Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The immunosuppressive effects of CD4⁺CD25⁺ regulatory T cells (Tregs) interfere with anti-tumor immune responses in cancer patients. In the first part of this work, we present a novel class of engineered Interleukin-2 (IL-2) analogues that antagonize the IL-2 receptor, for inhibiting Treg suppression. These antagonists are engineered for high affinity to the IL-2 receptor a subunit and low affinity to either the [beta] or [gamma] subunit, resulting in a signaling-deficient IL-2 analogue that sequesters the IL-2 receptor a subunit from wild type IL-2. Using this design, human and mouse IL-2 antagonists were generated with inhibition constants ranging from 200 pM to 5 nM in vitro. Genetic fusions with IgG2a Fc enhanced serum half-life up to 30 hours. In order to study the effects of IL-2 antagonism, Fc fragments with disrupted effector functions were used. Fc-antagonist fusions bound to but could not deplete peripheral Tregs. They downregulated CD25 on Tregs, but could not perturb Treg function in a syngenic tumor model, presumably due to the high sensitivity of the IL-2 receptor and a high threshold for antagonism in vivo. In the second part of this work, we present a novel multi-agent protein-based system for targeted siRNA delivery that provides potential advantages over other nanoparticle- and proteinbased delivery vehicles. In the first agent, the double stranded RNA binding domain (dsRBD) of human protein kinase R is used as an siRNA carrier, in fusion proteins that target epidermal growth factor receptor (EGFR). Targeted dsRBD proteins deliver large amounts of siRNA to endosomal compartments in an EGFR expressing cell line, but efficient gene silencing is limited by endosomal escape. The use of a second agent that contains the cholesterol dependent cytolysin, perfringolysin 0, enhances endosomal escape of siRNA. Targeted delivery of perfringolysin 0 induces gene silencing in a dose-dependent and EGFR-dependent manner. However, cytotoxicity of the cytolysin creates a narrow therapeutic window. Multiepitopic EGFR binders that induce EGFR clustering are explored as tools for enhancing gene silencing efficiency. Interestingly, they not only enhance gene silencing potency but also protect against toxicity from EGFR-targeted cytolysins, thus significantly widening the therapeutic window of this method. / by David Victor Liu. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2007. / Vita. / Includes bibliographical references. / Pretargeted radioimmunotherapy (PRIT) of cancer improves upon conventional radioimmunotherapy (RIT) by decoupling the pharmacokinetics of the targeting agent and the radioisotope. In order to improve upon PRIT, we have considered variables such as treatment setting and methodology, the transport and clearance characteristics of targeting agents, and the radionuclides used for therapy. PRIT has been modeled with the aim of examining the theoretical potential of PRIT under optimal conditions to kill every cell in malignant, avascular micrometastases. A mathematical model of PRIT was developed that combined a two-compartment pharmacokinetic model, antibody binding kinetics, diffusion and catabolism in tumor spheroids, and radiation dosimetry models for alpha- and beta-emitting radionuclides. This model demonstrated that it is theoretically possible to kill every cell in 100 tm radius micrometastases using 9Y- or 213Bi-based PRIT with acceptable toxicity as described. The therapeutic window for dosing radionuclide-carrying hapten was found to be strongly dependent on cell-specific parameters such as antigen concentration, void fraction, and the radiosensitivity parameter a, as well as on targeting agent molecular parameters such as the diffusivity and antigen-binding association rate. / (cont.) Surprisingly, the therapeutic window was insensitive to the radiosensitivity metric a/I, the targeting agent antigen-binding dissociation rate, and all pharmacokinetic parameters. Overall, 213Bi-based PRIT significantly outperformed 9Y-based PRIT in terms of the safe therapeutic time window for radiometal dosing and the degree of cell overkill that could be achieved. An attempt was made to isolate high-affinity scFv or linear peptide binders against the loaded metal chelate Ga-DOTA-biotin. Unfortunately, several different approaches led only to scFvs and linear peptides with at best micromolar affinity for Ga-DOTA-biotin. It is possible that Ga-DOTA-biotin is a difficult target against which to engineer high affinity binders due to the chelate's six-coordinate binding of the gallium ion, which may result in rapid exchange of the carboxyl arms of the chelate in solution. As an alternative approach to targeting agent design, an anti-CEA, anti-fluorescein single-chain bispecific diabody was designed, produced in S. cerevisiae and characterized. The full-length diabody (55 kDa) binds CEA expressed on the surface of colorectal cancer-derived SW1222 cells with a KI of 4.3 ± 2.5 nM, and also binds fluorescein while bound to CEA on the cell surface. / (cont.) Lastly, in order to assist in protein engineering via directed evolution, asymptotically optimal probability estimation was combined with numerical bootstrapping and non-linear curve fitting to make accurate predictions of the actual underlying diversities of populations based on small samples of data. / by Stefan C. Zajic. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 184-194). / Limiting the use of fossil fuels is vital to stemming climate change. Incorporation of renewable energy technologies into the grid, and the shift to electric vehicles for transportation increases the need for better energy storage media. Lithium-air (O₂) batteries are of great interest because they have high theoretical energy densities. However, conventional Li-O₂ batteries face challenges such as the use of volatile and flammable liquid electrolytes, side reactions between the electrolyte/electrode with oxygen reduction products, and high charging over-potentials that lead to poor cycle life. We address these challenges by developing non-flammable polymeric-based electrolytes and electrodes, and investigate their performance and stability in Li-O₂ batteries. In this thesis, we synthesized and studied the properties of a nonvolatile and nonflammable siloxane solid polymer electrolyte that can support Li-O₂ discharge, but show it is vulnerable to reaction with the desired Li2O₂ discharge product. We developed a screening tool that involves mixing commercial Li2O₂ with various polymers of interest for Li-O₂ batteries, and formulate polymer reactivity rules where the presence of electron-withdrawing groups on the polymer and adjacent hydrogen atoms make the polymer vulnerable to degradation. Of the polymers studied in contact with Li2O₂, poly(methyl methacrylate) was found to be stable, and then used as part of a gel polymer electrolyte with an ionic liquid (IL) and lithium salt. The Li/IL molar ratio in the GPE was shown to allow for a switch from a 2 e- to 1e- oxygen reduction chemistry, and the formation of ionic liquid-superoxide complexes as the discharge product. Exploiting this understanding of the influence of a bulky ionic liquid cation on the oxygen electrochemistry, we incorporate ammonium salts in a Li-O₂ battery and show it can also support discharge and lead to > 0.5 V reduction in charging overpotential when compared to lithium salts. Finally, we explore an electron conducting polymer electrode poly(3, 4- ethylenedioxythiophene) (PEDOT) as a Li-O₂ electrode and show the polymeric surface allows for oxygen reduction and Li2O₂ formation. Coupling fundamental understanding with material selection can empower the design of next generation Li-O₂ batteries. / by Chibueze Vincent Amanchukwu. / Ph. D.

Thesis (Sc.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2003. / Includes bibliographical references. / An experimental study was performed to characterize the effects of different patterns of membrane depolarization on undifferentiated pheochromocytoma (PC 12) cells. In response to chronic depolarization, cytoplasmic free Ca2+ ([Ca2+]i) levels increased transiently, then decreased to intermediate levels that were maintained for more than 90 minutes. Short pulses of depolarization, from 1 to 5 minutes in length, also resulted in transient increases in [Ca2+]i. Following a recovery period in a polarized state, a second increase in [Ca2+]i could be induced by repeated depolarization. The activity of signal transduction pathways was also characterized following patterns of depolarization. Chronic depolarization elicited transient (less than 10 minutes in duration) activation of the mitogen-activated protein (MAP) kinases Erkl and Erk2, while the cAMP-response element binding protein (CREB) remained active at intermediate levels for over 60 minutes. Pulsatile depolarization also stimulated Erkl/2 and CREB activation, and the rate of deactivation of the MAP kinases was not found to depend on pulse duration. Lastly, both the MAP kinases and CREB were successfully reactivated by pulsatile stimulation, following recovery periods of greater than 10 minutes in duration. Thus, pulsatile stimulation may be a means of maintaining signaling activity over long periods of time The effect of depolarization on gene expression was determined. Gene expression profiling of PC12 cells over the course of 8 hours following a single or double pulse of stimulation confirmed that a diverse set of genes were regulated by electrical activity. These genes included neural differentiation-specific genes, as well as genes involved in cell cycle control and intracellular signaling. / (cont.) Finally, the gene expression profiles of PC12 cells that were depolarized for one week were also measured. Prolonged depolarization induced a new set of diverse genes, and some of which also have roles in cell cycle control and differentiation. Thus, we conclude from our studies that different patterns of electrical stimulation can have numerous instructive influences on undifferentiated neuron-like cell lines. / by Amir H. Nashat. / Sc.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (p. 249-261). / State-of-the-art methods for simulating viscoelastic flows couple the conservation equations for mass and momentum with a model from kinetic theory that describes the microstructural state of the polymer. Introduction of appropriate numerical discretization and boundary conditions for these equations leads to a hybrid simulation for studying the dynamic behavior of polymeric liquids in complex geometries. This approach represents a rare example of a successful multiscale solution of a physical problem, as it allows investigation of arbitrary models of kinetic theory. The simulations, however, are not amenable to standard numerical techniques for system-level stability, bifurcation, and control analysis as this requires closed form equations. These simulation either use stochastic descriptions for the polymer microstructure that cannot be reduced to closed form, or involve equations for the evolution of a distribution of polymer conformations, which can only be written in closed form