Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 122-142). / A major challenge to communities across the world in the next century will be ensuring millions have access to adequate freshwater resources. Studies from the UN World Health Organization indicate that over 1.1 billion people currently lack access to reliable and secure freshwater supplies, with an estimated 2.5 million deaths per year from diseases associated with poor access and sanitation in 2007. Reverse osmosis (RO), a process through which water is desalted via pressurized flow past a salt-selective membrane, is an energy-efficient method to generate freshwater from oceanic, brackish, and waste water sources. However, there are a number of challenges to scaling up RO processes to large scale production, including the need to improve membrane selectivity and throughput. One method to assemble selective layers for RO membranes is layer-by-layer (LbL) assembly, which is a flexible, scalable assembly technique that enables the incorporation of a myriad of polyelectrolytes and inorganic nanoparticles into thin films. There is a gap in the scientific literature concerning the use of LbL to generate RO selective layers where previous approaches have not taken full advantage of the LbL process to incorporate nanomaterials that can generate ordered nanostructures for salt rejection. In particular, high-aspect ratio clay platelets are ideal for such a purpose; it was hypothesized that effective salt rejection could be achieved by hindering the diffusion of solvated ions through nano-channels formed by the platelets embedded within a polymer matrix. This body of work examines the application of spray layer-by-layer (spray-LbL) assembly with clay composite thin film architectures to generate nanostructured selective layers for use in RO membrane technology. First, appropriate substrates were identified as support layers for the deposition of spray- LbL assembled clay composite thin films. Both electrospun bisphenol-A polysulfone (PSU) mats of varying fiber diameter and polyethersulfone (PES) ultrafiltration (UF) membranes with varying pore diameters were examined. Second, a range of materials were investigated for the spray-LbL deposition of clay composite films. Laponite clay platelets were incorporated into several different film architectures including strong polyelectrolytes as well as cross-linkable weak polyelectrolytes to form both bilayer and tetralayer film architectures. The clay content was controlled via manipulating assembly conditions such as the pH and spray times of the film components. Assembled membrane architectures were tested at industrial RO operating conditions in dead-end permeation cells and evaluated for salt rejection, water permeability, and mechanical strength. Ultimately, it was determined the most uniform and robust films were those deposited on PES membranes with 30 nm pores, closely matching the characteristic length of the LAP clay platelets to reduce the impact of bridging. Although all the film architectures tested exhibited significantly greater water permeability than commercially available RO selective layers, the salt selectivity was found to be highly dependent on the film architecture and assembly conditions. The best performing film architecture consisted of a cross-linked clay composite tetralayer film, exhibiting salt rejection of 89% for aqueous 10,000 ppm NaCl solution with an order of magnitude increase in water permeability over a commercially-available thin film composite membrane. The key conclusion drawn from the studies indicate the presence of an optimal zone where the incorporation of clay platelets introduces additional salt selectivity via size exclusion, balanced with the cross-linked polymer component of the film to improve the mechanical strength and reduce the risk of critical defect formation during operation. Taken together, these investigations represent a new approach using structured nanomaterials to develop next generation clay composite RO selective layers. The increased water permeability of the clay composite selective layers offers an attractive advantage in desalting applications where high flux is desirable, such as with brackish water resources as well as in membrane unit operations near their thermodynamic limit. / by Jason Richard Kovacs. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 71-84). / Magnetic resonance imaging (MRI) is a powerful neuroimaging tool that allows non-invasive visualization of the brain with high spatial and temporal resolution. Research on MRI contrast agents and their application to problems in neuroscience is burgeoning, and there is particular interest in developing MRI agents that are sensitive to time varying components of neurophysiology. Relatively recent advances in biomolecular probes has demonstrated the potential and versatility of bioengineered MRI sensors for molecular imaging. However, a major limitation of these probes is the high concentration needed for imaging, which can lead to issues such as analyte buffering and toxicity, and restrict the applicability of the sensors. In this work, we explore two approaches for developing high relaxivity protein-based contrast agents to address the issues of low detectability. First, we coupled monoamine sensing with the disaggregation of superparamagnetic iron oxide nanoparticles (SPIOs). Ligand detection was imparted by integration of a monoamine sensing protein-based contrast agent derived from P450- BM3h (BM3). We demonstrated that this mechanism can produce robust signal changes of approximately 2-fold, while reducing the concentration of BM3 needed by 100-fold compared to the amount needed when only the protein is used for imaging. The second method demonstrated the feasibility of using semi-rational protein design to engineer a high relaxivity metalloprotein by tuning phenylalanine hydroxylase to bind gadolinium at high affinity. Mutations were found that increased the protein affinity by two orders of magnitude and enhanced relaxivity. The results of this thesis advance approaches for creating high relaxivity contrast agents which can be applied to the development of probes for other analytes, ultimately advancing and broadening the applicability of bioengineered probes in molecular functional neuroimaging. / by Vivian Hsieh. