Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / The human gastrointestinal tract is home to a dense and dynamic microbial community. The composition and metabolic output of the human gut microbiota have been implicated in many diseases: from inflammatory bowel disease, colorectal cancer, and diarrheal diseases to metabolic syndromes like diabetes. Treatment of these diseases will likely require targeted therapeutic interventions aimed at modulating the abundance and metabolism of specific commensal microbial species or probiotics. A promising avenue for such interventions is through diet, where the dietary components act as substrates for the species producing beneficial metabolites one wishes to enrich. In this thesis, I focus on a dietary intervention study in healthy individuals. Since the human gut microbiota is known for its highly heterogeneous composition across different individuals, it comes as no surprise that a more personalized approach is preeminent. / We first test effects of multiple micronutrients spiked into a fixed diet. Using a highly controlled diet within the cohort, we identify strong and predictable responses of specific microbes across participants consuming prebiotic spike-ins. However, select macronutrient spike-ins like unsaturated or saturated fat and protein, produce no predictable response. We next investigate prebiotic supplement in diet further as well as its downstream products, short chain fatty acids, in the digestive tract. We look to alleviate the stress of a highly controlled, low complexity diet on participants by testing the effect of different prebiotics simultaneously ex vivo. We show that individuals vary in their microbial metabolic phenotypes (as in they produce different quantities and proportions of short chain fatty acids from the same prebiotic inputs) mirroring differences in their microbiota composition. / Finally, we run a pilot study to elucidate how closely our ex vivo experiment results may reflect the in vivo changes following a short-term dietary fiber supplementation. In addition to obtaining preliminary data on this direct comparison, we also explore different parameters for generating high-throughput data on personalized dietary interventions. Together, these projects provide the framework for building a predicative model for the effect that prebiotic dietary supplementation will have on gut microbiota's composition. Such a prediction model would be equally helpful in both enhancing individuals' gut health and improving gut dysbiosis in cases of disease. / by Le Thanh Tu Nguyen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 147-173). / While organ transplantation is one of the greatest advances of modern medicine and provides immense therapeutic benefit to patients suffering from severe and fatal liver disease, donor tissue is scarce. Alternatives such as engineered cell-based therapies aim to restore tissue-specific functions of solid organs, but leave much to be desired. Key challenges hindering the translation of cell-based therapies relate to (1) cell sourcing, (2) graft scale-up, and (3) vascularization, all of which contribute to therapeutic performance. The performance of an implantable graft is a function of the underlying cell-cell and cell-matrix interactions. These grafts typically consist of a multicellular ensemble in which combinations of epithelial, stromal, and immune cells give rise to physiologic function. Currently, precise, spatiotemporal control of these interactions is experimentally intractable. / This thesis introduces a technique termed CAMEO (C̲ontrolled A̲poptosis in M̲ulticellular tissues for E̲ngineered O̲rganogenesis), in which we can non-invasively actuate the removal of a desired cell population from a pre-established multicellular ensemble. As an exemplar, we use CAMEO to study the contribution of supportive stromal cells to the phenotypic stability of primary human hepatocytes. 3D hepatic ensembles, in which stromal cells enhance phenotypic stability of spheroids, were found to rely only transiently on fibroblast interaction to support multiple axes of liver function, such as protein secretion and drug detoxification. Importantly, CAMEO revealed crucial cell-cell and cell-material interactions that occur in the first 24 hours of co-culture that drive the stabilization and enhancement of hepatic phenotype. / Due to its modularity, we expect that CAMEO is extendable to other applications that are tied to the complexity of 3D tissues, including in vitro organoid models and in vivo integration of cell therapies. As such, we also employed CAMEO and our strategy of engineering-via-elimination in an implantable device containing both hepatic ensembles and engineered vasculature, and demonstrate our ability to engineer desired function and cell composition. With an improved understanding of cell-cell interactions in vitro in hand, the next step toward the clinic is to assess the performance of 3D hepatic ensembles in vivo. Here, we lay the groundwork for defining a final product lock for our hepatic cell therapies, and specifically explore the role of fibroblasts in in vivo integration, incorporate vasculature to meet the metabolic demands of