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.

Estuaries are often challenging to manage, as management must strike a balance between the needs of the users and the ecological and economic values within the context of multiple legislative drivers. To help facilitate integrated management, a novel Estuarine Planning Support System (EPSS) framework, using the Humber Estuary in Eastern England as a case study, has been developed. This integrated EPSS framework goes beyond previous approaches as it brings together the legislative drivers, management tools and other mechanisms for controlling plans (formal/legal management, action or work plans (e.g. shoreline management plans) and projects (a new structure or activity such as extending a port). It thereby enables managers and users to assess and address both the current environmental state, and the way in which the new project could impact an estuary in an accessible and understandable tool. This study has been primarily completed by desk based research using peer reviewed literature, technical and research reports, marine licence applications and legislation, with correspondence to several sources to determine the baseline information and existing knowledge gaps. Further to the framework, an EPSS tool was developed to provide a practical application of these requirements. The GIS-based tool ensures that the information is accessible for regulators, managers, the scientific community, developers and the public. Whilst the tool is adaptable for regions within and outside the United Kingdom (UK), the research presented in this thesis focussed on the Humber Estuary. The successful application of the tool for a complex socio-economic and environmental system such as the Humber Estuary shows that the tool can efficiently guide users through the complex administrative requirements needed to implement a management plan, and therefore support sustainable development. In addition, the tool can be used as a scoping mechanism to identify potential stressors which are to be addressed in an environmental impact assessment (EIA). The tool was validated against four case studies and was also tested by a number of stakeholders to determine the utility and accuracy of the tool. The tool was subsequently updated to reflect feedback from the stakeholders. This project should be viewed as a ‘proof of concept’ in that its primary purpose is to demonstrate the potential for developing and operationalizing an approach in the field. The method has the potential to integrate highly technical knowledge from scientists, and the views of non-scientists, to make better-informed management and planning decisions and to provide reasonable assurance to justify those decisions. The tool can be used to prevent conflicts among stakeholders and/or between uses and users and the environment, and makes it possible to integrate all the existing background data in thematic maps and identifies the human activities that use the areas, resources and services. The EPSS tool can save time and resources, aid in the decision-making process and make the decision process more transparent and consistent. It has the potential to make the governance of the marine area more logical, simple, fast and therefore more cost effective. The tool has been developed to be flexible in its approach, this means that it can be easily adapted to be used internationally and to allow for it to be adaptable to future changes. It combines the many aspects required for a holistic approach to marine management, from the inclusion of governance and stakeholder views, to the need for, and use of, monitoring information. In marine management, there will always be a need for robust and scientifically and legally defendable science to inform management. The increasingly large spatial scales that are addressed by policy makers, and the reduction in funding, means that new methods are needed to provide the evidence base which this tool helps to provide, and can be applied worldwide. The developed toolbox is an important step towards such an integrated, holistic ecosystem based approach for marine management, demonstrating that a single tool can bring together the legislative, environmental and economic considerations. The tool is a method to undertake the assessments that are currently being carried out by separate organisations, to combine in a single process that is consistent and transparent and on a quicker timescale helping to reduce costs.

Tissue factor (TF) is the main initiator of blood coagulation. In addition to its procoagulant property, TF has the ability to regulate various functions within cells including proliferation, angiogenesis and apoptosis. These outcomes appear to depend on the amount of TF with which the cell comes into contact with. In this study, human dermal blood endothelial cells (HDBEC) were transfected to express wild-type TF which is released following the activation of PAR2 in a normal physiological response. In addition, a model for the accumulation of TF in vascular disease and cancer was used by expressing a mutant form of TF (TFAla253-tGFP) which although expressed is not released by the cells and therefore it accumulates intracellularly. Initially, the phosphorylation of Src1 and Rac1 were monitored in order to determine any difference in phosphorylation patterns following PAR2 activation of cells. Phosphorylation of Src1, but not Rac1 was prolonged on expression of TF and was further enhanced on intracellular accumulation of TF. Therefore, the role of Src1 as a mediator of TF-induced apoptosis was examined next. Either inhibition of Src using pp60c-srcpeptide, or suppression of Src1 expression using siRNA prevented the TF-induced p38 MAPK activation and subsequent cellular apoptosis. Following confirmation of the role of Src1 in this process, an attempt was then made to delineate upstream intermediaries involved in this pathway. By using an inhibitory antibody (AIIB2), β1-integrin was shown to participate in TF-induced Src1 activation. In contrast, prevention of Src1-FAK complex formation using FAK inhibitor-14 did not interfere with the TF-mediated Src1 activation, despite a clear reduction in Src1 phosphorylation. Furthermore, TF-induced apoptosis did not appear to require Src1-FAK binding. In conclusion, this study has established further steps in the pathway by which TF can induce cellular apoptosis, and suggests a mechanism by which the increased amount of TF during inflammation can have detrimental outcome on the vascular system.

Nitric oxide (NO), being a free radical, has a unique biological chemistry that, since its elucidation as a signaling molecule in the cardiovascular system, has been implicated in a range of diverse biological functions. Both cardiovascular disease, exacerbated by hypertension, and antibiotic-resistant bacterial infections are major yet unavoidable public health concerns. Atypical NO synthesis may be a factor these pathologies and exogenous NO may provide an alternative therapeutic strategy when endogenous NO synthesis is atypically low. Furoxans are an old, yet largely untapped resource of nitric oxide donor compounds. A panel of twelve such compounds were developed through heterocyclic synthetic chemistry and assessed for their potential biological activity. The compounds have had their NO release characterised via the Griess assay and have been investigated for both novel antibacterial and vasodilatory effects. Direct toxicity was explored using an optimised MTT assay to assess cell viability post incubation in three bacterial species: Staphylococcus epidermis, Escherichia coli, and Pseudomonas aeruginosa. The lead compound RJP06A was then exposed to bacterial biofilms, investigating its ability to prevent initial biofilm formation and promote its dispersal before determining its mechanism. Vasodilatory potential of the compounds was studied using rat thoracic aorta, pulmonary artery and renal artery. Results demonstrate that these compounds elicit a toxic effect against all three bacterial species with IC50 values ranging from 31-745 uM, which was enhanced in the presence of S-nitrosoglutathione, despite having negligible toxicity itself. Biofilm investigations of RJP06A demonstrate its ability to disperse mature single-species bacterial biofilms in a dose-dependent manner. Isometric tension data show that three quarters of the compounds produced strong, sustained relaxation of contracted aorta, and two in particular caused almost complete reversal of contraction at concentrations of 250 and 350 nM respectively. Mechanism studies suggest that the lead furoxan, RJP06A exerts its effects primarily through a cGMP-mediated NO-activated pathway, but may also have a small component whereby thiols can bypass this step and activate calcium channels directly. RJP06A has been shown to be better at inducing relaxation of resistance vessels, such as aorta, rather than pulmonary or renal artery. In both investigations, nitric oxide from RJP06A appears to be the key molecule involved in the observed effects, with scavenging evolved NO using haemoglobin attenuating its effect, and importantly the two mechanisms identified in very different models imply an evolutionarily conserved pathway of nitric oxide biology.