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1998. / Includes bibliographical references (leaves 223-234). / by Brian D. Phenix. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015. / Page 11 out of sequence; inserted between page 4 and page 5. Page 209 blank. Cataloged from PDF version of thesis. / Includes bibliographical references. / Although petroleum and other fossil fuels have traditionally been used to fulfill our energy needs, rising concerns over energy security and the climate-changing effects of our continual greenhouse gas emissions have led to great interest in developing a domestic source of renewable fuel with low net carbon emissions. Biodiesel is an attractive option for replacing petroleum-based fuels used in the transportation sector due to its compatibility with existing infrastructure. Single cell oils from heterotrophic oleaginous microorganisms as a source of bio diesel allow for high productivity from a wide array of potential feedstocks, including agroindustrial and municipal waste streams. The goal of this work is to use the tools of rational metabolic engineering to improve lipid production in the non-conventional oleaginous yeast Yarrowia lipoytica on two representative carbon sources, glucose and acetate. Previous work in this area achieved considerable success with the simultaneous overexpression of the native acetyl-CoA carboxylase (ACC 1) and diacylglycerol acyltransferase (DGA2) genes; the resulting strain was used as a benchmark to evaluate our own efforts. We began with the compilation of a set of 44 genes and evaluated the effects of the individual overexpression of each gene on the ability of the resulting strain to produce lipids in fermentations of glucose and acetate. The genes tested here represent many different functions potentially important to lipid production, including the Kennedy pathway, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport. Our results demonstrate that a diverse subset of gene overexpressions led to significant improvements in lipid production on at least one substrate. The largest improvements unsurprisingly came from overexpressing genes directly related to triacylglycerol synthesis, such as diacylglycerol acyltransferase DGAI, which on glucose increased the lipid titer, content and yield by 236%. 165%, and 246%, respectively, over our wild-type control strain, and the acylglycerolphosphate acyltransferase SLC1 gene, which increased titer/content/yield on glucose by 86%/73%/87% and on acetate by 99%/91%/151%. Significant improvements were also detected from genes that more indirectly effect lipogenesis, such as glycerol-3-phosphate dehydrogenase GPD (which produces head groups for triacylglycerol molecules) and the 6-phosphogluconolactoase SOL3 (catalyzing the middle step of the NADPH-producing oxidative pentose phosphate pathway). We next chose the aforementioned SLCl, GPD, and SOL3 genes for use in continued rational engineering of our benchmark strain due to the significance of their effects and the lack of redundancy in their likely mechanism of improving lipogenesis when overexpressed along with ACC I and DGA2. The results of this investigation indicate that the strain overexpressing ACC 1, DGA2,'and SLC 1 may be superior to our benchmark strain, increasing lipid content and yield by 24% and 20%, respectively, with a statistically equivalent titer on acetate. This strain produces the highest reported overall lipid yield of an oleaginous yeast on acetate, at 0.207 g lipids/g acetate. / by Andrew Michael Silverman. / Ph. D.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1941. / Vita. / Includes bibliographical references (leaves 303-306). / by David Stover Weddell. / Sc.D.

Thesis (B.S.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1958. / MIT copy bound with: Fluid flow conditions within a model blast furnace / Conrad Simon Revak, Richard Herbert Hough. 1958. / Includes bibliographical references. / by Jack Shelton and Larry Ogden. / B.S.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1998. / Includes bibliographical references (p. 249-257). / by Taeshin Park. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / According to the American Cancer Society, approximately 1,479,000 new cases of cancer were expected to be diagnosed, while 562,340 Americans were expected to die from cancer in 2009 alone. Even though advances in early diagnosis and therapy over the past few decades have led to continual decreases in incidence and mortality, cancer remains the second leading cause of death among all Americans. Consequently, further technological development in all areas of cancer detection and treatment are still of great importance not only to the scientific community, but to society itself. To address the shortcomings in current cancer diagnosis and treatment, a novel, highly adaptable, targeted nanoparticle system based on alternating amphiphilic copolymers has been developed having a variety of potential clinical applications. These polymers consist of an alternating copolymer backbone composed of hydrophilic polyethylene glycol-900 (PEG900) and dimethyl 5-hydroxyisophthalate (linker) monomer units. The linker within the backbone polymer has a free hydroxyl group to which a variety of sidechains can be attached, including hydrophobic groups to impart amphiphilicity, targeting ligands, as well as contrast agents for imaging applications. Three major areas of investigation were addressed to develop and evaluate the performance of the proposed amphiphilic alternating copolymers: (1) backbone polymer synthesis, (2) attachment of radioiodine, and (3) targeted delivery in vitro and in vivo. The first step in the production of the alternating amphiphilic copolymers is a chemo-enzymatic condensation polymerization of