Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 151-165). / Immunization is a powerful approach for the prevention and control of infectious disease, however despite the successes of modem vaccine development, there remain several notable obstacles for the advancement of vaccine-mediated improvements in global healthcare. Many of the current limitations in vaccine availability and administration are the result of obligate needle-based delivery, which in addition to contributing to reduced speed, ease, and compliance in administration, has been shown to contribute to reduced overall safety due to needle re-use and needle-based injuries. Needle-based vaccine delivery to immunologically passive tissues such as muscle may limit efficacy, thus motivating the targeting of more inherently potent immune-competent sites. These inherent limitations of needle-based vaccination on global health have led to a strong impetus to develop needle-free vaccination strategies which have the potential to improve vaccine efficacy and availability, enhance the ease, speed, and safety of vaccine administration, and reduce vaccination associated costs world-wide. Here we present the design and preclinical testing of several parallel materials strategies for the noninvasive delivery of subunit vaccines to the skin. We have utilized laser ablative micro-molding of poly(dimethylsiloxane) to generate bio resorbable poly(lactide-co-glycolide) micro-structured skin patches bearing -100 micron-scale needles arrayed across their surface. Upon topical application, these 'microneedle arrays' are able to safely, and painlessly insert into the immune-competent epidermal skin layers to generate microscopic conduits through which otherwise impermeant vaccines and therapeutics are able to passage into the body. We have leveraged this approach in combination with layer-by-layer (LbL) directed assembly to generate vaccine-loaded conformal coatings on the surface of these microneedle arrays, which are then delivered into the skin through topical patch application. The construction of coatings containing antigen-expressing plasmid DNA (pDNA), together with immune-stimulatory RNA, and degradable cationic polymers provided tunable control over vaccine dosage, rapid and effective vaccine delivery in murine and primate skin models, and potent immunogenicity against a model HIV antigen in mice. In this case, DNA vaccine delivery was able to elicit strong functional CD8' T cell and humoral responses matching or exceeding the potency of in vivo electroporation, currently the most promising approach for clinical DNA delivery in humans. Further efforts have explored the use of LbL for encapsulation and delivery of soluble and particulate protein subunit vaccines, giving enhanced generation of diverse and potent humoral responses in mice. In other work, we have developed an approach enabling rapid delivery of micron-scale degradable polymer matrices or hydrogel depots using dissolvable composite microneedle structures for the delivery of vaccines with programmable kinetics. These efforts have demonstrated the potential of persistent vaccine release on tuning immune potency following non-invasive microneedle delivery, including induction of potent effector and memory CD8* T cell responses and more powerful and diverse antigen-specific humoral responses. Finally, we have developed an approach for simple loading and delivery of clinically advanced recombinant adenoviral vaccine vectors from sugar-glass coatings on bioresorbable microneedles. Formulation in microneedle coatings improved vaccine stability at room temperature and preclinical testing of these vaccine patches in mice and nonhuman primates demonstrated equivalent immunogenicity compared to parenteral injection, eliciting strong systemic and disseminated mucosal CD8' and CD4* T cell responses to a model HIV antigen. These cellular responses were correlated with a similarly potent systemic and mucosal humoral response, together suggesting the utility of this approach for non-invasive adenoviral immunization in a model close to humans. Together these results strongly demonstrate the potential of materials engineering strategies for the effective formulation, delivery, and release of recombinant vaccines by microneedle patches targeting the skin. In addition to the significant practical advantages enabled by microneedle delivery including improved safety, convenience, and storage, we have shown that advanced formulation strategies paired with controlled release are able to initiate humoral and cellular adaptive immunity more potently than possible through parenteral injection. Comprehensive tests in both mice and primates have suggested that these principles may be broadly applied to enhance various recombinant vaccination strategies potentially targeting numerous disease targets. Finally, initial tests performed in nonhuman primates have indicated the promise of engineered microneedle approaches for successful translation to humans. Overall, these findings provide a strong basis for the continued development of similar vaccination strategies for the comprehensive transformation of conventional vaccination enabling significant vaccine-mediated improvements in global health. / by Peter C. DeMuth. