Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008. / Vita. / Includes bibliographical references. / Tumor targeting drugs that selectively treat cancerous tissue are promising agents for lowering the morbidity and mortality of cancer. Within this field, antibody treatments for cancer are currently being developed for both imaging and therapeutic applications. A major limitation with this class of drugs is the poor distribution and low uptake in tumor tissue. Poor distribution leaves some cells completely devoid of treatment, while others experience marginally toxic concentrations that could foster drug resistance. The low overall uptake in vascularized tumors constrains the therapeutic index and lowers signal to noise ratios for imaging applications. Since antibody therapies are currently used to treat both bulk tumors and residual disease, an understanding of the limitations in targeting prevascular metastases and vascularized tumors is required. In order to circumvent the current limitations with antibody therapies, the underlying causes must first be determined. In this thesis, the various steps in tumor localization of antibodies are analyzed in order to determine which steps are limiting uptake and distribution. Mathematical models are developed that indicate the distance antibodies and other binding macromolecules will penetrate into tumors and micrometastases. These models can also estimate the maximum uptake and time course of antibody concentration in tumors. The experimental distribution of a CEA binding antibody is measured in tumor spheroids and a mouse xenograft system to validate the model predictions. Using dimensional analysis of the fundamental transport rates that occur in tumors and micrometastases, two main groups determine the distance antibodies will penetrate in tumor tissue. / (cont.) The clearance modulus indicates whether antibody persistence in the blood is sufficient to allow the drug to reach all cells in the micrometastasis or vascularized tumor. The Thiele modulus, defined for antibody transport in tumors, relates the internalization and catabolism of bound antibodies on cancer cells to the maximum distance the antibodies will reach in the tissue. These groups are related to the overall time course and maximum uptake in tumors, indicating when all cells will be targeted, and what factors determine this limit. These models can aid in experimental design, data interpretation, and strategies to improve uptake. / by Greg M. Thurber. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1996. / Includes bibliographical references (p. 145-152). / by Brian Hylton Cumpston. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004. / Includes bibliographical references (p. 289-302). / (cont.) The effects of recombinant protein production were observed through expression analysis of induced, uninduced, and Empty-Vector cultures. As expected, recombinant α₁AT production led to increased expression of heat-shock genes, including proteases and chaperones that are known to be involved in α₁AT degradation. Based on expression analysis data, production of recombinant α₁AT also resulted in catabolite repression and decreased amino acid biosynthesis. This work demonstrates the utility of DNA microarrays in analyzing and improving microbial fermentations. Global expression studies have suggested several strategies for increasing the resistance of bioprocesses to the damaging effects of oxygen and recombinant protein production. / Both exposure to oxygen and recombinant protein production are known to have adverse effects on microbial fermentation, including increased proteolytic and oxidative damage to the product. In an effort to characterize the effects of these stresses on the cell, DNA microarrays were used to monitor global gene expression of E. coli producing recombinant human αl-antitrypsin (α₁AT) during exposure to defined aeration conditions. Recombinant α₁AT has been shown to undergo oxygen-dependent degradation during production in E. coli, due in part to activation of the heat-shock response. The goal of this work is to better understand the effects of oxygen in order to improve this recombinant protein production process. In order to study the effects of oxygen extremes, global expression analysis was performed on α₁AT-producing cultures exposed to pure nitrogen, air, and pure oxygen. The most notable effects of oxygen exposure were those of superoxide. This reactive oxygen species is generated upon oxygen exposure and is known to oxidize iron-sulfur clusters. In response to hyperoxic conditions, the SoxRS stress response was activated, as were genes involved in iron uptake and the Isc and Suf repair systems for Fe-S clusters. Supplementation of iron in the growth medium resulted in expression changes consistent with improved formation of Fe-S clusters. Iron supplementation also decreased superoxide stress at the expense of a short-term increase in the peroxide (OxyR) stress response. In addition, iron supplementation dramatically reduced the oxygen dependence of recombinant α₁AT degradation. Regeneration of Fe-S clusters is proposed to improve protein folding and clusters is proposed to improve protein folding and limit activation of the heat-shock response. / by William Bryon Perry. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1994. / Includes bibliographical references (leaves 174-179). / by Charles Winslow Krueger. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007. / Includes bibliographical references (p. 93-97). / Assays that predict toxicity are an essential part of drug development and there is a demand for efficient models to better predict human responses. The in vivo micronucleus (MN) assay is a robust toxicity test that assesses the genotoxic effect of drugs on adult bone marrow (BM) using the metric of genotoxic damage to the reticulocyte population in mice. An in vitro correlate to this assay might facilitate extension to human cells and thus provide a highly predictive genotoxicity assay. As first steps in developing a toxicity assay, this thesis work (a) adapted a fetal liver-based in vitro erythropoietic culture system to induce optimized erythropoietic growth from the lineage-marker-negative (Lin) population in adult BM, as adult hematopoietic tissue is ultimately a feasible source of cells; and (b) demonstrated that exposure to alkylating agents induces physiological MN-formation in erythroid populations derived in vitro. The potential for increased efficiency in this in vitro model depends on the ability to stimulate terminal erythroid differentiation at an optimal level from adult BM. / (cont.) With this goal in mind, this thesis work employed experimental design strategies, erythroid-specific growth measurements, and multi-linear regression to model erythropoietic growth in this system and thus estimate the relative sensitivity of Lin7 BM to erythropoietic growth parameters, including Erythropoietin, Stem Cell Factor, p02, and Fibronectin, among others. From these erythroid-specific growth measurements, it is estimated that >1500 MN assays can be conducted using the BM of a single mouse. This throughput represents a significant improvement over the current in vivo test, which assays a single condition per mouse. This thesis work then quantified the genotoxic response to three alkylating agents (1,3-bis(2-chloroethyl)- 1-nitrosourea [BCNU], N-methyl-N -nitro-N-nitrosoguanidine [MNNG], and methylmethane sulfonate [MMS]) in this culture system and detected a significant cytotoxic response and concomitant increase in MN incidence in reticulocytes. / (cont.) This increase in MN frequency provides a clear signal of the genotoxic events that likely lead to global toxicity, and thus mimics the physiological hematopoietic response to alkylating chemotherapeutics. In addition, this thesis work determined that DNA repair-deficient (MGMT -/-) BM displayed sensitivity to genotoxic exposure in vivo compared with wild-type (WT) BM, and that this phenotypic response was reflected in erythropoietic cultures. These findings suggest that this in vitro erythroid MN assay is capable of screening for genotoxicity on BM in a physiologically reflective manner. Finally, responses to genotoxicants during erythroid differentiation varied with exposure time, facilitating the study of genotoxic effects at specific developmental stages. / by Joseph F. Shuga. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 161-177). / The demand for clean energy in portable applications is driving the development of high specific energy batteries, which will enable automobiles powered by electricity derived from renewable energy sources such as solar and wind. Lithium-air batteries are a promising avenue for advancing the energy storage capabilities beyond that of current lithium-ion technology. These batteries face a number of challenges which prevent their practical implementation in devices. This thesis explores possible mitigations for two of these challenges: (1) the high charging overpotential and (2) the volatility and poor oxygen conduction of liquid electrolytes in Li-air batteries. In the first part, Vulcan carbon-based electrodes were developed where chemically-synthesized lithium peroxide was included during the electrode preparation process. Variants of these electrodes which further included noble metal catalyst nanoparticles (Au, Pt, and Ru) were also prepared, and Pt and Ru were both demonstrated to begin oxidizing Li₂O₂ 500 mV lower than required for carbon-only or Au-containing electrodes. Using a differential electrochemical mass spectrometer (DEMS) designed and built over the course of this thesis, we showed that Ru-containing electrodes produce oxygen throughout the oxidation of Li₂O₂, while Pt generated both carbon dioxide and oxygen, indicative of electrolyte decomposition. These results served as a foundation for future efforts to develop solid catalysts for the oxidation of Li₂O₂ in Li-air