Thesis (B.S.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1961. / MIT copy bound with: Transient responses in gaseous fractionation by adsorption - analogue computer study / Donald D. Easson. 1961. / Includes bibliographical references (leaves 48-49). / by Nelson E. Stefany, Paul H. Fricke. / B.S.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Nanoparticles (NPs) are poised to have a tremendous impact on the treatment of many diseases, but their broad application is limited because currently they can only be administered by parenteral methods. Oral administration of NPs is highly preferred because of the convenience and compliance by patients, but remains a significant challenge because of the barriers presented by the gastrointestinal tract. In particular, transport across the intestinal epithelium limits efficient oral delivery of NPs. The neonatal Fc receptor (FcRn) mediates IgG antibody transport across epithelial barriers. It was discovered as the receptor in the neonatal intestine that transports IgG in breast milk from mother to offspring. However, FcRn is expressed into adulthood at levels similar to fetal expression. FcRn interacts with the Fc portion of IgG in a pH-dependent manner, binding with high affinity at acidic (<6.5) but not neutral pH (7.4). Targeting NPs to FcRn using IgG Fc fragments was hypothesized to enable orally administered NPs to be transported across the intestinal epithelium. FcRn-targeted NPs were formulated using poly(lactic acid)-b-polyethylene glycol (PLA-PEG) block copolymers and engineered to have particle sizes less than 100 nm with IgG Fc conjugated to the surface. Transepithelial transport of the NPs was first evaluated in an in vitro cell monolayer transport model using Caco-2 cells. FcRn-targeted NPs were transported across the monolayer at a rate twice that of non-targeted NPs. The transport rate was reduced significantly when excess IgG was added along with the FcRn-targeted NPs. Next, FcRn-targeted NPs were then evaluated using in vivo mouse models. Fluorescent FcRn-targeted NPs were observed with fluorescent microscopy crossing the intestinal epithelium and entering the lamina propria after oral administration. Using radiolabeled NPs, orally administered FcRn-targeted NPs were detected in the liver, lungs, and spleen with a mean absorption efficiency of 13.7% for FcRn-targeted NPs compared with only 1.2% for non-targeted NPs. Finally, insulin was encapsulated in the NPs to evaluate the FcRn-targeted NPs as a NPbased therapeutic. In wild-type mice, orally administered FcRn-targeted NPs containing insulin were able to generate a prolonged hypoglycemic response using a clinically relevant insulin dose of 1.1 U/kg. The response was specifically due to FcRn, as studies in FcRn knockout mice mitigated the enhanced response of the FcRn-targeted NPs. This technology has the potential to have an impact on the treatment of many diseases by enabling NP-based therapies to be administered orally. In addition, the encapsulation of drugs or biologics that are currently limited by low bioavailability into FcRn-targeted NPs may enable markedly more efficient oral delivery of the therapies. / by Eric M. Pridgen. / Ph.D.

Massachusetts Institute of Technology. Dept. of Chemical Engineering. Thesis. 1966. Sc.D. / Bibliography: leaves 229-234. / Sc.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2013. / "September 2013." Page 173 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 152-163). / Although tumor cells obtained from human patients by surgical biopsy, image-guided intervention, blood draws or fluid drainage (paracentesis, thoracentesis) are a valuable source for analyzing tumor cells, conventional means of proteomic analysis are limited. Highly sensitive and quantitative technologies for point-of-care and multiplexed analysis on small sample sizes are in great demand. To this end, we developed three technologies to improve our understanding of the molecular signatures of cancer in clinical samples. In the first section, we describe a diagnostic magnetic resonance (DMR) device that was developed for point-of-care analyses of human tumors. We optimized a magnetic nanoparticle assay to improve sensitivity and robustness of the DMR approach. The DMR device was tested by analyzing samples from 50 patients. The results were then validated in an independent cohort of 20 additional patients. DMR enabled quantification of multiple protein markers in all patients. Using a four-protein signature enabled us to achieve 96% accuracy for establishing cancer diagnosis, surpassing conventional clinical analysis by immunohistochemistry. Results also show that protein expression patterns decay with time, underscoring the temporal need for rapid sampling and diagnoses. Also, a surprising degree of heterogeneity in protein expression both across different patient samples and even within the same tumor was observed, which has important implications for molecular diagnostics and therapeutic drug targeting. In the second section we molecularly profiled tumor cells in ascites - peritoneal fluid frequently drained for symptomatic relief in advanced ovarian cancer (OvCA) patients. First, we profiled a comprehensive panel of 85 biomarkers in ovarian cancer and benign cell lines. From this data set, 31 markers were identified and profiled in a training set of human ascites samples (n=1 8). We identified an ascites-derived tumor signature termed ATCdx containing four markers which was then validated in a cohort of 47 patients (33 ovarian cancer and 14 control) and correctly identified all 33 ovarian cancer patients. Serial samples were obtained from a subset of patients' serial samples (n=7) and profiled, demonstrating that ATCs can be used to measure treatment response and differentiate responders from