polyethylene glycol (PEG) and dimethyl 5-hydroxyisophthalate (linker) to produce backbone polymer. Because of their generally low equilibrium constants, condensation polymerizations require effective removal of the condensation byproduct (in this case, methanol) in order to achieve significant increases in molecular weight. The increased viscosities at higher molecular weights not only increase the difficulty of byproduct removal, but may also affect the mixing characteristics as well as the mass transfer of other species in the reaction. The enzymatic polymerization was investigated using both predictive modeling and experiment. The ultimate goal was to increase the molecular weight of the synthesized polymer to allow for increased substitution of the polymer backbone. Key experimental variables were tested in glass flasks typically used in organic synthesis. In these reactions, 4A molecular sieves had the greatest affect on the backbone polymer molecular weight. In particular, addition of sieves, which can act as sinks for both water and methanol, led to a twofold increase in weight-average molecular weight above that observed previously for the enzymatic polymerization. The Protherm, a novel, thin-film reactor was employed in order to improve methanol mass transfer and mixing within the polymer melt. Three separate reactions in the Protherm produced the highest Mw backbone polymer (approximately 20 kDa). A blade speed of 500 rpm with molecular sieves present was able to achieve this Mw in 48 hr. Two separate models were proposed to describe the polymerization, including a homogeneous kinetic model and a Fick's Law mass transfer model. Significant differences were observed between the experimental results and the predictions of the homogeneous model. The mass transfer modeling, which estimated the increase in reactant and methanol surface concentration relative to the concentration in the bulk, was unable to bridge the gap between experiment and model results. Limited knowledge of key model parameters, including the equilibrium constant and methanol solubility, was one proposed explanation for the observed discrepancy. In order to assess the performance of a nanoparticle delivery system in biological applications, a label that is detectable under a wide range of conditions and concentrations must be present within the molecule. Radioiodine was selected because of its multiple potential applications depending on the selected isotope, including 124I for positron emission tomography, 131I for radiotherapy, and 125I for inexpensive, quantitative research applications. A standard protein-labeling technique was adapted for application to the copolymers in this work. The successful adaptation of this procedure for use with our polymers represented the first demonstration in the field of a nanoparticle-forming polymer that was directly labeled with radioiodine without any additional chemicalalterations or intermediate reactions. The process was characterized using a variety of chromatographic techniques and radiometric measurements to confirmed covalent, stable attachment of iodine in a product with high radiochemical purity. The alternating amphiphilic copolymers were combined with an engineered peptide having an extremely high binding affinity for the epidermal growth factor receptor (EGFR), a biomarker prevalent in a variety of human cancers. This high-affinity binder, the E13.4.3 peptide, was developed by collaborator Dr. Benjamin Hackel under the guidance of Professor K. Dane Wittrup. A number of polymer design variables were considered, including the targeting ligand density, identity of the hydrophobic sidechain, polymer molecular weight, and length of the spacer connecting the peptide to the backbone. The ligand density and hydrophobic sidechain identity were chosen for study. Initial studies demonstrated selective uptake of E13.4.3-conjugated polymers into a target-bearing, EGFR-positive human cancer cell line relative to untargeted controls. Preparative gel permeation chromatography (GPC) was used to create high molecular weight, low polydispersity fractions of backbone polymer. Polymers synthesized from these fractions achieved the greatest increase in selective uptake in vitro with a four- to sixfold increase in uptake for E13.4.3-conjugated polymers relative to untargeted controls. Animal studies measured the biodistribution, blood circulation, and tumoral accumulation of various polymer formulations. Statistically significant selective tumor accumulation was observed for two different targeted polymers, each having different targeting ligand density and different hydrophobic sidechains. The E13.4.3-polymers have proven a rich platform for study. Their demonstrated ability to selectively accumulate in targeted tumors combined with their potential use in diagnostic and/or therapeutic clinical applications makes them an attractive option for intensified investigation. / by Kevin P. Brower. / Ph.D.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1990. / Vita. / Includes bibliographical references (leaves 441-479). / by Do Hyun Kim. / Sc.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, June 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Nanoengineered glycan sensors may help realize the long-held goal of accurate and rapid glycoprotein profiling without labeling or glycan liberation steps. Current methods of profiling oligosaccharides displayed on protein surfaces, such as liquid chromatography, mass spectrometry, capillary electrophoresis, and microarray methods, are limited by sample pretreatment and quantitative accuracy. Microarrayed platforms can be improved with methods that better estimate kinetic parameters rather than simply reporting relative binding information. These quantitative glycan sensors are enabled by an emerging class of nanoengineered materials that differ in their mode of signal transduction from traditional methods. Platforms that respond to mass changes include a quartz crystal microbalance and cantilever sensors. Electronic response can