batteries. In the second part, Li-O₂ devices using a solid electrolyte based on poly(ethylene oxide) (PEO) were developed. The discharge performance at room temperature and 60 °C was characterized, with dramatically higher discharge capacity and rate capability achievable at the elevated temperature. DEMS was used to show that the gases evolved during charging in argon were sensitive to the temperature of charging, with additional carbon dioxide observed at and above 50 °C. Finally, the autoxidation of PEO at 60 °C in Li-O₂ environments was studied, where NMR and DEMS measurements showed that the rate of PEO autoxidation increases with increasing applied potential, and that this reaction has a significant impact after only one charging cycle, identifying another condition that must be met for stable and practical Li-air batteries. / by Jonathon R. Harding. / Ph. D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2000. / Includes bibliographical references. / Catalytic combustion of methane has been widely studied as an alternative to gasphase homogeneous combustion. It allows combustion to occur at high levels of excess air, leading to more complete reaction and reduced hydrocarbon emissions. It further enables combustion to proceed at lower temperatures, significantly reducing the NO" production. Noble metal systems, such as platinum and palladium, have been studied as combustion catalysts. However, noble metal clusters tend to sinter or vaporize at the high combustion temperatures. Recently, complex oxides have been examined for methane combustion due to their enhanced thermal resistance. Barium hexaaluminate (BHA) was chosen for this research, since its unique crystalline structur~ has the potential to suppress grain growth at high temperatures. A novel reverse microemulsion-mediated sol-gel processing technique was developed to synthesize non-agglomerated BHA nanoparticles with high surface areas and thermal stability. The reverse microemulsion also provided a unique medium to achieve highly dispersed active species on BHA nanoparticles to enhance the catalytic performance for methane combustion. Reverse microemulsions of water/i30-octane and water/cyclohexane were successfully stabilized with a non-ionic surfactant system consisting of polyethoxylated and linear alcohols. The water/iso-octane system was found to be ideal for the sol-gel mediated synthesis, since it required only a small amount of surfactants for stabilization. Quasi-elastic light scattering and small-angle neutron scattering showed that at low water contents, the reverse microemulsions consisted of slightly polydisperse discrete aqueous domains with a core-shell structure. Systems with higher water contents could be best described with a bicontinuous structure with intermixed water and oil domains. The water/iso-octane system was found to possess excellent stability under the conditions required for reverse microemulsion-mediated sol-gel processing of BHA materials. The composition of the reverse microemulsion governed the morphology of the aqueous domains, which in tum determined the shape and aggregation of the BHA particles derived. Non-agglomerated nanospheres were recovered from reverse microemulsions with water volume fractions of 0.05-0.15. At higher water contents, percolation between aqueous domains in the system became significant, yielding BHA particles with filament-like morphologies. The water:alkoxide ratio in the sol-gel process determined the relative rates of hydrolysis and polycondensation reactions. At a relatively high water:alkoxide ratio of ~100 times the stoichiometric value, the stability of the reverse microemulsion was preserved throughout the aging process. Well-defined, high surface area BHA nanoparticles were successfully recovered from the medium by freeze drying. Residual surfactants and volatiles were best removed by supercritical drying. The resulting materials were crystallized at a relatively low temperature of 1050°C due to their superb chemical homogeneity. Surface areas of >160 m2/g and ultrafine grain sizes of S30 nm were retained by these BHA nanoparticles after calcination at l 300°C. Active transition metal and rare earth oxides could be deposited with ultrahigh dispersion on BHA nanoparticles during their aging in the reverse microemulsion medium. BHA nanoparticles coated with Mn02 and Ce02 clusters showed light-off (defined as 10% conversion of an air stream containing 1 vol% CH4) at remarkably low temperatures of ~400°C, rivaling noble metal systems. These novel materials sustained their activity for extended periods at temperatures in excess of 1000°C, demonstrating a thermal stability superior to other existing combustion catalysts. The performance of BHA-based materials was evaluated in an atmospheric burner operated under realistic industrial conditions. Catalyst systems were washcoated onto monoliths of different compositions and microstructures. Nickel foams and fiber reinforced honeycombs demonstrated