscriptome in regulating cellular adaptation to physiological stringency, and (3) provides mechanistic insights into how the epitranscriptome can be engineered for the development of new RNA-targeted medicines. / by Nick K. Davis. / Sc. D. / Sc.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: M. Eng., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages. 85-97). / Tau neurofibrillary tangles or aggregates are a common neuropathological feature found in a number of neurodegenerative conditions, including Alzheimer's disease. Understanding the kinetics of this aggregate build up, how it varies across patients, and how aggregation might be influenced by intracellular pathways is critical for both a deeper knowledge of these disorders as well as identification of potential therapeutic targets. To this end, I employed an in vitro tau aggregation assay to study the kinetics of tau aggregation as it relates to aggregates in sporadic Alzheimer's disease. I found that the formation of aggregates was a logistic process, with a lag phase, an exponential rise phase, and a plateau phase. Aggregation kinetics varied significantly between different cases of sporadic Alzheimer's disease, paralleling the heterogeneity that is observed in the clinical presentation of Alzheimer's disease. Likewise, I found that inhibition of intracellular pathways of macroautophagy and endosomal microautopahgy heterogeneously increased tau aggregation and changed tau aggregation kinetics, dependent upon the case of Alzheimer's disease. These results inform that tau aggregates vary significantly not just between disorders, but even within disorders, and that protein degradation pathways uniquely process aggregates, perhaps potentiated by further molecular differences in aggregate structure or composition. / by Tarun Kamath. / M. Eng. / M.Eng. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from PDF version of thesis. / Includes bibliographical references. / T cells occupy essential roles throughout the immune system to prevent and limit disease. As such, breakdowns in their function and recognition underlie poor clinical outcomes across diverse maladies including pathogen infection, cancer, autoimmunity, allergies, and transplant rejection. Yet, when properly directed, T cells drive potent protective and therapeutic responses in prophylactic vaccinations and novel immunotherapies. Therefore, understanding and harnessing T cell function and recognition is of great importance to improving patient care and addressing currently unmet clinical needs. The function and recognition of T cells are driven through their T cell receptors (TCRs), which bind with great specificity to peptide-MHCs (pMHCs), Major Histocompatibility Complex proteins displaying tissue- and disease-specific peptide antigens derived from their host cell or its surroundings. / However, to specifically and comprehensively present and surveil antigens across highly divergent maladies, extreme diversity is required of both the population-level TCR and pMHC repertoires. However, this same diversity which drives T cell function also confounds generalized understanding of these repertoires, as well as their recognition. Therefore, there has been considerable recent interest in the development and application of tools to comprehensively define, predict, and screen these repertoires and their recognition at high throughput. In this thesis, I both utilize and build upon these tools to define TCR and pMHC repertoires and explore their recognition, particularly with yeast-displayed pMHC libraries for CD4⁺ T cell recognition of class II pMHCs, and especially in the context of cancer. / Using these technologies, I empirically define pMHC repertoires, explore the antigenic basis of TCR repertoire convergence in a preclinical tumor model, and explore the antigen reactivity of human T cells with clinical relevance. While these results provide detailed insights into the specific TCRs and pMHCs studied, they also provide guidance for future avenues in the exploration of TCR and pMHC repertoires and their recognition. / by Charles Garrett Rappazzo. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 107-112). / Heterogeneity of the immune system has increasingly necessitated the use of high-resolution techniques, including flow cytometry, RNA-seq, and mass spectrometry, to decipher the immune underpinnings of various diseases such as cancer and autoimmune disorders. In recent years, high-throughput single-cell RNA sequencing (scRNA-seq) has gained popularity among immunologists due to its ability to effectively characterize thousands of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Incorporation of TCR sequencing into scRNA-seq data could identify cells with shared antigen-recognition, further elucidating dynamics of antigen-specific immune responses in T cells. / In the