be detected from electrochemical, field effect transistor, and pore impedance sensors. Optical methods include fluorescent frontal affinity chromatography, surface plasmon resonance methods, and fluorescent single walled carbon nanotubes-(SWNT). Advantages of carbon nanotube sensors include their sensitivity and ability to multiplex. The focus of this work has been to develop carbon nanotube-based sensors for glycans and proteins. Before detailing the development of these new sensors, the thesis will begin with a very brief primer on glycobiology, its connection to medicine, and the advantages and limitations of existing tools for glycan analysis. In the second chapter we model the use of quantitative nanosensors in a weak affinity dynamic microarray (WADM) to simulate practical uses of these sensors in bioprocessing and clinical diagnostics. There is significant interest in developing new detection platforms for characterizing glycosylated proteins, despite the lack of easily synthesized model glycans or high affinity receptors for this analytical problem. In the third chapter we experimentally demonstrate 'proof of concept' of carbon nanotubebased glycan sensors. This is done with a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni2l complexes that act as fluorescent quenchers for SWNT embedded in a chitosan hydrogel spot to measure binding kinetics of model glycans. We examine as model glycans both free and streptavidin-tethered biotinylated monosaccharides. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (KD) for these pairs as free glycans (106 and 19 [mu]M respectively) and streptavidin-tethered (142 and 50 [mu]M respectively) were found. The absolute detection limit for the first-generation platform was found to be 2 pg of glycosylated protein or 100 ng of free glycan to 20 pg of lectin. Glycan detection (GlcNAc-streptavidin at 10 [mu]M) is demonstrated at the single nanotube level as well by monitoring the fluorescence from individual SWNT sensors tethered to GafD lectin. Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (KD of 250 ± 10 [mu]M). We are also able to identify the locations of "strong-transducers" on the basis of dissociation constant (4 sensors with KD < 10 [Mu]) or overall signal modulation (8 sensors with > 5% quench response). We report the key finding that the brightest SWNT are not the best transducers of glycan binding. SWNT ranging in intensity between 50 and 75% of the maximum show the greatest response. The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a high throughput method to profile glycans without protein labeling or glycan liberation pretreatment steps. In the fourth chapter we move from detection of model glycoproteins (streptavidin with biotinylated glycans) to a more applied problem: detection of antibodies and their glycosylation. We do this with a second generation array of SWNT nanosensors in an array format. It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (KD), using three different examples of binding IgG with Protein-A as the recognition site, including polyclonal human IgG (KD [mu] = 19 [mu]M, [sigma]2 = 1000 [mu]M2 ). murine IgG (KD = 4.3 [mu]M, 2= 3 [mu]M 2), and human IgG from CHO cells (KD [mu] = 2.5 nM, [sigma]F2 = 0.01 RM2). Second, we show that an array of nanosensors can uniquely monitor weakly-affined analyte interactions via the increased number of observed interactions. One application involves monitoring the metabolically-induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself. One great limitation to these practical applications, common to other new sensor developments, are the constraints of large, bulky, and capital-intensive excitation sources, optics, and detectors. In the fifth chapter we detail the design of a lightweight, field-portable detection platform for SWNT based sensors using stock parts with a total cost below $3000. The portable detector is demonstrated with antibody detection in our lab and onsite at a commercial facility 3700 miles away with complex production samples. Along the course of developing these sensors, there was a need to analyze noisy data sets from signal nanotubes (Chapter 3) to determine distinct binding states. NoRSE was developed to analyze highfrequency data sets collected from multi-state, dynamic experiments, such as molecular adsorption and desorption onto carbon nanotubes. As technology improves sampling frequency, these stochastic data sets become increasingly large with faster dynamic events. More efficient algorithms are needed to accurately locate the unique states in each time trace. NoRSE adapts and optimizes a previously published noise reduction algorithm (Chung et al., 1991) and uses a custom peak flagging routine to rapidly identify unique event states. The algorithm is explained using experimental data from our lab and its fitting accuracy and efficiency are then shown with a generalized model of stochastic data sets. The algorithm is compared to another recently published state finding algorithm and is found to be 27 times faster and more accurate over 55% of the generalized experimental space. This work is detailed in Chapter 6. Future uses of these sensors include in vivo reporters of protein biomarkers. In Chapter 7, three-dimensional tracking of single walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated for this purpose. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map corral volumes (0.27-1.32 Im 3), determine an active transport velocity (455 nm/s), and calculate local viscosities (54-179 cP) within the cell. With respect to the future use of SWNTs as sensors in living cells, we conclude that the sensor must change the fluorescence signal by at least 4-13% to allow separation of the sensor signal from fluctuations due to rotation of the SWNT when measuring with a time resolution of 32 ms. In the final chapter we draw conclusions from the development of this carbon nanotube-based sensor for glycan analysis and show the start of future work with arrays of SWNT sensors for glycoprofiling. / by Nigel F. Reuel. / Ph. D.