scaled-up tissue grafts, and tune tissue microarchitecture to enhance engraftment, function, and persistence in vivo. / Taken together, the efforts contained in this thesis represent a significant advance in tools and biology that enable clinical applications of tissue engineering and regenerative medicine. / by Amanda X. Chen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. "February 2021." / Includes bibliographical references (pages 153-168). / Ovarian cancer is the fifth leading cause of cancer death for women in the United States, with only modest improvements in patient survival in the past few decades. Standard-of-care consists of surgical debulking followed by a combination of platinum and taxane agents, but relapse and resistance frequently occur. To identify genes that confer sensitivity or resistance in tumor cells treated with platinum chemotherapeutics, I performed genome-wide screens combining cisplatin or oxaliplatin with CRISPRi pooled gene knockdowns. Screens were analyzed at 9-days to mimic patient care, and at 48-hours to isolate the short-term DNA damage response. Genes whose knockdown caused sensitivity to the platinum chemotherapeutics were identified through a multi-objective optimization approach to account for knockdown efficiencies and variances in sequencing depth. / To filter the noise in the genome-wide screen and more confidently identify 'hits,' a smaller pooled CRISPRi screen of four hundred targets was designed, and a few 'hits' were validated. Interestingly, knockdown of FAAP24, a component of the FA core complex, was found to sensitize multiple ovarian cancer cells to platinum compounds, and thus may be a promising candidate for a combination treatment with oxaliplatin and cisplatin. Chapter 5 details an implementation of a combination therapy with cisplatin using peptide nanoparticles. Peptide nanoparticles are a promising therapeutic for the delivery of siRNA and allow for targeting of specific proteins that are difficult to inhibit with small molecular inhibitors; specifically, nanoplexes allowed for the targeting of the REV3 protein, the catalytic component of the translesion synthesis polymerase. / Interfering with REV3 expression through siRNA has a synergistic effect with cisplatin treatment in both human and mouse models of lung cancer, indicating that REV3 is an excellent target to combine with cisplatin therapies. This REV3 knock-down sensitivity was also extended to human ovarian cancer cell lines, indicating the potential of the combination treatment for both lung and ovarian cancers. / by Erika Daphne Handly. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 77-89). / Antibiotics, such as beta-lactams, are essential medical tools for the treatment of bacterial infections. Unfortunately, clinical treatment efficacy is declining over time as bacteria adapt to and evade antibiotic treatment through mechanisms called antibiotic resistance, tolerance, and persistence. Antibiotic tolerance and persistence, in particular, are often context-dependent phenotypes: environmental factors can influence bacterial physiology and alter antibiotic efficacy. Optimal antibiotic use, as well as strategies to enhance antibiotic efficacy, can therefore be informed by studies of context-dependent antibiotic action. In this thesis, I present three vignettes about beta-lactam antibiotic efficacy and how environmental context alters in vitro treatment outcomes. First, I explore bacterial killing in multi-drug contexts, focusing on how different beta-lactams can have different effects in combination with antibiotics of other classes. Second, I present a new counter-tolerance method using metabolic stimulation to sensitize tolerant, stationary phase bacteria to beta-lactam antibiotics. Third, I present an extension of this metabolic counter-tolerance strategy, now combining metabolic and target-specific stimulation to further enhance beta-lactam efficacy. I demonstrate that this combined approach, when coupled with beta-lactamase inhibitors, restores beta-lactam sensitivity to simultaneously tolerant and resistant cultures of clinically relevant pathogens. I conclude by discussing opportunities for future study into antibiotic context-dependence and the application of counter-tolerance approaches such as the one described in this thesis. / by Sarah Christine Bening. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. "February 2021." / Includes bibliographical references (pages 95-106). / Living organisms universally encode amino acids with three-base codons specifying the twenty canonical amino acids. A genetic code entirely based on four-base codons would answer many questions about the origin of life and have profound implications for expanding the genetic code to include novel amino acids. However, the task of assembling enough quadruplet-tRNAs (qtRNAs) to implement an all-quadruplet code remains a major hurdle. Here, we create qtRNAs that decode canonical amino acids by modifying E. coli tRNAs that continue to rely upon endogenous aminoacyl-tRNA synthetases (AARSs) for charging. We find that AARSs generally tolerate quadruplet anticodons, resulting in efficient, selectively charged qtRNAs for eight of the twenty