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 135-148). / Polymeric microparticles have a wide variety of uses, ranging from traditional applications in paints and coatings, to specialized applications in medical therapeutics and diagnostics. For biological applications - including drug delivery, analytical assays, and tissue engineering - it is important to tailor the interactions between the microparticles and their external environment. To do this, it is necessary to precisely control the physical and chemical properties of the engineered microparticles. Recently, it has become apparent that in addition to particle chemistry, the physical properties of a particle - for example, size, shape, internal structure, and mechanical deformability - play an important role in determining particle behaviour in biological environments. However, it remains largely unknown exactly how these various physical properties influence particle behaviour and function, and how these properties should be exploited for different applications. This thesis focuses on the development and characterization of polymeric hydrogel microparticles with well-controlled physical and chemical properties, and shows several applications of these custom microparticles. In particular, we explore particle motifs inspired by biological entities, designing particles with different shapes, internal structure, and mechanical deformability, functionalized with proteins and nucleic acids. We employ microfluidic tools for synthesis and characterization of these hydrogel microparticles, and also investigate the interaction of functionalized particles with nucleic acids and cells, in the context of biomolecule detection and specific cell capture, respectively. Based on the microfluidic particle synthesis technique, stop flow lithography, we fabricate custom particles - including non-spherical 3D capsules and 2D extruded cylindrical rings with systematically varied internal architecture. We design microfluidic channels to study the flow and deformation of these particles, investigating the effects of internal structure, size, and stiffness on passage through microfluidic constrictions. We expand on this work, designing a microfluidic platform to specifically position particles in hydrodynamic traps, based on particle physical properties. This platform enables subsequent encapsulation of immobilized particles in monodisperse, isolated aqueous droplets. We demonstrate the platform's utility with chemically functionalized microparticles enabling sensitive, multiplexed microRNA detection. To further explore the interactions of functionalized microparticles with biological systems, we study how antibody-functionalized microparticles of varying shape can capture specific cells for future diagnostic applications. / by Lynna Chen. / Ph. D.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / With constantly increasing demand for lightweight power sources, electrode architectures that eliminate the need for conductive and organic additives will increase mass specific energy and power densities. The increased demand for lightweight power is coupled with increasing device miniaturization. As the scale of devices decreases, current battery technologies add mass on the same scale as the device itself. A dual functional electro-mechanical material that serves as both the device structural material and the power source would dramatically improve device integration and range for powered movement. To address the demand for lightweight power with the objective of a dual functional electro-mechanical material, the M 13 bacteriophage was used to create novel 3-dimensional nano-architectures. To synthesize 3-dimensional nanowire scaffolds, the M13 virus is covalently linked into a hydrogel that serves as a 3-dimensional bio-template for the mineralization of copper and nickel nanowires. Control of nanowire diameter, scaffold porosity, and film thickness is demonstrated. The nanowire scaffolds are found to be highly conductive and can be synthesized as free-standing films. To demonstrate the viability of the 3-dimensional nanowire networks for electrical energy storage, copper nanowires were galvanically displaced to a mixed phase copper-tin system. These tin based anodes were used for lithium rechargeable batteries and demonstrated a high storage capacity per square area and stable cycling approaching 100 cycles. To determine the viability of the 3-dimensional nanowire networks as dual functional electro-mechanical materials and the mechanical stability of processing intermediates, phage hydrogels, aerogels, and metal nanowire networks were examined with nano-indentation. The elastic moduli of the metal networks are in the range of open cell metal foams The demonstration of 3-dimensional virus-templated metal nanowire networks as electrically conductive and mechanically robust should facilitate their implementation across a broad array of device applications to include photovoltaics, catalysis, electrochromics, and fuel cells. / by F. John Burpo. / Sc.