by invoking mathematical closure approximations that can have a significant qualitative impact on the predictive ability of these simulations. The focus of this thesis was to develop a novel numerical method that can enable hybrid simulations to perform system-level analysis of polymeric flows. This numerical approach has been applied directly to kinetic theory models and hybrid simulations to obtain stationary states and associated bifurcations and stability information. The method is general in its applicability in that it treats kinetic theory models and hybrid simulations as black boxes that are then used to obtain system-level information without any modification. The methods developed here are illustrated in a variety of problems. / (cont) Steady state results have been obtained for the non-interacting rigid dumbbell model in steady shear, and for the free-draining bead-spring chain model in both steady shear and uniaxial elongation that are in excellent agreement with previous studies and steady state computed from direct integration. The method is also applied to a hybrid simulation for the pressure-driven flow of non-interacting rigid dumbbells in a planar channel with a linear array of equally spaced cylinders. The computed steady state is in agreement with direct integration and qualitatively matches previous computations with closed models. Bifurcation analysis has been performed for the Doi model at equilibrium with the Onsager excluded volume potential. This analysis agrees with previous studies and accurately predicts the isotropic-nematic transition and turning point for the unstable to stable transition on the prolate solution branch. Bifurcation analysis has also been performed for the Doi model in the weak shear flow limit for the Maier-Saupe excluded volume potential. It is found that stable stationary solutions are lost at a limit point beyond which time-periodic tumbling orbits are the only stable solution. This transition occurs via an infinite period global bifurcation, while the limit point approaches a threshold value as the shear rate approaches zero. This result matches a recently published scaling analysis and demonstrates the ability of the method to provide general bifurcation analysis of kinetic theory models. Stability analysis of the fiber-spinning process for polymeric fluids has also been performed by using a hybrid simulation that couples the one-dimensional conservation equations for mass and momentum with a stochastic description for the configuration fields of the Hookean dumbbell model. The steady-state velocity profiles are in good agreement with previous studies with the Oldroyd-B model. / (cont) The analysis predicts onset of the draw resonance instability via a Hopf bifurcation and subsequent stabilization via second Hopf bifurcation in draw ratio parameter space. This result is in good agreement with experimentally observed behavior during polymer fiber-spinning. / by Zubair Anwar. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 22-26). / Genetically encoded ratiometric sensors can provide valuable mechanistic understanding of biological systems. Characterization of cellular response of these sensors is the first step in validating their use. Here, we characterize the response of a genetically encoded H₂O₂ sensor, HyPer, expressed in HeLa cells. Using quantitative fluorescence microscopy, we found significant heterogeneity in HyPer response among the cell population. Further analysis showed that the variation in HyPer response was dependent on expression of HyPer protein as well as on cell cycle phase. Cells with higher levels of expressed HyPer protein showed a stronger HyPer response to H₂O₂. Cells synchronized in S-phase showed a weaker HyPer response than unsynchronized cells. It was determined that this weaker response could be a function of higher antioxidant capacity in S-phase cells. The dependence of HyPer response on these factors needs to be accounted for to avoid experimental artifacts. / by Sohail Feroz Ali. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1964. / Vita. / Includes bibliographical references (leaves 194-195). / by Gary L. Falkenstein. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 210-229). / New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigations. The ultimate challenge lies in replicating the native cell/tissue environment ex vivo. Certain key features of living tissues such as the three dimensionality, biophysical and biochemical microenvironment cannot be readily replicated in traditional culture platforms. Moreover, the capability for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small populations is lacking. In this thesis, we developed a novel multiparameter microfluidic-based cell culture platform. The system permits 2D or 3D culture of cells on/in biologically-derived or synthetic hydrogel scaffolds and allows for controlled flow rates, pressure and concentration gradients while directly visualizing cellular response. In addition to the realtime and post-fixation imaging using optical microscopy, methods were developed to extend post-fixation analysis to transmission electron microscopy (TEM). The platform was subsequently used to demonstrate for the first time, two microfluidicbased 3D in vitro assays with direct relevance to tumor development and glaucoma. For the first assay, biochemical induced sprouting was demonstrated. Endothelial cells sprout from an intact monolayer to form multicellular capillary-like structures. Furthermore, using time-lapse microscopy the cellular dynamics during sprouting angiogenesis