non-responders. Finally, we specifically designed a novel microfluidic enrichment chip that allows rapid visualization of cancer cells in heterogeneous ascites fluid. This chip requires small sample volumes (< 1 mL) and has single cell detection sensitivity. Furthermore, it is inexpensive to construct and can be easily fabricated using soft lithographic techniques, providing a point-of-care method that could potentially find widespread use for ATC analyses and diagnosis. In the final section, a multiplexed proteomic assay using a photocleavable DNA barcoding method was developed to multiplex protein detection in single cells. We tested 94 antibodies against common cancer markers to examine different treatment responses and heterogeneity at the single cell level. We then extended our analysis to human clinical samples to demonstrate the potential of protein-based measurements to assist in monitoring cancer therapy through differential changes before and after treatment. We show that protein based tumor profiles can provide sufficient information to predict treatment response. Finally, we examined interpatient variability and intratumoral heterogeneity of single cells with this highly sensitive assay. Together, these technologies can help overcome current clinical limitations and expedite advancements in cancer treatment. / by Vanessa M. Peterson. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009. / Includes bibliographical references. / This thesis is about identifying genetic interventions that improve the performance of targeted pathways in the metabolism of the bacterium Escherichia coli. Three case studies illustrate three disparate approaches to identifying genetic interventions: (i) combining metabolomic measurements with thermodynamic calculations to identify rate-limiting reaction steps in a target pathway; (ii) use of stoichiometric, optimization-based models of metabolism to predict target genetic interventions in silico; and (iii) the mutagenesis of promoter sequences to fine-tune the expression level of rate-limiting genes. These techniques can be classified by both the number of strain modifications created, and the number of variables measured in each. Taken together, the cases suggest that the best methods for identifying genetic interventions balance the number of strain modifications with the number of measured variables. The first case is butyrate production in recombinant E. coli. A strain of E. coli deleted for the production of lactate, ethanol, and acetate was designed to minimize competing pathways for carbon, and was unexpectedly found to exhibit oxygen auxotrophy. Expression of genes from Clostridium acetobutylicum resulted in production of 3-hydroxybutyric acid, but not butyric acid. / (cont.) The clostridial genes ptb and buk were capable of producing S-3-hydroxybutyric acid from the butyrate pathway intermediate metabolite S-3-hydroxybutyryl-CoA. In parallel, the intracellular concentrations of pathway metabolites was measured for a set of strains expressing the clostridial butanol biosynthesis pathway in various configurations. Comparison of measured pool sizes and pool sizes for thermodynamic equilibrium pinpointed the butyryl-CoA dehydrogenase step, encoded by bcd, as a bottleneck enzyme. Thus, points for genetic intervention are ptb, buk, and bcd. The second case is tyrosine overproduction in E. coli. Constraints-based models of E. coli metabolism proved incapable of predicting gene knockout targets. Therefore, to understand factors underlying tyrosine overproduction, the intracellular concentrations of amino acids were measured. In tyrosine overproducers, the intracellular concentrations of most proteinogenic amino acids were vastly perturbed relative to non-producing strains. This fact and thermodynamic considerations suggested that the transamination of p-hydroxyphenylpyruvate to tyrosine was near equilibrium, and thus that nitrogen supply may be limiting tyrosine production. Culture media amended with glutamate or glutamine, but not with a-ketoglutarate or other organic acids, increased tyrosine production in these strains more than 8-fold, showing that interventions which affect nitrogen supply are attractive targets for engineering tyrosine overproduction in E. coli. The last case addresses the question of what types of intervention are best. A series of 22 promoters with well-characterized, variable strengths was created by mutagenesis. This library was used to replace promoters for key genes in the biosynthesis of lycopene or biomass from glucose. These metabolic phenotypes exhibited strain-dependent optima with respect to the expression levels of the key rate-controlling genes genes. Promoter engineering thus shows that subtle genetic interventions can have profound effects on pathway function. / by Curt R. Fischer. / Ph.D.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1997. / Includes bibliographical references (leaves 160-171). / by Hongming Chen. / Sc.D.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1985. / Bibliography: leaves 123-132. / by Sorab R. Vatcha. / M.S.