excellent thermal shock resistance; the latter were preferred for high-temperature operations since they would give rise to negligible pressure drops. In our catalytic combustor design, nanocrystalline PdO/Ce02-BHA was used as the low-temperature ignition catalyst to initiate the reaction by 250°C. A mid temperature catalyst, such as MnOi-BHA or Ce02-BHA nanocomposite, was utilized to promote reaction in the range of 600-1000°C. A flame-supporting catalyst, consisting of pure nanostructured BHA was employed to stabilize the flame at temperatures up to 1300°C. Using this multi-stage catalyst design, flames of ultra-lean methane:oxygen ratios (0.2S~0.5) were ignited and sustained for extended periods over multiple heating-cooling- restarting cycles. This system successfully eliminated NOx production with no unburned hydrocarbon emissions in an effective catalytic methane combustion process. / by Juan Andrey Zarur Jury. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1992. / Includes bibliographical references. / by David James Mooney. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 127-138). / The optimization, kinetic investigation, or scale-up of a reaction often requires significant time and materials. Silicon microreactor systems have been shown advantageous for studying chemical reactions due to their small volume, rapid mixing, tight temperature control, large range of operating conditions, and increased safety. The primary goal of this thesis is to expand the capabilities of automated microreactor systems to increase their scope and efficiency. An automated optimization platform is built utilizing continuous inline IR analysis at the reactor exit, and a Paal-Knorr reaction is chosen as the first example chemistry. This reaction, where both the first and second reaction steps affect the overall rate, leads to a more complex conversion profile. A steepest descent algorithm is first used to optimize conversion and production rates. The steepest descent algorithm tends to move slowly up the production rate ridge, significantly reducing efficiency. This issue is overcome by using a Fletcher-Reeves conjugate gradient method, which finds the constrained optimum in much fewer experiments. The conjugate gradient algorithm is then further improved upon by incorporating a hybrid Armijo line search and bisection contraction method. However, the conversion is only about 40% at the maximum in production rate. A further optimization is performed using a quadratic loss function to penalize conversions of less than 85%. This optimization of production rate led to an optimum at higher residence time, where a conversion of 81% is achieved. In the conventional view of reaction analysis, batch reactions are thought to be significantly more efficient in generating time-course reaction data than flow reactions, which are generally limited to steady-state studies. By taking advantage of the low dispersion in microreactors, successive fluid elements of the reactor may be treated as separate batch reactors. By continuously manipulating the reaction flow rate and tracking the total reaction time of each fluid element, time-course data analogous to that conventionally derived from batch reactors are generated and shown to be in agreement with steady-state results. Palladium-catalyzed carbonylation and CN-coupling reactions are used extensively in laboratory synthesis and industrial processes. The primary reaction studied involves the coupling of bromobenzene and morpholene with the addition of one or two carbonyl groups. The dependence of reaction conversion and selectivity on temperature, CO pressure, and Pd concentration are investigated using GC and IR analysis. A temperature ramp method is employed to rapidly investigate temperature effects on reaction rate and selectivity. The experiments reveal a change in the rate determining step at approximately 120 °C and corresponded well with GC data taken at several setpoints. In addition, the activation energy of the lower temperature regime as determined by this IR analysis is found to be very similar to that found by GC analysis, the experiments for which took significantly longer both to perform and analyze. Furthermore, the data collected from these experiments are used to fit a kinetic model. Multicomponent reactions (MCRs) are important to drug discovery by affording complex products in only a single step. By linking two of these MCRs, a Petasis boronic acid-Mannich reaction and an Ugi reaction, six different components could be incorporated in a relatively short time. The kinetics of each reaction are investigated with online UPLC analysis, allowing for quantification of a number of reaction components, including monitoring the formation of side products that were unknown prior to experimentation. A simple microcalorimeter is built using thermoelectric elements and a silicon microreactor to experimentally determine the heats of reaction during flow to allow for understanding the