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 103-123). / Objectives: 1) To perform a quantitative comparison of proteins released to media on combination with cytokine (IL-1[beta[ or TNF-[alpha]) and Injury as compared to either treatment alone, and to thus identify proteins which may be responsible for the synergism seen between cytokine and injury in causing catabolism of cartilage in vitro. 2) To identify proteins which contribute most to some commonly observed phenotypes on treatment of cartilage with cytokine or injury or both. Methods : Cartilage explants from calves were treated with (i)IL-1 (10 ng/ml), (ii)TNF[alpha] (100 ng/ml), (iii)Injurious compression (50% strain at 100%/sec) and IL-1[beta] (10 ng/ml) or (iv)Injurious compression (50% strain at 100%/sec) and TNF-[alpha](10 ng/ml), cultured for 5 days post treatment, and the pooled media collected, labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time of flight instrument. Peptides were identified and quantified using Protein PilotTM, and MATLAB scripts used to obtain protein ratios. These results were analyzed using different statistical techniques. Data from two iTRAQ experiments were combined to generate data for all possible injury and cytokine treatment conditions, and proteins on which injury and cytokines acted synergistically identified. PLSR analysis was performed using Unscrambler®X software with the combined data set to determine which proteins are most relevant to some observed phenotypes. The phenotypes chosen were sGAG released to media in 5 days post treatment, proline and sulfate incorporation rates on day 6 post treatment, and nitrite accumulation in media in 5 days post treatment Results and Discussion: TNF-[alpha]+injury and IL-[beta] +injury treatment conditions show a very high correlation with each other. Most cytosolic, ER lumen and nuclear protein levels are highly elevated with both cytokine+injury conditions, while ECM proteins are either highly down regulated or marginally elevated. Many collagen telopeptides are down regulated, possibly indicating reduced anabolism. However, attempts at repair exist, as shown by increased levels of TGF-[beta] and activin A, and reduced levels of LTBP1. Also, biglycan and lumican, SLRPs known to be involved in early development are significantly increased, possibly indicating repair attempts. Other SLRPs such as PRELP and chondroadherin are also highly elevated, with one or both injury+cytokine treatments. While MMPs are mildly down regulated or remain the same, ADAMTS1 increases with TNF-a+injury, indicating increased catabolism. Among ECM structural proteins, COMP shows high down regulation with TNF-[alpha]+injury, possibly due to reduced synthesis. Proenkephalin, a signaling molecule possibly involved in tissue/repair and apoptosis, AIMPI, a multifunctional proapoptotic, inflammatory and pro-repair cytokine and Annexin A5, a protein indicating mineralization and apoptosis are all highly elevated with cytokine+injury indicating heightened apoptosis and/or repair. When results of two 4-plex iTRAQ experiments are combined to obtain data for all possible combinations of injury and cytokine, we again find a very high correlation between TNF-a+injury and IL-1 +injury (-95%), slightly higher than the correlation between TNF-[alpha] alone and IL-[beta] alone (-90%), and much higher than the correlation of either cytokine+injury condition with cytokine alone (-70%) or injury alone (-75%). / (cont.) This shows that IL-1[beta] and TNF-[alpha] in combination with injury act through very similar pathways in chondrocytes to produce their effect on cartilage tissue. TNF-a and injury were seen to act synergistically in a positive fashion on aggrecan, CILP-2, COL6A3 and histone H4, and in a negative fashion on SPARC and IGFBP7, suggesting that these proteins may be involved in causing synergism between injury and cytokine in releasing sGAG to the media. A PLSR analysis shows that SPARC and IGFBP7 project close to proline and sulfate incorporation, and far away from sGAG, indicating that SPARC and IGFBP7 may be proteins involved in anabolism. The highest phenotype-protein positive correlations obtained using PLSR are sGAG with Perlecan, SAA3, Complement factor B, CILP-2 and pleiotropin, indicating that all these 5 proteins are associated strongly with catabolism and can serve as markers of catabolism. The correlation of inflammatory proteins SAA3 and complement factor B with sGAG indicates the role of inflammation with catabolism. Conclusion: The combination of injury and cytokine affects tissue differently at a molecular level as compared to either chemical or mechanical stresses alone. Increased catabolism and increased attempts at tissue repair are observed due to a combination of injury and cytokine, and a combination of injury and cytokine may thus serve as a useful model to study OA in vitro. / by Krishnakumar Swaminathan. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The degradation of articular cartilage is the hallmark in the pathogenesis of osteoarthritis (OA). It still remains largely unknown which precise mechanisms initiate cartilage degradation. However, risks factors include traumatic joint injury that results in immediate upregulation of inflammatory cytokines within the joint, as well as direct mechanical damage to the cartilage, factors known to contribute to the onset of OA and its progression. The first aim of this thesis focused on elucidating the importance of post-injury mechanical loading of cartilage. An in vitro model was used to simulate aspects of joint injury: mechanically damaged cartilage was co-cultured in the presence of inflammatory cytokines (TNF-Q and IL-6). Intermittent dynamic compression was then applied to simulate different strain levels known to exist in vivo after joint injury. Strain-dependent modulation of aggrecan biosynthesis and degradation, aggrecanase cleavage of aggrecan, chondrocyte gene expression profiles and changes in cell viability (apoptosis) were observed. Results imply that appropriate biomechanical stimuli can be beneficial during rehabilitation for post traumatic OA (PTOA) treatment. In the second aim, a combination therapy of insulin-like growth factor-1 (IGF-1) and the glucocorticoid dexamethasone (Dex) was tested as a potential therapeutic for PTOA. The effects of this combination were examined at the transcriptional and protein levels in the presence of IL-i a. Our results showed that the combination of IGF- 1 and Dex significantly improved aggrecan biosynthesis, blocked aggrecan and collagen proteolysis and loss, and rescued cell viability. These dramatic results could not be achieved by using either IGF-1 or Dex alone, thus providing strong support for the concept and use of a combination therapy for PTOA treatment. Dex is used to relieve inflammation and pain for short term OA treatment; however, it has not been studied as a potential disease-modifying drug for OA. In the last aim, the pro-survival role of Dex was investigated at the signaling, gene expression, and protein levels. Results suggest that Dex inhibits caspase-dependent apoptosis pathways, possibly through suppression of the phosphorylation of JNK and NF-kB/ixB signaling pathways. Taken together, these studies support the use of glucocorticoid treatment for inflammation-related cartilage cell death such as that found in PTOA. / by Yang Li. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / Cataloged from PDF version of thesis. "September 2012." / Includes bibliographical references (p. 97-110). / Vaccines have benefited global health by controlling or eradicating life threatening diseases. With better understanding of infectious diseases and immunity, more interest has been placed on stimulating mucosal immune responses with vaccines as mucosal surfaces function as a first line of defense against infections. Progress made in nanoparticle research, in particular the successful use of liposomes for drug delivery, has made liposomes an attractive candidate for vaccine delivery. Here, we investigate the efficacy of using a novel nanoparticle system, Interbilayer Crosslinked Multilamellar Vesicles (ICMVs), as a mucosal vaccine to stimulate mucosal and systemic CD8 immunity. We first assessed the ability of ICMVs to elicit mucosal CD8 response, against the model antigen ovalbumin (OVA), by administration of the nanoparticles through the lungs. We explored the use of 2 different Toll-like receptor agonists (TLRa), monophosphoryl lipid A (MPLA) and Polyinosinic:polycytidylic acid (poly (I:C) or pIC) added to ICMVs as adjuvants. Pulmonary administration of ICMV with both adjuvants was found to give the most potent CD8 T cell response in both systemic and mucosal compartments. We looked further into the quality of the immune response and detected the presence of antigenspecific memory CD8 T cells in the system at ~2.5 months after immunization. The majority of these cells were found to be effector memory cells (CD44hiCD62Llo) and expressed markers for long term survival (CD127hiKLRG1lo), suggesting that long term protection against infection can be induced by pulmonary delivery of ICMVs. We also explored using this system to deliver a model HIV peptide epitope, AL 1, and ICMV successfully induced CD8 response against this epitope. Animals immunized against AL 11 were challenged with a live virus expressing the same epitope and protection was seen only in the pulmonary ICMV treatment group. Virus was delivered via the lungs and viral titre was decreased in both the lungs and ovaries. Neither the soluble form of the vaccine or ICMV delivered via parenteral injection conferred protection. Safety of the ICMV system was also assessed and no significant negative effects were observed in body weight and histological analysis on lungs. Finally, mechanism of using nanoparticles as pulmonary vaccines was investigated to gain better understanding in how particulate vaccine and route of immunization improved the efficacy of a vaccine. Overall, this thesis describes a comprehensive study of systemic and mucosal CD8 responses generated by pulmonary delivery of a novel nanoparticle system. This data provides evidence that mucosal delivery of ICMVs can safely and effectively stimulate disseminated mucosal CD8+ T cells at sites relevant for protection against mucosal infection. A better understanding of nanoparticles for pulmonary immunization was also gained. / by Adrienne Victoria Li. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 140-160). / Despite great progress over the past several decades in the development and application of computer-aided tools for engineering enzymes for a vast array of industrial applications. rational enzyme design remains an ongoing challenge in biotechnology. This thesis presents a set of novel applications and methods for the computer-aided understanding and design of enzyme activity. In the first part. we apply biophysical modeling approaches in order to design non-native substrate specificity in a key enzymatic step (the thiolase-catalyzed condensation of two acyl-CoA substrates) of an industrially useful de novo metabolic pathway. We present a model-guided. rational design study of ordered substrate binding applied to two biosynthetic thiolases. with the goal of increasing the ratio of C6/C4 products formed by the 31HIA pathway, 3-hydroxyhexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly ten-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elonglation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes. In the second part, we apply methods from machine learning to an ensemble of reactive and non-reactive, but "almost reactive" molecular dynamics trajectories in order to elucidate catalytic drivers in another industrially important model enzyme system, ketol-acid reductoisomerase. Using a small number of molecular features, we show that we can identify conformational states that are highly predictive of reactivity at specific time points relative to the progress of the prospective catalytic event and also that provide mechanistic insight into the reaction catalyzed by this enzyme. We then present a novel theoretical framework for evaluating the contribution to the overall catalytic rate of the conformational states found in the previous part to be predictive of reactivity. Leveraging a computational enhanced sampling technique called transition interface sampling, we show that trajectories sampled in such a manner as to selectively visit the conformations predicted to be characteristic of reactivity exhibit rate constants many orders of magnitude greater than trajectories not required to visit these reactive conformations. The results of this analysis illustrate the importance of incorporating dynamical information into existing frameworks for biocatalyst design. / by Brian M. Bonk. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 169-179). / Optical traps have played a central role in the exploration of biological systems through the examination of molecular motors, biopolymers, and many other interactions at the nano and micro length scales. This thesis seeks to extend the applications of optical trapping instrumentation and the knowledge of biological systems by building new tools, expanding traditional measurements and developing new assays. First, an economical design of a high-end optical trap is presented as a teaching implement for an undergraduate lab. In addition to equipment specifications and construction directions, three experimental modules highlighting concepts in biology and physics are put forward including single molecule measurement of protein motor torque and the mechanical properties of DNA. A second optical trap design is developed to promote the integration of optical forces and semiconductor materials. This project provides a non-invasive method for control, construction, and measurement that leverages existing semiconductor fabrication techniques while retaining the nanometer position resolution and piconewton force sensitivity of an optical trap encouraging applications in MEMS, microfluidics, and single molecule studies. To better understand the properties of components of biological assembly, assays for single molecule measurement of adhesion force and kinetic off rate are established and carried out for short 12 amino acid sequences previously selected to adhere to glass surfaces and sapphire substrates. Finally, the mechanism of motility for the biological motor kinesin is investigated in depth using the optical trap in two assays. One researches motility in a heterodimeric kinesin with one motor head unable to hydrolyze ATP. The second establishes the force generation mechanism of kinesin through selective mutation of the N-terminal coverstrand segment of the enzyme. / by David Collins Appleyard. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 122-131). / This work investigates the roles that peptides play in the fields of antimicrobials, surface functionalization, carbon capture, and biocatalysis. The results demonstrate that peptides, sometimes dismissed for their lack of complexity, can have a breadth of applications. First, the killing kinetics of a pore-forming, engineered antimicrobial peptide (CM15) were imaged using a high-speed atomic force microscope (HS-AFM). The fast time resolution of the HS-AFM (13 seconds per image) enabled characterization of the initial stages of the killing of live Escherichia coli cells. The results suggested that the killing process of CM15 is a combination of a time-variable incubation phase and a more rapid execution phase, offering an interesting parallel between antimicrobial-peptide-induced death and mammalian cell apoptotic death. As a follow-up, an engineered peptide (2K1) with high affinity toward oxide surfaces was used to functionalize a diverse set of materials, including titanium