Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 150-157). / The RNA-guided bacterial nuclease Cas9 can be reengineered as a programmable transcription factor by a series of modifications to the Cas9 protein and the direct fusion of a synthetic transcriptional activation domain (AD). However, the modest levels of gene activation achieved by first generation Cas9 activators limited their potential applications. We describe the development of a versatile, improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-RTA (VPR), fused to Cas9. We demonstrate its utility in activating expression of endogenous coding and non-coding genes, and showcase the ability to target several genes simultaneously. As a further demonstration of the tools efficacy, we stimulate neuronal differentiation of induced pluripotent stem cells (iPSCs), validating the biologically relevant levels of activation attained by this tool. Beyond applications to cellular programming, Cas9 transcriptional and epigenetic activators hold tremendous promise for in vivo gain of function studies as well as therapeutics. However, the most convenient and only approved vector for human delivery is the Adeno-Associated Virus (AAV). This convenient virus allows for targeting of various tissue types with high efficiency and little risk of integration or immunogenicity. Unfortunately, the virus is limited to a genomic payload of 4.7kb, which can be pushed to 5kb but not much further. The most commonly used Cas9 ortholog from S. pyogenes is 4.2kb alone, leaving very little room for an additional promoter let alone an sgRNA expression cassette or accessory activation domains. Here, we detail a single-vector system for delivery of a miniaturized, potent Cas9 activator for AAV mediated transcriptional regulation in neuronal, hepatic, gonadal, and muscle derived cell lines on a panel of genomic targets. / by Suhani Vora. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 116-126). / One specific thesis of pathogenesis holds that diseases are dependent upon the complex interactions of multiple compounds and cofactors. While one agent may dominate, its powerful effects are mediated by ancillary factors that alone may have no discernible action. We examined this paradigm by comparing catecholamine-infusion induced cardiomyopathy with the organ disease observed by catecholamine-secreting pheochromocytoma cells. Pheochromocytomas are widely believed to induce cardiomyopathy via hypersecretion of catecholamines including norepinephrine (NE). NE can have direct cardiomyocyte toxicity and can stimulate myocardial remodeling secondary to the induction of hypertension. Yet, cardiomyopathy development is not entirely related to catecholamine dose. To explore these effects we engineered a polymeric cell encapsulation system to control PC12 cell kinetics and NE release in vitro and in vivo. We demonstrated that pheochromocytoma-conditioned media induced greater cardiomyocyte contractility and cytoskeletal remodeling than NE alone. We next examined the cardiomyopathy development in animals implanted with agarose- encapsulated pheochromocytoma (PC12) cells, DOPA decarboxylase knock-out PC12 cells deficient in norepinephrine (PC12-KO), or norepinephrine-secreting pumps. Implanted PC12 cells induced a greater degree of cardiomyopathy than animals dosed only with norepinephrine. Elimination of norepinephrine secretion in PC12-KO cells eliminated the effects of PC12 cells, while reintroduction of NE secretion via NE secreting pumps implanted alongside PC12-KO cells induced a cardiac pathology similar to intact PC12 cells. In the second portion of this work, we examined the role of pregnancy in our disease model and its ability to exacerbate pheochromocytoma-induced cardiomyopathy. Gravid pheochromocytoma-bearing mice experienced a greater loss of cardiac function and increased expansion of cardiac volume compared to non-gravid pheochromocytoma- bearing mice. Our data suggest that it is the confluence of these factors that enable the synergistic induction of myocardial injury. The implications of our work lie not only in a better understanding of pheochromocytomas and their secretory products but also in the differences between cell-secreted substances and their exogenous analogues in isolation. / by Hector R. Mobine. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 86-91). / Metabolic engineering efforts have so far focused on strain optimization through careful metabolic modeling and tinkering with host genomes, through gene knockouts or knockins, to direct flux in desired channels. These efforts have borne fruit with the development of large manufacturing processes for numerous chemicals. The next challenge for metabolic engineering, however, lies in tackling issues associated with construction of more complex pathways, such as those that directly interfere with host metabolism, have branchpoints with promiscuous enzymes, or synthesize toxic intermediates or products. Dynamic metabolic engineering has emerged as a new frontier for tool development to allow regulation and control of native and cellular pathways during the course of a production run. Advantages in dynamic strategies are especially apparent in the aforementioned examples where traditional static strategies of gene knockouts or knockins are not an option. Instead, it is necessary to be able to control when certain genes are expressed, such as to build biomass before switching on growth-limiting production pathways, or accumulating intermediates to drive the reaction of a promiscuous enzyme along a certain branch. In this thesis, we propose enzyme control strategies that are independent of any biosynthetic pathway of interest. Therefore, they can theoretically be applied to a wide