Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references. / Systems biology represents a powerful method to describe and manipulate phenotypes of interest by incorporating biological information from various levels of cellular organization. Such an approach is illustrated from a library of both rationally-directed and combinatorial gene knockout strains of E. coli recombinantly producing the small molecule lycopene. Global genomic and proteomic expression changes associated with increased lycopene production of mutant E. coli constructs were discovered using whole-genome DNA microarrays and a novel LC-MS technique, respectively. While most genes and proteins showed few expression changes, key differences were identified, including targets distal to the non-mevalonate and precursorsupplying pathways. Based upon the expression data sets, it was hypothesized that the following may be associated with lycopene overproduction: histidine biosynthesis (hisH); the quinone pool (wrbA); acid resistance (ydeO and gadE); the glyoxylate pathway (iclR); NADPH redox balance (pntB); growth rate reduction; and membrane composition. In the pre-engineered background strain, deleting pntB (~20-25%) and ydeO (~30%) each led to moderately increased production; overexpressing wrbA led to 50-100% more production at 8 hours and 5-15% more production at later time points; deleting iclR caused small production increases (~5-10%); and supplementing media with histidine caused the parental and mutant strains to have similar production. / (cont.) From these observations, several themes emerged. First, reduced cellular growth and energy conservation appear to be important tradeoffs for increasing lycopene production. Second, reducing overflow metabolism to acetate and corresponding acid stress as well as providing a gluconeogenic flux to increase lycopene precursors appeared beneficial. Next, NADPH availability and balance seemed to be critical production factors. The sS factor is known to affect lycopene accumulation, and it was observed to have far-reaching effects on both the transcriptomic and proteomic data sets. While expression changes were not strictly additive between the five mutant strains examined in comparison to the pre-engineered background strain, a number of these common factors appear to be responsible for the high lycopeneproduction phenotype. This work serves as an important example of incorporating multiple layers of complementary biological information to define a basis for an observed phenotype, demonstrating a powerful paradigm for realizing production increases via systems metabolic engineering. / by Brian E. Mickus. / Ph.D.

Thesis (B.S.) Massachusetts Institute of Technology. Dept. of Chemical Engineering, 1957. / MIT copy bound with: Laboratory preparation of deuteroammonia / R. Bruce Grover, Jr. 1957. / Bibliography: leaf 43. / by Herbert E. Klei. / B.S.

Thesis (B.S.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1980. / Includes bibliographical references (leaves 60-62). / by Diane Lynn Medved. / B.S.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1992. / Science hard copy is bound in 1 v. / Includes bibliographical references (leaves 364-377). / by Nicholas L. Abbott. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Contamination of marine sediments and water environments by urban runoffs, industrial and domestic effluents and oil spills is proving to be of critical concern as they affect aquatic organisms and can quickly disperse to large distances as highlighted by the recent Gulf oil spill disaster. Polycyclic aromatic hydrocarbons (PAHs), poly chlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and heavy metals like mercury, lead and manganese are among the ubiquitous trace contaminants of marine and freshwater systems. Presence of these contaminants raise concerns as small quantities of the organic chemicals have been shown to be carcinogenic to mammals and can pose a risk to both human health and the aquatic biota. We have proposed a remediation technique based on a magnetically enhanced separation technology as an alternative to existing methods to separate the target contaminants from a sediment matrix or wastewater stream. This technology uses specifically tailored surface modified magnetic nanoparticles (MNP) that are capable of a high uptake of trace metals. These particles have a magnetic core that facilitates their recovery, a shell that provides stability, protection from oxidation and a surface to which contaminant specific ligands are attached. The advantages of this alternative are that it involves low cost chemicals and magnets, can be implemented in continuous manner and is target specific. To evaluate the feasibility of this project, we have explored thermodynamics of adsorption of contaminants on particles and transport of these particles through their medium of application (water and porous media). This work focuses on treating effluents contaminated with heavy metals, in particular, mercury. For the treatment, dithiocarbamate functionalized magnetic nanoparticles