dioxide, zinc, and stainless steel. After demonstrating that 2K1 works as affinity tag for small molecules and fusion proteins, a 2K1-CM15 peptide was made in an attempt to develop a single-step, facile antimicrobial functionalization of oxide surfaces. Second, motivated by the role of peptides in mineralization processes, the yeast Saccharomyces cerevisae was engineered to display peptides and proteins that enhanced the capture of CO2 . An industrial-scale CO2 mineralization process was designed using this engineered yeast with an associated cost of $52 per tonne of CO2 . The effect of the engineered yeast on the process was significant - the cost of CO2 capture was decreased by 8.5-13.5%, as compared to a process with no biological components. Finally, M13 bacteriophage (M13 phage) was established as a temperature stable, highlymultivalent biocatalytic scaffold through display of engineered histidine-biased peptides. A protocol for generating histidine-biased peptide libraries displayed on the major coat protein (pVII) of M13 phage was developed. By analogy to known histidine-based active sites, seven sequences were chosen from amongst hundreds of sequenced histidine-biased pVIII peptides. Two demonstrated esterase activity with a ... 170 that matches, and a ... 4 mM that is only 20-fold lower than, that reported for a commputationally designed esterase. / by Roberto Juan Barbero. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 50-53). / Purpose: This research develops a modeling approach and an implementation toolset to simulate reticular lamina displacement in response to excitation at the ear canal and to characterize the cochlear system in the frequency domain. Scope The study develops existing physical models covering the outer, middle, and inner ears. The range of models are passive linear, active linear, and active nonlinear. These models are formulated as differential algebraic equations, and solved for impulse and tone excitations to determine responses. The solutions are mapped into tuning characteristics as a function of position within the cochlear partition. Objectives The central objective of simulation is to determine the characteristic frequency (CF)-space map, equivalent rectangular bandwidth (ERB), and sharpness of tuning (QERB) of the cochlea. The focus of this research is on getting accurate characteristics, with high time and space resolution. The study compares the simulation results to empirical measurements and to predictions of a model that utilizes filter theory and coherent reflection theory. Method We develop lumped and distributed physical models based on mechanical, acoustic, and electrical phenomena. The models are structured in the form of differential-algebraic equations (DAE), discretized in the space domain. This is in contrast to existing methods that solve a set of algebraic equations discretized in both space and time. The DAEs are solved using numerical differentiation formulas (NDFs) to compute the displacement of the reticular lamina and intermediate variables such as displacement of stapes in response to impulse and tone excitations at the ear canal. The inputs and outputs of the cochlear partition are utilized in determining its resonances and tuning characteristics. Transfer functions of the cochlear system with impulse excitation are calculated for passive and active linear models to determine resonance and tuning of the cochlear partition. Output characteristics are utilized for linear systems with tone excitation and for nonlinear models with stimuli of various amplitudes. Stability of the system is determined using generalized eigenvalues and the individual subsystems are stabilized based on their poles and zeros. Results The passive system has CF map ranging from 20 kHz at the base to 10 Hz at the apex of the cochlear partition, and has the strongest resonant frequency corresponding to that of the middle ear. The ERB is on the order of the CF, and the QERB is on the order of 1. The group delay decreases with CF which is in contradiction with findings from Stimulus Frequency Otoacoustic Emissions (SFOAE) experiments. The tuning characteristics of the middle ear correspond well to experimental observations. The stability of the system varies greatly with the choice of parameters, and number of space sections used for both the passive and active implementations. Implication Estimates of cochlear partition tuning based on solution of differential algebraic equations have better time and space resolution compared to existing methods that solve discretized set of equations. Domination of the resonance frequency of the reticular lamina by that of the middle ear rather than the resonant frequency of the cochlea at that position for the passive model is in contradiction with Bekesys measurements on human cadavers. Conclusion The methodology used in the thesis demonstrate the benefits of developing models and formulating the problem as differential-algebraic equations and solving it using the NDFs. Such an approach facilitates computation of responses and transfer functions simultaneously, studying stability of the system, and has good accuracy (controlled directly by error tolerance) and resolution. / by Samiya Ashraf Alkhairy. / M.Eng.