were observed with great detail, showing tip cell dynamics, cell division events and lumen formation. Of particular relevance to tissue engineering community, we demonstrated that endothelial cells when cultured for several days can assemble into vascular networks with open, perfusable lumen. Using this new system, we present novel findings and results supporting a potential mechanism for flow-mediated mechanical regulation of angiogenesis by transendothelial fluid flow. We demonstrate that flow direction is sufficient to define an angiogenic ON or OFF state. The balance is tipped by forces generated at mechano-sensitive cell-matrix adhesions involving FAK-mediated signaling. These results provide one explanation for the bias towards angiogenesis occurring from the venous side of the circulation. For the second assay, an aqueous humor (AH) outflow model was developed. Subsequent proof-of-concept experiments confirmed its capability for studying the role of the inner wall endothelium in the regulation of AH outflow dynamics. / by Vernella V. V. Vickerman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 89-93). / T cells are orchestrators for adaptive immunity. Antigen recognition by T cells is mediated by the interactions between T-cell receptors (TCRs) and peptide-MHC (pMHC) molecules. How T cells can translate stimulatory external cues (e.g., TCRpMHC interactions where the peptides are derived from foreign proteins) to functional responses (e.g., proliferation), while not responding to self-pMHC has been a puzzle for decades. The ability to discriminate foreign antigens from self antigens demands extraordinary intricacy for the design and operation of signaling pathways. This problem is a special challenge since fluctuations at the microscopic level are ubiquitous in biochemical networks, due to stochastic nature of reactions and uncertainties in protein expressions. The prevalence of noise imposes further challenges for T cells to deliver biological functions reliably. The overarching theme of this thesis is to understand the role of stochasticity in T-cell signaling. Four problems have been selected for presentation in this thesis: 1. Fluctuation-driven transitions can drive cellular systems out of stable states and lead to spurious responses. We proposed a theoretical and computational framework to identify key reactions or species that are responsible for regulating such stochastic transitions. The identification of these critical components for network stability not only pinpoints key deleterious protein mutations, but also helps intelligently select drug targets. The semi-analytical method we derived using large-deviation theory and calculus of variations agrees well with computational costly brute-force simulations. Additionally, our framework unveils qualitative characteristics of key reactions regulating stochastic transitions. We believe that we have developed the first method to carry out fully stochastic sensitivity analyses using analytical calculations. 2. In collaboration with Dr. Jeroen Roose's lab at UCSF, we investigated the roles of RasGRP and SOS in the activation of ERK and P38 MAP kinase pathways. We extended established computational models developed in our lab and predicted that SOS' allosteric pocket is important for the magnitude and bimodal pattern of ERK activation, which was confirmed experimentally. The synergy between computational modeling and experimental studies enabled us to propose mechanistic models that incorporate features such as co-operativity and non-linearity thresholding to study P38 activation. These models were consistent with the experimental findings that SOS is preferentially more important than RasGRP for Rac-P38 activation and SOS' allosteric pocket has little effect on Rac-P38 pathways, and also generated numerous experimentally testable hypotheses. 3. While rare events, such as escapes from stable basins, take a long time (waiting time) to occur, they take little time to complete once they have started. We showed that for Markov processes characterized by detailed balance, successful transitions, on average, complete exactly as quickly as transitions in the opposite (non-rare) direction. We first provide a general proof by invoking time reversibility, and then elaborate the proof by considering two specific dynamics, namely, continuous-time Markov Chains with detailed balance and one-dimensional Langevin Dynamics. We employ ideas from measure theory and stochastic calculus. We conclude that rare events, once they happen, happen quickly, and speculate about extensions to nonequilibrium systems, such as viral escape. 4. While microscopic fluctuations complicate reliable functioning of biochemical networks, stochastic noise also offers enormous information about the underlying network that generates such noise. We present an effort to exploit the non-random structure of random noise for network topology identification. In particular, we applied linear noise approximations to two three-node network motifs, namely, incoherent feed-forward loop (IFF) and negative feedback loop (NFB), and obtained correlation functions governing the fluctuations of species copy numbers at steady state. We identified two signatures that can be used to discriminate IFF from NFB. This endeavor represents a first step toward understanding how, and to what extent, time-series data with fine time and length resolutions can be used to infer network structures. / by Ming Yang. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1998. / Includes bibliographical references. / by Lei Zhang. / Ph.D.