were synthesized and their adsorptive properties for mercury at different pH conditions, ionic strengths and in presence of salinity and competing ions were explored. A competitive adsorption model based on mercury speciation was developed to explain the experimental results. In addition to the adsorption experiments, theoretical models to determine binding constants of the functional group on these particles to the mercury species were evaluated using Gaussian. Transport properties through porous (representing sediment like structures) and nonporous (representing effluents) media were studied using finite element models. The simulations provided a fundamental understanding of how magnetic nanoparticles would behave differently under magnetic field gradients and in porous media. In addition, parametric results of a continuous separation model that quantifies the trend in separation as a function of system parameters were also investigated. Bench scale runs for treating wastewater-containing mercury with these particles were demonstrated. Apart from adsorption, this process uses a well-studied high gradient magnetic separation (HGMS) system to capture the magnetic nanoparticles. Breakthrough analysis of mercury and particles through the entire system, capture on particles by the HGMS system, recovering magnetic nanoparticles by stripping off the contaminant were studied in this work. As part of the PhDCEP Capstone paper, commercialization prospects of this technology have been examined for industrial applications, particularly heavy metal removal. An in-depth market analysis of North America's water and wastewater treatment chemicals market was carried out to determine market attractiveness. This was followed by competitor analysis and evaluation of this technology's value proposition based on economics and technical applicability. A roadmap of strategies that need to be adopted based on key market insights was discussed. This chapter concludes with the verdict on magnetic nanoparticles' potential as a disruptive game changer in industrial wastewater treatment market / by Asha Parekh. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 159-167). / Transdermal drug delivery offers many advantages over traditional drug delivery methods. However, the natural resistance of the skin to drug permeation represents a major challenge that transdermal drug delivery needs to overcome in a safe and reversible manner. One method for enhancing transdermal drug delivery involves the application of ultrasound (US) to skin to physically overcome the skin's barrier properties. To advance this method, the focus of this thesis has been to develop novel experimental strategies and data analyses that can be utilized in in vitro investigations of passive and US-enhanced transdermal drug delivery. US treatment is often combined with a chemical enhancer such as the surfactant sodium lauryl sulfate (SLS). The simultaneous application of US and SLS (referred to as US/SLS) to skin exhibits synergism in enhancing transdermal drug delivery and has been utilized in clinical settings. In order to study the delivery of therapeutic macromolecules into US/SLS-treated skin, e.g. vaccine delivery to the Langerhans cells or drug delivery to the blood capillaries near the epidermis-dermis junction, it would be desirable to conduct in vitro US/SLS-enhanced transdermal diffusion experiments using split-thickness skin (STS) models, in which much of the dermis is removed in order to simulate the in vivo transdermal diffusion to the desired skin component. Therefore, STS was evaluated as an alternative to the well-established US/SLStreated full-thickness skin (FTS) model for the delivery of hydrophilic permeants. The skin permeabilities and the aqueous pore radii of US/SLS-treated pig FTS, 700-pm-thick pig STS, human FTS, 700-pm-thick human STS, and 250-pm-thick human STS were compared over a range of skin electrical resistivity values. The US/SLS-treated pig skin models were found to exhibit similar permeabilities and pore radii, but the human skin models did not. Furthermore, the US/SLS-enhanced delivery of gold nanoparticles and quantum dots (two model hydrophilic macromolecules) was found to be greater through pig STS than through pig FTS, due to the presence of less dermis that acts as an artificial barrier to macromolecules. In spite of greater variability in correlations between STS permeability and resistivity, the results strongly suggest the use of 700-pm-thick pig STS to investigate the in vitro US/SLS-enhanced delivery of hydrophilic macromolecules. After the validation of the pig STS for US/SLS studies, this skin model was used to study the transdermal delivery of nanoparticles. While nanoparticles have potential as transdermal drug carriers, many studies have shown that nanoparticle skin penetration is limited. Therefore, the US/SLS treatment was evaluated as a skin pre-treatment method for enhancing the passive transdermal delivery of nanoparticles. Quantitative and qualitative methods (elemental analysis / (cont.) and confocal