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The adaptive immune response is very important for our survival in that it gives us the capability of detecting a wide variety of foreign material, allows for the elimination of pathogens, and provides memory to protect against future attacks by the same pathogen. Key mediators of the adaptive immune response are CD4+ T cells, which depending on the cytokine milieu, and the activating conditions during antigen recognition, can differentiate into different effector T cells. One particular type of effector T cell, Th 17, is highly inflammatory and has been implicated in various autoimmune diseases, such as Multiple Sclerosis. Three chapters within this thesis investigate the conditions which lead to Th17 differentiation and the mechanisms involved in their regulation. Th 17 cells can be obtained in vitro by culturing naive CD4+ T cells with IL-6 and TGF-p under activating conditions. IL-6 was thought to primarily activate the transcription factor STAT3, which has been shown to be necessary for ThI7 differentiation. Numerous cytokines activate STAT3, but IL-6 is the most potent inducer of TH17 cells, so we sought to find out what is special about IL-6's induction of STAT3. In the first of these three chapters, we propose a simple genetic network which is capable of translating IL-6's high amplitude, transient STAT3 signal into a pro-inflammatory response and IL-10's low amplitude, sustained STAT3 signal into an antiinflammatory response. This network is able to predict that IL-6 and IL-10 would induce an indistinguishable anti-inflammatory response in SOCS3-/- cells where IL-6's STAT3 signal is sustained. In the second of these chapters, we continue our research into the origin of signal specificity in cytokine signaling by systematically characterizing the activation of STAT1 and STAT3 by IL-6, IL-10, IL-21, IL- 27, and different combinations of cytokines in CD4+ T cells. In this analysis we find that the ratio of STAT3 to STAT1 activated is the important quantity in determining whether or not a cytokine will be an inducer of TH17 differentiation (IL-6, IL-21) or an inhibitor (IL-27). We show that in the absence of STAT1, that IL-6 and IL-27 are both potent inducers of TH17 differentiation since they have similar STAT3 activation profiles. In the third of these chapters, we develop a simple algorithm for clustering gene activation profiles for intermediate numbers of genes measured (10-50) and use it to analyze a 96- hour time course of gene activation during Th 17 differentiation for a number of genes of interest. In order for T cells to differentiate into effector cells, they must first recognize antigen which is presented on the surface of an antigen presenting cell by a membrane-bound extracellular complex called MHC. The MHC have a groove which peptide fragments (antigen) are bound in. Without peptide loaded in the pocket, the MHC are quite unstable so they are synthesized with a generic peptide fragment loaded. A protein, DM, is responsible for stabilizing the MHC while the generic peptide is ejected and the peptide fragment of interested is loaded. Two chapters within this thesis investigate the role of DM in peptide loading / unloading and attempt to characterize the interaction of DM with MHC. / by Kevin D. Fowler. / Ph.D.

Thesis (M.S.) Massachusetts Institute of Technology. Dept. of Chemical Engineering, 1947. / Bibliography: leaf 109. / by David W. Bareis. / M.S.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2002. / Includes bibliographical references (p. 166-184). / Metabolic engineering refers to the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant DNA technology. It has been used to investigate and modify intermediary metabolism in a variety of microbial organisms of biotechnological interest. An emerging area of application for metabolic engineering is medicine, in particular the study of metabolic disorders, where analysis and manipulation of metabolic pathways have obvious relevance. Central to metabolic engineering is the notion that metabolism results from the concerted and coordinated activities of biochemical pathways connected through shared intermediates in the form of common reactants, products, and catalysts. According to this "metabolic network" concept, an enhanced understanding of metabolism and cellular function is obtained by considering the component biochemical reactions together, rather than individually. In this light, this thesis work was motivated by the idea that the application of metabolic engineering analysis to biological systems relevant to human disease has the potential to provide valuable insight into the biochemical underpinnings behind metabolic disorders. In the present dissertation, this idea was explored by investigating a metabolic disorder known clinically as hypermetabolism that is associated with the systemic inflammatory response to severe injury. At the whole body level, hypermetabolism is characterized by elevated resting energy expenditure and increased turnover of proteins, fatty acids, and carbohydrates. / (cont.) If this state persists over a period of days to weeks, the patient is predisposed to muscle wasting, progressive organ dysfunction, multiple organ failure, and ultimately death. Unfortunately, existing nutritional therapies are inadequate for preventing the onset of persistent hypermetabolism, because many of the mechanistic details of this process are poorly understood. An important player in the hypermetabolic response to injury is the liver, which responsible for synthesizing healing factors from muscle protein derived amino acids, converting carbohydrate and lipid fuel resources to useful energy substrates, and eliminating waste products generated by these processes. In order to better understand the biochemical underpinnings behind injury derived hypermetabolism in the liver, the following specific aims were addressed: 1) to develop and validate tissue and organ models of injury for the liver; 2) to delineate activity changes in the major metabolic pathways in the liver during the developmental period of hypermetabolism; and 3) to build diagnostic tools for detecting and grading the injury derived metabolic abnormalities in the liver. A particularly useful metabolic engineering tool is metabolic flux analysis (MFA), which refers to a methodology whereby intracellular reaction fluxes are estimated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of a network of reactions ... / by Kyongbum Lee. / Ph.D.