first part of this thesis work, we develop a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 32 barcoded scRNA-seq samples. This approach is compatible with common 32 scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with clonal T cells from murine and human samples. In both cases, we observed preferential phenotypes among subsets of expanded T cell clones, including cytotoxic T cell states associated with immunization against viral peptides. In the second part of the thesis, we apply the strategy to a 12-patient study of peanut food allergy to characterize T helper cell responses to oral immunotherapy (OIT). We identified clonal T cells associated with distinct subsets of T helper cells, including Teff, Treg, and Tfh, as well as Th1, Th2, and Th17 signatures. / We found that though the TCR repertoires of the patients were remarkably stable, regardless of their clinical outcomes, Th1 and Th2 clonotypes were phenotypically suppressed while Tfh clonotypes were not affected by therapy. Furthermore, we observed that highly activated clones were less likely to be suppressed by OIT than less activated clones. Our work represents one of the most detailed transcriptomic profiles of T helper cells in food allergy. In the last part of the thesis, we leverage the simplicity and adaptability of the method to recover TCR sequences from previously processed scRNA-seq samples derived from HIV patients and a nonhuman primate model of TB. In the HIV study, we recovered expanded clonotypes associated with activated T cells from longitudinal samples from patients with acute HIV infections. In the TB study, we modified the primers used in the method to T cells from TB granulomas of cynomolgus macaques. / We identified not only expanded clonotypes associated with cytotoxic functions, but also clonotypes shared by clusters of activated T cells. In total, these results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology. / by Ang A. Tu. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 253-268). / The ability to fabricate silica materials with highly organized nanostructures is of increasing demand across the medical, optical, energy, and mechanical fields. Diatoms, a class of eukaryotic algae, produce intricately-patterned silica structures under ambient conditions through a process initiated by post-translationally modified silaffin peptides that nucleate silicic acid. Designing these peptides would enable the production of silica nanostructures with desired properties; however, the functional effects of the modifications are poorly understood. In this thesis, I use Escherichia coli to express and modify recombinant silaffin R5 peptide from the diatom Cylindrotheca fusiformis. A library of 38 enzymes is tested for R5 modifications in vitro, from which active methyltransferases, kinases, acetyltransferases, oxidases, and myristoyltransferases are identified from diatoms, humans, yeast, and bacteria. Modified R5 peptides are used for silica precipitation and the impacts on particle size, shape, porosity, and surface area are quantified. I then used these individually characterized modifications to build synthetic enzyme pathways in vitro and in vivo, and demonstrate that introducing multiple modifications to R5 has additive effects on silica morphology. In the second part of this thesis, I apply the R5 peptide to synthesize silica coated core-shell nanoparticles for a range of core materials (Fe₃O₄ TiO₂, ZnO, HfO₂, and Ta₂O₅), and show that silica shell thickness can be tuned (2.3 - 120 nm) by altering the concentration of R5 used in the reaction. Together, these projects illustrate a design-driven approach for rapidly engineering and synthesizing silica nanostructures and multifunctional composite nanomaterials under ambient conditions, with potential applications in biomedicine, electronics, and photonics. / by Andrea Kimi Wallace. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

From the bacterium Streptomyces sp. SCC-2136 (ATCC 55186), two angucycline natural products are produced, designated Sch 47554 and Sch 47555. These compounds are produced through a type II polyketide biosynthetic pathway. The early biosynthetic steps to these molecules were confirmed. These include the minimal polyketide synthase (PKS), the C-9 keteoreductase, the first-ring aromatase, the subsequent ring cyclase, and two oxygenases. Also confirmed were the biosynthetic genes responsible for production of the first amicetose moiety, as well as the glycosyltransferase that creates a C-glycosidic bond between the angucyclic scaffold and the amicetose moiety, In confirming these patways, two new natural products were produced: GG31, an amitosylated rabelmycin, and GG53, rabelmycin hydroxylated at C-12b. Future work will be to understand the late biosynthetic steps and generate new angucyclines through combinatorial biosynthesis.