variety of contexts in a "plug-and-play" fashion to control pathway enzyme expression. After initial work to understand the limitations of nutrient starvation strategies to induce genetic circuits, we decided to use quorum sensing circuitry to create circuits that can be autonomously induced. We used parts of the Esa QS system (derived from Pantoea stewartii) to create circuit variants in the Lscherichia cohi genome, which switch off expression of the targeted gene at various times and cell densities. Switching times were varied by modulating the expression of the AHL synthase, and therefore the production rate of AHL, the quorum sensing molecule. Switching dynamics were characterized and ranked for the entire library of circuit variants using fluorescent reporters. The characterized device was used to identify optimal switching times for redirection of glycolytic fluxes into heterologous pathways, resulting in a 5.5-fold boost in myo-inositol (MI) and increasing glucaric acid titers from unmeasurable quantities up to >0.8 g/L. With a focus on industrial application, consistency of device performance was verified in benchtop bioreactors, achieving nearly 10-fold and 5-fold boosts in specific titers of myoinositol and glucaric acid, respectively. To demonstrate broad utility and "off-the-shelf" applicability, the control module was applied to dynamic downregulation of flux into aromatic amino acid biosynthesis to accumulate the industrially-relevant intermediate, shikimate, resulting in an increase in titers from unmeasurable quantities to >100 mg/L. Finally, this QS device was coupled with a MI-biosensor circuit to institute two layers of dynamic regulation and further improve glucaric acid titers. Production trials in these composite strains resulted in the highest glucaric titers (-2 g/L) reported to date from E. coli K-strains. This work reports the first completely autonomous dynamic regulation module and its application in bioproduction of multiple products from different metabolic pathways. We envision that the strategy presented here may be adapted to any pathway context and gene of interest. With increased prevalence of dynamic regulation, the relevant strategies may become standardized for general use. / by Apoorv Gupta. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 117-119). / The increased precision offered by developments in suspended microchannel resonator (SMR) technology opens the possibility for measuring small mass changes in cells. Mass accumulation rate (MAR) measurements in single suspended cells over short periods of time have the potential for characterizing heterogeneous collections of tumorigenic cells and serve as a functional marker for the effects of anti-cancer drugs. In this thesis we adapt mass accumulation measurements for use in Glioblastoma Multiforme (GBM) patient-derived cell lines, exploring the heterogeneity between and within patient tumors, and validating the measurement as a predictor of drug susceptibility with response times on the order of 24 to 48 hours using an experimental MDM2 inhibitor. While MAR measurements can be performed on suspended single cells with high precision, it has not yet been adapted for measuring the growth of adherent cells. We develop a technique to measure mass accumulation in cells adhered to the inner surface of the resonator channel. To overcome challenges inherent in such a measurement, we use infrared imaging and multiple resonant modes to reveal the cell's position in the SMR, and utilize differential measurements from a second cantilever to account for frequency drift. / by Nigel Shijie Chou. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The high mortality rates associated with uncontrolled bleeding motivate hemostatic material development for traumatic injuries. Uncontrolled, or hemorrhagic, internal bleeding requires hemostatic materials that can be directly delivered to or target the bleeding locations. To address these needs, injectable systems are being developed that: (1) generate artificial clots independent of the coagulation cascade or (2) interact with blood components to accelerate or otherwise improve coagulation processes. Hemostatic materials designed for internal bleeding can save lives in the battlefield, en route to emergency rooms, and in the operating room. This thesis first focuses on developing a shear-thinning hydrogel for injection onto bleeding surfaces and into ruptured vasculature. Based on in vitro assays of hydrogel performance, it was amenable to clinical delivery methods and reduced whole blood clotting times by 77%. In vivo bleeding models showed reduced blood loss and improved survival rates following a lethal liver injury. The hydrogel was also used as an embolic agent, where its occlusive potential in an anticoagulated model was demonstrated. Next, recombinant protein-based hemostatic materials were expressed to modulate clotting kinetics and performance. By incorporating clot interacting peptide sequences (CIPs) into a protein scaffold, a family of multifunctional fibrinogen like proteins (MFLPs) was developed and assayed. Clot turbidity, an indication of fibrin clot formation, was increased among enzyme-interacting CIPs. Mimicking the polymerization mechanism of fibrinogen, knob sequences were shown to be procoagulant at low concentrations by increasing clot turbidity, reducing clotting times, and inhibiting plasmin lysis. Finally, to understand the impact of hemostats on clot structure, imaging procedures were developed to systematically assess hemostatic materials and their influence on clot architecture. Static and dynamic approaches were developed to quantify the activity of hemostats based on the spatial distribution of fibrinogen, red blood cells, and platelets around hemostat surfaces. Quantification of these hemostat-blood component interactions resulted in a unique pattern of interactions for each hemostat studied. These techniques could serve as a screening technique for hemostats and improve characterization prior to in vivo assays. Taken together, the results highlight multiple approaches to address internal bleeding and opportunities to improve in vitro characterization of hemostats using microscopy. / by Reginald Keith Avery. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student submitted PDF version of thesis. / Includes bibliographical references (p. 123-134). / Twenty five percent of all the deaths worldwide are caused by infectious diseases. They are also the biggest cause of mortality among children under five years of age. Among them diarrheal diseases alone cause as many deaths as AIDS or TB and malaria combined. Also up to 80% of traveler's diarrhea is bacterial in nature. Vibrio cholerae (cholera), Salmonella spp (typhoid fever), Shigella spp (shigellosis) and a variety of enteropathogenic Escherichia coli strains are among the principle bacterial agents that cause this type of diarrhea. Improvements in hygiene and access to adequate nutrition are good strategies but immunization against specific diseases still offers the best solution to fight these infections. Unfortunately the wide diversity of bacterial and viral agents that cause diarrhea complicates accurate diagnosis and makes the development of vaccines difficult. Antibiotics used in timely manner and in appropriate doses can be effective but the diagnosis is usually made too late for the therapy to be effective. Moreover frequent use of over-the-counter drugs without any medical supervision has led to multidrug resistance in most of the bacterial strains. To counter this problem I demonstrate a proof of principle of a novel cell therapy against Pseudomonas Aeruginosa (major cause of urinary tract disease and hospital infections). Using principles of Synthetic Biology I genetically modified a probiotic strain of E. coli to specifically detect PAO₁ and respond by secreting a novel, pathogen-specific engineered toxin. Additionally, I translated the bacterial system into mammalian cells and established a foundation for an adaptive system where the sentinel cells secrete an alternate toxin if the pathogen becomes resistant to the first one. Finally, based on this system I proposed designs against highly pathogenic strains of Shigella, Salmonella and Vibrio cholerae. This cell therapy remains inert until a threat is detected, and then serves as an early detection and rapid response agent. Furthermore this platform can be tuned to release minimum but sufficient amounts of very narrow spectrum antimicrobial proteins to control the early stages of infection before the disease becomes systemic. Therefore this system's rapid, automated and highly specific response can be helpful in reducing the occurrence of dose dependent resistance. This approach offers a single integrated solution to eradicating multiple threats with a strategy that is a rapid, selective, and highly sensitive. / by Saurabh Gupta. / Ph.D.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 69-75). / The multiple "hits" carcinogenesis models are extensions of the cancer incidence theory developed by researchers from Nordling (1953), Armitage-Doll (1954 and 1957), Knudson (1971), Moolgavkar and Verzon (1979), to Moolgavkar and Knudson (1981), among others. These studies relate to the evolutionary process of normal tissue cells in an individual's organ from a normal stage to an initiated pre-neoplastic stage, and finally promoted to a neoplastic stage, resulting in tumorigenesis. The most significant impact of this type of research is to gain insight into the complex process of cancer development in humans. In the case of skin cancer, epidemiological and molecular data clearly indicate that sunlight is a carcinogen, the primary cause of skin cancer, in which forms of point mutation are associated with ultraviolet radiation. Furthermore, sunlight is attributed both as a tumor initiator and a tumor promoter by favoring the clonal expansion of p53 mutated cells. By utilizing studies of the multiple genetic hit model of oncomutation and inference that preneoplasia appears to be a clonal continuation of juvenile growth in adult tissues from which one stem cell creates an embryonic organ with lethal consequences, this thesis is devoted to the analysis of the process of skin cancer development and distribution of mutant cells in human skin, as well as the calculation of and inferences based on Gostjeva and Thilly's hypothesis on mutational clone frequency is restricted to the fetal-juvenile period. / by Leslie E. Kao. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / "February 2009." Cataloged from PDF version of thesis. / Includes bibliographical references (p. 161-178). / Numerous therapeutics, such as viral gene therapy vectors, have unintended toxicity in part due to interactions with inflammatory cytokine signaling to elicit hepatocyte death, thus limiting their clinical use. Although much is known about how cytokines and certain therapeutics individually induce hepatotoxicity, there is little understanding of how they jointly regulate the complex cellular signaling network governing hepatocellular death. In this thesis, we explored the signaling mechanisms governing the cytokine-induced hepatocellular death in the context of adenoviral vector (Adv) infection and pharmaceutical compounds with idiosyncratic hepatotoxicity. Initially, we examined the role of autocrine and intracellular signaling pathways in governing the synergistic induction of hepatocyte apoptosis by the cytokine tumor necrosis