heat transfer needs for scale up. The result from the nitration of benzene, which has a heat of reaction of -117 kJ/mol, is -118.6 +/- 2.4 kJ/mol. The experimentally determined values are close to the known values; however, there is significant noise in the output during the reaction due to the two-phase nature of the reaction. The Paal-Knorr reaction is further investigated to determine the limits of sensitivity of the microcalorimetry system. A continuous concentration ramp experiment is performed with online IR analysis, enabling the thermoelectric output to be adjusted for reaction rate to determine the sensitivity to the heat of reaction. Below approximately 2 M, the sensitivity decreases rapidly, largely due to noise in the temperature control and concentration. To attempt to correct for the former, a calorimetry system with larger thermal mass is constructed and shown to decrease the sensitivity limit to 1 M, corresponding to a heat flow of approximately 0.05 W. / by Jason Stuart Moore. / Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2001. / Includes bibliographical references (leaves 183-210). / A central goal in metabolic engineering is the design of more productive biological systems by genetically modifying metabolic pathways. In this thesis we report such an optimization in the bacterial strain Corynebacterium glutamicum that is employed for the fermentative production of various amino acids such as lysine. The main goal of the research presented here was the application of metabolic and genetic engineering tools in order to investigate the role of the pyruvate node in cellular physiology. This was achieved by integrating the tools of bioinformatics, recombinant DNA technology, enzymology and classical bioengineering in the context of control and genetically engineered strains of C. glutamicum. First, the main anaplerotic pathway responsible for replenishing oxaloacetate, namely pyruvate carboxylase was targeted. After fruitless attempts to establish an in vitro enzymatic activity for this enzyme, our efforts were directed towards its gene identification. This was achieved by designing PCR primers corresponding to homologous regions among pyruvate carboxylases from other organisms. Utilizing these primers, a PCR fragment was isolated corresponding to part of the gene of the C. glutamicum pyruvate carboxylase. The sequence of the complete gene was finally obtained by screening a C. glutamicum cosmid library. In order to investigate the physiological effect that this enzyme has on lysine production, recombinant strains and deletion mutants were generated. The presence of the gene of pyruvate carboxylase in a multicopy plasmid is not sufficient to yield a significant overexpresssion of this enzyme in C. glutamicum. Contrary to our expectations, overexpression of pyruvate carboxylase has a negative effect on lysine production but improves significantly the growth properties of C. glutamicum. A metabolic model was developed according to which pyruvate carboxylase overexpression increases the carbon flux that enters the TCA cycle, thus the higher growth. However due to the presence of a rate-limiting step in the lysine biosynthesis pathway this increased carbon flux does not translate into higher lysine production. The role of aspartokinase, the first step in lysine biosynthesis, was explored as such a potential bottleneck. Its overexpression proves to increase the amount of lysine produced, however it leads to a lower growth and finally a lower productivity. Since pyruvate carboxylase and aspartokinase have opposite effects on cell physiology, the combination of the overexpression of these two enzymes was finally studied. By this simultaneous overexpression, we achieved to create a C. glutamicum recombinant strain with similar growth as that of the control but higher lysine production and productivity. In the context of exploring the physiological role of pyruvate carboxylase, a biotinylated enzyme, two other enzyme that utilize biotin were also investigated namely acetyl-CoA-carboxylase and biotin ligase. The first enzyme was purified to completion and its N-terminal as well internal amino acid sequences were obtained. A cosmid from the C. glutamicum cosmid library was identified that most likely contains the gene of the latter enzyme. In summary, in the present work we have achieved to prove unequivocally the presence of pyruvate carboxylase in C. glutamicum. We have also achieved to characterize the second biotinylated enzyme in this organism, namely acetyl-CoAcarboxylase. The physiological effect of both pyruvate carboxylase and aspartokinase was established and a metabolic model was developed based on these experimental results. This model finally led us to the construction of a new recombinant strain with improved lysine productivity. As such, this work stands as one of the few examples of a primary metabolite production improvement using metabolic engineering techniques. / by Mattheos A.G. Kofas. / Ph.D.