Roughly the same volume of water that rushes over the Niagara Falls is produced as wastewater in North America. This wastewater is treated through a variety of means to ensure that it can be safely returned to the natural ecosystem. This thesis examines two novel means for this treatment, one biological and one physical-chemical in nature, namely, Rotating Algae Biofilm Reactor treatment and expanded shale augmented coagulation-flocculation. Rotating algae biofilm reactors (RABRs) support biofilm algae growth, and in turn, the algae take up harmful contaminants from the wastewater. This system was tested in wastewater from petroleum refining operations. The efficacy of the RABR system was compared with a traditional method of wastewater treatment, open pond lagoons, where wastewater is open to sunlight and algae growth occurs in suspension as compared to the biofilm formed by the RABR system. The RABR treatment demonstrated a statistically significant increase in removal of three constituents in wastewater that are harmful to the environment: nitrogen, phosphorus, and suspended solids. Additionally, the RABR treatment demonstrated increased biomass production. This biomass can be converted into a variety of bioproducts including biofuels, agricultural feed, and nutraceuticals. This study is the first demonstration of this system in petroleum refining wastewater. Currently, many wastewater treatment facilities use coagulation-flocculation to remove suspended solids from the wastewater. To achieve this removal, coagulants are added to the wastewater, which removes surface charges of the suspended particles, allowing particles in solution to coalesce and settle by gravity out of solution. One common coagulant added to wastewater is ferric sulfate. This study demonstrated that the addition of a new compound, expanded shale, to ferric sulfate could greatly improve the efficacy of the existing ferric sulfate coagulation system.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 108-115). / The inability of articular cartilage to heal after damage or disease has motivated investigation of novel cartilage tissue engineering technologies. The objective of this thesis was to advance the use of self-assembling peptide hydrogel scaffolds for cartilage repair by encapsulating bone-marrow-stromal cells (BMSCs) and incorporating chondrogenic cues to stimulate differentiation and neotissue production. To test the hypothesis that self-assembling peptide hydrogels provide cues which enhance the chondrogenic differentiation of BMSCs, a technique for rapid, high-viability BMSC encapsulation was developed. BMSCs were cultured in two peptide hydrogel sequences and compared to agarose hydrogels. BMSCs in all three hydrogels underwent TGF-3 1-mediated chondrogenesis as demonstrated by comparable gene expression and ECM biosynthesis. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher sulfated-glycosaminoglycan content and more spatially uniform proteoglycan and type II collagen deposition. These data showed that self-assembling peptide hydrogels enhance chondrogenesis compared to agarose. To evaluate the capacity for BMSCs from young and adult equine donors to produce cartilage-like ECM, neotissue formation was compared to that for animal-matched primary chondrocytes. Young chondrocytes stimulated by TGF-PlI accumulated ECM with higher sulfated-glycosaminoglycan content than adult chondrocytes and BMSCs of either age. BMSCs produced neotissue with higher dynamic stiffness than young chondrocytes. Measurement of aggrecan core-protein and chondroitin-sulfate length by atomic-force microscopy revealed BMSCs produce longer core protein and chondroitin-sulfate, and fewer catabolic-cleavage products than chondrocytes. Therefore, BMSC-produced aggrecan appears to have a younger phenotype than chondrocyte-produced aggrecan. These advantages make BMSCs a potentially superior cell source for peptide-hydrogel-based cartilage repair. To deliver TGF-pl to BMSCs via a bioactive scaffold, BMSCs were encapsulated in peptide hydrogels with both tethered and adsorbed TGF-p1 and cultured in TGF-p 1-free medium. Chondrogenesis was compared to that of unmodified peptide hydrogels with medium-delivered TGF-p1. Adsorbed-TGF-plI peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like ECM accumulation, while tethered TGF-p1 was not different from TGF-pl -free controls. TGF-p1 adsorbed to self-assembling peptide hydrogels can stimulate BMSC chondrogenesis. BMSC-seeded self-assembling peptide hydrogels, modified for controlled delivery of pro-chondrogenic factors, generate cartilage-like neotissue and are compatible with a single-surgery, autologous therapy for cartilage repair. / by Paul Wayne Kopesky. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 127-140). / Checkpoint blockade with antibodies against CTLA-4 or PD-1 has demonstrated that an endogenous adaptive immune response can be stimulated to elicit durable tumor regressions in metastatic cancer, but these dramatic responses are confined to a minority of patients¹-³. This outcome is likely due in part to the complex network of immunosuppressive pathways present in advanced tumors, which necessitates the development of novel therapeutics and combination immunotherapies to generate a counter-directed network of pro-immunity signals⁴-⁸. In Chapters 2 and 3 of this thesis, we describe methods for enhancing T cell priming against tumor antigens via covalent modification of molecular vaccines to enhance lymphatic drainage, serum stability, or cytosolic access to improve presentation on MHC class I. In Chapter 4, we demonstrate a combination immunotherapy that recruits a diverse set of innate and adaptive effector cells, enabling robust elimination of large tumor burdens that to my knowledge have not previously been curable by treatments relying on endogenous immunity. Maximal anti-tumor efficacy required four components: a tumor antigen targeting antibody, an extended half-life IL-2⁹, anti-ƯPD-1, and a powerful T-cell vaccine¹⁰. This combination elicited durable cures in a majority of animals, formed immunological memory in multiple transplanted tumor models, and induced sustained tumor regression in an autochthonous BRraf[superscript V600E]/Pten[superscript -/-] melanoma model. Finally, in Chapter 5, we show preliminary data on combination immunotherapies used to treat antigenically heterogeneous tumors. Taken together, these data define design criteria for enhancing the immunogenicity of molecular vaccines and elucidate essential characteristics of combination immunotherapies capable of curing a majority of tumors in experimental settings typically viewed as intractable. / "During my doctorate by the John and Fanny Hertz Foundation Fellowship (specifically the Wilson Talley Hertz Fellowship), the NSF Graduate Research Fellowship Program, and the Siebel Scholarship"--Page 141. "This thesis work was supported in part by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute, the US National Institutes of Health (NIH) grant CA174795, the Bridge Project partnership between the Koch Institute for Integrative Cancer Research and the Dana Farber-Harvard Cancer Center (DF-HCC), the V Foundation, the Ragon Institute, and the Howard Hughes Medical Institute"--Page 141. / by Kelly D. Moynihan. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references. / Molecular design of novel pharmaceutical agents depends on a mechanistic paradigm. A library of compounds was designed to mimic some features of cisplatin that are believed to be in part responsible for its success as a chemotherapeutic agent against testicular cancer. The new compounds are designed to utilize proteins such as steroid receptors that are abnormally expressed in cancers to enhance toxicity and specificity. The synthetic phase of this project successfully produced a compound, 11P, that when tested against cancer cell lines demonstrated remarkable potency, including the induction of apoptosis in normally apoptosis-resistant prostate cancer lines. This drug candidate, comprising an aniline mustard tethered to a steroid moiety, is intended to form covalent adducts with DNA that, through association with the androgen receptor over-expressed in some prostate cancers, are likely to be shielded from repair, generating lethal crosslinks. Moreover, the titration of androgen receptor away from its normal function as a transcription factor may further inhibit tumor growth. While the mechanistic features of I1P are still under investigation, the potent anticancer activity warrants consideration of its possible clinical use, specifically against hormone-refractory, metastatic prostate cancer, for which current therapeutic options are extremely limited. One important step toward that goal involves characterizing the pharmacokinetics of the compound, including: a) understanding the rate of hydrolytic decomposition, b) evaluating the metabolic conversion by intracellular enzymes to active or inactive derivatives, and c) exploring the potential for contraindications between this drug candidate and other medications a prostate cancer patient may be taking concurrently. Herein are described the experiments that elucidate the pharmacokinetics of 1I1, showing that the drug candidate is a) subject to hydrolysis at rates that depend on the presence of plasma protein; b) extensively metabolized by the cytochromes P450 to products demonstrating reduced capacity for forming covalent adducts with DNA; and c) a potent inhibitor of the metabolism of known substrates of the 2D6 and 3A isoforms of cytochrome P450. / by Charles Ingalls Morton, IV. / Ph.D.