microscopy, respectively) were utilized to compare the delivery of 10-nm and 20- nm cationic, neutral, and anionic quantum dots into US/SLS-treated and untreated pig STS. The findings include: (a) ~0.01% of the quantum dots penetrated the dermis of untreated skin (which was quantified for the first time), (b) the quantum dots fully permeated US/SLS-treated skin, (c) the two cationic quantum dots studied exhibited different extents of skin penetration and dermal clearance, and (d) the quantum dot skin penetration is heterogeneous (which was determined using a novel application of confocal microscopy). Routes of nanoparticle skin penetration are discussed, as well as the application of the methods described herein to address conflicting literature reports on nanoparticle skin penetration in the context of nanoparticle skin toxicity. US/SLS treatment is concluded to significantly enhance quantum dot transdermal penetration by 500 - 1300%. The findings suggest that an optimum surface charge exists for nanoparticle skin penetration, and motivate the application of nanoparticle carriers to US/SLS-treated skin for enhanced transdermal drug delivery. The final investigation of this thesis focused on chemical penetration enhancers, which are used to enhance drug delivery through several biological membranes, particularly the stratum corneum of the skin. However, the fundamental mechanisms that govern the interactions between penetration enhancers and membranes are not fully understood. Therefore, the goal of this work was to identify naturally fluorescent penetration enhancers (FPEs) in order to utilize well-established fluorescence techniques to directly study the behavior of FPEs within the skin. In this study, 12 FPE candidates were selected and ranked according to their potency as skin penetration enhancers. The best FPEs found compared well to SLS, a well-known potent skin penetration enhancer. Based on the ranking of the FPEs, FPE design principles are presented. In addition, to illustrate the novel, direct, and non-invasive visualization of the behavior of FPEs within skin, three case studies involving the use of two-photon fluorescence microscopy are presented, including visualizing glycerol-mitigated and US-enhanced FPE skin penetration. Previous two-photon fluorescence microscopy studies have indirectly visualized the effect of penetration enhancers on skin by using a fluorescent permeant to probe the transdermal pathways of the penetration enhancer. These effects can now be directly visualized and investigated using FPEs. The combination of FPEs with fluorescence techniques represents a useful new approach for elucidating the mechanisms involved in penetration enhancement and membrane irritation, and for improving structure-activity relationships for penetration enhancers. The new physical insights obtained using FPEs will aid in designing effective penetration enhancers for drug delivery applications, including penetration enhancers to be combined with US for synergistically enhancing transdermal drug delivery. The experimental strategies presented in this thesis pave the way for investigations in several transdermal fields, including evaluating nanoparticle skin toxicity, designing nanoparticle drug delivery carriers, evaluating ultrasound-assisted transdermal vaccination, elucidating mechanisms of chemical penetration enhancer-induced skin irritation, designing topical formulations with penetration enhancers, and elucidating mechanisms of ultrasound and penetration enhancer synergism in enhancing skin permeability. / by Jennifer Elizabeth Seto. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006. / Includes bibliographical references (leaves 38-44). / The process of building accurate chemical mechanisms for hydrocarbon oxidation systems is difficult since these mechanisms can have hundreds of species and thousands of reactions. Computer programs have recently been developed to construct these models automatically, but until this work, these programs did not include tools for the propagation of uncertainty. Rate constants and thermodynamic properties are not known precisely, and this can lead to large errors in model predictions. This work presents tools for sensitivity analysis and uncertainty propagation within an automatic reaction mechanism generator. A function for calculating first-order sensitivity coefficients with respect to rate and thermodynamic parameters and initial conditions is implemented in the MIT Reaction Mechanism Generator (RMG). An algorithm for generating error bounds on model output using first-order sensitivity coefficients and uncertainties in model parameters is also implemented. These tools are applied to an automatically generated model for the oxidation of the neopentyl radical, and results are compared to experimental observations. / (cont.) Comparison of the model with experimental data allowed identification of two rate constants. At 673 K and 60 Torr, kC5H11+O2-->OH+C5HI0O = 1.9x 10-14 ± 6x 10-15 cm3/molecule-s, and kOH+C5H1I-C5HOI+H20 = 3.1 x 10-12±1 .5 x 10-2 cm3/molecule-s.The computer-generated model is consistent with two prior literature studies. / by Sarah V. Petway. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 127-134). / Antibodies are higher order protein structures produced by a subset of lymphocytes (B cells) in the immune system for protection against pathogenic species. These homodimers of heterodimers form highly specific interactions with their cognate antigens and hence have become very important for the development of prophylactic or therapeutic agents against different disease pathogens. A key step in the development of human monoclonal antibodies as therapeutics is identification of candidate antibodies either by direct screening of human antibody repertoires or by filtering through combinatorial libraries of human variable genes using display technologies. Combinatorial libraries of human variable genes afford the flexibility to pursue many targets of interest, but often result in the selection of low affinity antibodies or unnatural heavy and light chain pairings that would have been selected against in vivo. With direct screening of the human B cell repertoire, the challenge is how to efficiently isolate clones of interest. Presented in this thesis is a high-throughput, integrated, single-cell methodology based on microengraving that allows the rapid recovery of antigen-specific human B cells. Microengraving is an analytical technique wherein secreted molecules from individual cells seeded into a dense array of subnanoliter wells are captured on the surface of a glass slide, generating a microarray from which desirable cells can be identified and recovered. Combined with high resolution epifluorescence microscopy and single-cell RT-PCR, we have developed assays for the simultaneous profiling of surface-expressed phenotypes of primary antibodyproducing cells, as well as functional characteristics of their secreted antibodies and germline variable gene usage. Using clinical samples from HIV- and West Nile virus-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies from both plasmablast/ plasma cell and memory B cell populations, and is ideal for the detailed characterization of cells from anatomical sites where sample sizes are often limited and disease pathophysiology is poorly understood (e,g. gut tissue, bone marrow). / by Adebola O. Ogunniyi. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1996. / Includes bibliographical references. / by Sung Min Park. / Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2000. / Includes bibliographical references (p. 189-206). / Metabolic flux and population heterogeneity analysis were used to develop relations between mammalian cell physiology and specific culture environments and to formulate strategies for increasing cell culture performance. Mitochondrial characteristics associated with respiration, membrane potential, and apoptosis along with physiological state multiplicity involving both metabolism and apoptotic death played a key role in this research. Research involving the accurate calculation of metabolic flux and the analysis of cellular behavior occurring in continuous cultures set the stage for subsequent research on physiological state multiplicity. This phenomena was observed in continuous cultures when at the same dilution rate, two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations. Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the tri-carboxylic (TCA) cycle for energy generation. This metabolic adaptation was unlikely due to favorable genetic mutations and was implemented in subsequent research aimed at improving cell culture performance. A hypothesis stating that mitochondrial physiology and cellular physiology are correlated was tested and confirmed. A mammalian cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123. The MMP sorted subpopulations were subjected to apoptosis inducers, and the apoptotic death was characterized both morphologically through the determination of apoptosis related chromatin condensation and also biochemically through the measurement of caspase-3 enzymatic activity. The results showed dramatic differences in apoptotic death kinetics with the higher MMP subpopulations demonstrating a higher resistance to apoptotic death. These results were applied in the development of novel fed-batch feeding and operating strategies. The first strategy showed that overfeeding cells later in culture leads to an increase in culture viable cell concentration, viability, and productivity. The second strategy showed that cell populations with a higher mean MMP are able to resist apoptosis during fed-batch culture. These results indicate that mammalian cell populations have considerable flexibility in their ability to redistribute metabolic flux in central carbon metabolism. Furthermore, these cell populations contain subpopulations that vary in their resistance to apoptotic death. The analysis of mitochondrial physiology and metabolic flux led to these discoveries, and these areas will play a key role in future mammalian cell culture research. / by Brian D. Follstad. / Ph.D.