canonical amino acids, as well as candidate qtRNAs for the remaining 12 amino acids. We develop a directed evolution technique based on Phage Assisted Continuous Evolution and use it to improve the translation efficiency of qtRNAs. In order to address the large number of necessary evolutions, we execute these evolutions using a high-throughput evolution platform we developed. We find that directed evolution of qtRNAs can substantially improve quadruplet codon translation efficiency, often by 10x or more, without compromising amino acid selectivity. We use the evolved qtRNAs to implement an 10-amino acid all quadruplet codon code and processive quadruplet codon translation of a small peptide within a standard bacterial chassis, without the need for genome recoding. / by Erika Alden DeBenedictis. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 87-104). / Biological systems depend on numerous molecular messengers that transduce information across large distances. Understanding the spatial and temporal dynamics of molecular signaling networks is crucial for the construction of systems- and organism-level models of biological function. Molecular imaging, a technique that employs chemical probes to relay molecular events into spatially-resolved signal changes, is a promising strategy for studying complex molecular signaling networks in situ. Magnetic resonance imaging (MRI) is a leading noninvasive imaging modality that allows for imaging of large volumes of deep tissue with high spatiotemporal resolution. Paramagnetic molecular sensors enable detection of molecular phenomena with MRI (molecular MRI). The scope of molecular MRI experiments thus far, however, has been limited by the modest sensitivity and signal changes provided by existing probes. / In this dissertation, I introduce Ḻiposomal Ṉanoparticle Ṟeporters (LisNRs), a novel class of MRI-detectible sensor that utilizes an innovative contrast mechanism in which reversible modulation of the water permeability of liposomal bilayers simultaneously modulates water access to a large, concentrated pool of conventional T1-weighted MRI contrast agents. This architecture gives rise to significant signal amplification with respect to first-generation MRI probes that rely on stoichiometric sensing mechanisms in which binding of one analyte molecule modulates water access to a single paramagnetic metal ion. I employ two strategies for the signal-dependent modulation of liposomal water permeability. The first approach uses reversible modulation of lipid bilayer fluidity to induce changes in passive bilayer water permeability. To demonstrate this concept, I build Light- LisNR, a photosensitive MRI contrast agent, which I use to map light distribution in the rat brain. / The second approach utilizes ligand-gated water-permeable channels to modulate bilayer water permeability. I demonstrate the potential of this strategy for molecular sensing using biotin/streptavidin as a model system. Together, this work introduces and demonstrates a novel platform for sensing with MRI that addresses longstanding challenges of low sensitivity and signal change with existing MRI-detectible probes. / by Jacob Cyert Simon. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 90-95). / Bacteria and humans have a long-standing symbiotic relationship. As the details of their symbiosis continue to be elucidated, it has become clear that these host-resident bacteria are much more than spectators within the body. The ability of bacteria to manipulate human biology has inspired the notion that bacteria can be harnessed as "probiotics" to combat disease; in other words, as living therapeutics. Synthetic biology takes this concept one step further through genetically introducing novel functions into bacteria to provoke a targeted therapeutic effect in humans. However, none of the engineered living therapeutics that have progressed into clinical trials have been approved for therapeutic use. I pursued two approaches in an effort to reverse this trend. In my first approach, I developed a platform to overcome practical challenges of therapeutic strain design. This platform enables high protein expression levels from the genome of E. coli Nissle through the development of a genetic "landing pad" system and characterization of genetic regulators that can be controlled through the addition of chemical inducers. In my second approach, I developed a method for screening a diverse panel of bacteria for their ability to receive and express biosynthetic gene clusters encoding for antimicrobial peptides. After identifying bacteria that were capable of expressing these peptides, I explored their potential to prevent infection of the opportunistic pathogen Clostridium difficile and to serve as a bioproduction chassis of the C. difficile-targeting peptide. Together, this work outlines the development of a platform for creating the next-generation of living therapeutics and a unique method for engineering collections of bacteria to identify new chassis strains for heterologous protein expression. / by Alexander John Triassi. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages [137]-[153]). / Alzheimer's disease (AD) is a form of dementia characterized by the appearance of amyloid-[beta] plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Despite numerous clinical trials, a limited understanding of disease pathogenesis has prevented the development of effective therapies. Several lines of genetic and biomolecular evidence indicate that AD progression involves cellular signaling through neuronal and glial protein phosphorylation networks. In order to understand which phosphorylation networks are dysregulated, I use mass spectrometry to characterize the phosphoproteome of post-mortem brain tissue from AD patients and multiple mouse models of AD. Using computational analysis, I identified several signaling pathways that are dysregulated before neurodegeneration occurs. Many of these signaling factors were expressed primarily in non-neuronal cell types, including microglia, astrocytes, and oligodendrocytes. / My results highlight potential therapeutic targets in the signaling responses of glial cells and are split into two parts. In the first part of this thesis, I have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. I identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also found to be upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFN[gamma]) in BV-2 cell line and human stem-cell derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. / Collectively, my results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration. In the second part of this thesis, I performed a combined analysis of the tyrosine, serine, and threonine phosphoproteome, and proteome of temporal cortex tissue from AD patients and aged matched controls. I identified several co-correlated peptide modules that were associated with varying levels of Tau, oligodendrocyte, astrocyte, microglia, and neuronal pathologies in different patients. I observed phosphorylation sites on known Tau-kinases and other novel signaling factors that were correlated these peptide modules. Finally, I used a data-driven statistical modeling approach to identify individual peptides and co-correlated signaling networks that were predictive of AD pathologies. Together, these results build a map of pathology-associated phosphorylation signaling events occurring in AD. / by Nader Francis Morshed. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 206-221). / Synthetic biology has become one of the most rapidly evolving research fields, with impacts on all aspects of our daily life. Through applying engineering principles to programming biological systems, synthetic biology provides advanced techniques to program organisms to perform desired tasks, similar to machines created by humans. Today, it has enabled the development of alternative meat substitutes, biosensors for water contamination, and living fertilizers that promote plant growth. The grand challenge to bridge the concept-to-product gap is twofold: scalability and safe deployment. First, most model microorganisms cannot produce a macroscale matrix to sustain themselves as standalone devices. The field of engineered living materials (ELMs) aims to recapitulate the remarkable properties of natural biology to create novel, growable, multifunctional materials using genetically engineered organisms. / Nevertheless, most relevant pioneering work was created using nano- to microscale biofilm, which has rather small yields and usually requires costly modification. Second, releasing genetically modified microorganisms (GMMs) into the field for food, water, or agricultural applications is often considered risky due to the uncertainty of wild-type organisms acquiring undesirable traits, such as antibiotic resistance, from the GMMs. A significant effort in addressing these unmet needs is called for. This Thesis starts with an introduction of genetic circuits and an in-depth review of the current trends in materials synthetic biology, which includes two major categories of ELMs: self-organizing functional materials and hybrid living materials. The following chapters describe the technologies developed to achieve high scalability and safe deployment of ELMs in these two categories and living devices suitable for real-world applications. / Finally, a detailed outlook summarizes the challenges and prospects for materials synthetic biology and engineering living functional materials. / by Tzu-Chieh Tang. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 43-47). / Artificially photosynthetic systems aim to store solar energy and chemically reduce carbon dioxide. These systems have been developed in order to use light to drive processes for carbon fixation into biomass and/or liquid fuels. We have developed a hybrid-biological system that manages both genetically controlled generation of products along with the photoactivability of a semiconductor system. We show an increase in the production of ethanol, a common biofuel, through the electron transfer stimulated by biologically produced cadmium sulfide nanoparticles and light. This work provides a basis on which to improve the production of many metabolites and products through endogenously produced nanoparticles. / by Shalmalee Pandit. / S.M. / S.M. Massachusetts Institute of Technology, Department of Biological Engineering