factor-a (TNF) in the presence of Adv infection in a primary rat hepatocyte cell culture model. We demonstrated that Adv/TNF-induced hepatocyte apoptosis is regulated by a coupled and self-antagonizing autocrine signaling cascade involving the sequential release of anti-apoptotic transforming growth factor-a (TGF-a), pro-apoptotic interleukin- 1 a/p (IL-l a/), and anti-apoptotic IL- 1 receptor antagonist (IL- Ira). This three-part autocrine cascade regulates multiple intracellular signal pathways, including ERK and JNK, that serve to integrate TNF- and Adv-induced signals and govern the resultant hepatocellular death response. Following this, we demonstrated that numerous idiosyncratic hepatotoxins, whose hepatotoxicities are not evident in standard cell preclinical screening models, elicit synergistic induction of hepatocellular death upon multi-cytokine co-stimulation in primary rat and human hepatocyte cell culture models. We showed that this drugcytokine co-treatment model could be usefully scaled to the high-throughput demands of pharmaceutical screening while maintaining idiosyncratic hepatotoxicity prediction accuracy. To identify the signaling mechanisms regulating these drug/cytokine hepatocellular death synergies, we collected multi-pathway signal-response data compendia from two human hepatocyte donors. Through the use of partial least-squares regression modeling, we showed that hepatocytes integrate signals from four pathways -- ERK, Akt, mTOR, and p38 -- to specify their cell death responses to toxic drug/cytokine conditions and that accurate prediction of hepatocellular death responses can be made across human hepatocyte donors. Together, these findings demonstrate that cytokine-induced hepatocellular death in the context of hepatotoxic therapeutics is governed by integrated network activity of multiple autocrine and intracellular signaling pathways. / by Benjamin D. Cosgrove. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 173-175). / Lysine acetylation is a prevalent post-translational modification whose multi-varied biological roles have recently emerged. While having all the necessary components of a signaling network, lysine acetylation studies have been limited to a small subset of proteins and pathways. Using a quantitative unbiased mass spectrometry approach, we explored the role of growth factor stimulation on lysine acetylation. Although the growth factors bind receptor tyrosine kinases, growth factor stimulation resulted in rapid and dynamic changes in lysine acetylation. Furthermore, we demonstrated that short-term HDAC inhibition alters phosphotyrosine-signaling networks. To better understand this behavior, a suite of biochemical and computational methods were developed. Bromodomains were engineered to explore binding preferences using degenerate peptide arrays as well as develop acetyllysine affinity reagents as an alternative to anti-acetyllysine antibodies. Additionally, bioorthogonal proteomics were employed to identify acetyltransferase substrates. Taken together, the knowledge generated and the methods developed provide a toolkit for the analysis of lysine acetylation networks in the context of many biological processes as well as diseases. / by Bryan David Bryson. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 105-123). / In vitro multi-cellular 3D cultures can provide tremendous insight into pathologies arising from dysregulation of extracellular communication by recapitulating key features of the microenvironment, such as ECM-cytokine interactions, spatially regulated cell-cell interactions, and matrix biophysical cues. Relevant 3D in vitro systems have been limited by the difficulty to assay the local microenvironment that cells directly experience, and by the lack of modularity of naturally-derived hydrogels such as Matrigel and collagen gels. Here, we exploit the modularity of synthetic hydrogels to overcome these limitations by designing a novel 3D culture system that can be locally and accurately interrogated. We begin by investigating the underlying principles directing morphogenesis in 3D synthetic PEG hydrogel cultures by screening the relevant parameter space - specifically, the intertwined relationship between adhesion and matrix degradation, the effect of cell-cell contacts, and cell-mediated matrix deposition. We use vasculogenesis of human iPSC-derived endothelial cells as a case study. Understanding vasculogenesis is a crucial requirement for engineering tissue models exceeding 100 gm due to nutrient diffusion limitations. Here we define microenvironment conditions that are permisive of the formation of 3D interconnected structures of iPSC endothelial cells in PEG hydrogels. Lastly, we adapted the synthetic hydrogels to enable local interrogation of the 3D microenvironment -a hitherto unavailable feature. Here we report a novel hydrogel system that can be used to both encapsulate complex co-cultures, and recover intact cells and local cytokines from the 3D matrix by repurposing a mutant of sortase A (SrtA). We demonstrate SrtA-mediated gel dissolution preserves important cell morphological features and can be used to measure recovered cytokines much more accurately compared to other proteolytic degradation methods. We observe discrepancies between the cytokine concentrations inside the gel and those in the culture medium. These local measurements reveal behaviors in response to an inflammatory stimulus that cannot be captured by monitoring only the cytokine concentrations in the medium. This system allows for better understanding of protein communication networks in relevant complex 3D multicellular cultures and can aid in identifying and testing targets in drug discovery and development. / by Jorge L. Valdez Macias. / Ph. D.