Dissertations / Theses on the topic 'Biological engineering design'

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Investigating protein location and concentration is critical to understanding function. Reagentless biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can detect analytes of interest with high temporal and spatial resolution. However, because these biosensors require laborious empirical screening to develop, their adoption has been limited. Hence, we establish design principles that will facilitate development. In this thesis, we first develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. Following these guidelines, we use the human tenth type III fibronectin domain to engineer new binders against several ligands of the EGFR receptor. Using these binders and others, we design and characterize biosensors based on various target analytes, scaffolds and fluorophores. We observe that analytes can harbor specific binding pockets for the fluorophore, which sharply increase the fluorescence produced upon binding. Furthermore, we demonstrate that a fluorophore conjugated to locally rigid surfaces possesses lower background fluorescence. Based on these newly identified properties, we design biosensors that produce a 100-fold increase in fluorescence upon binding to analyte, about a 10-fold improvement over the previous best biosensor. In order to improve the methodology of reagentless biosensor design, we establish a method for site-specific labeling of proteins displayed on the surface of yeasts. This procedure allows for the screening of libraries of sensors for binding and fluorescence enhancement simultaneously. Finally, we explore an alternative sensor design, based on competitive inhibition of fluorescence quenching.
by Seymour de Picciotto.
Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, February 2012.
"February 2012." Cataloged from PDF version of thesis.
Includes bibliographical references (p. [153]-191).
The focus of my research is the development of technology for building compound biological systems from simpler pieces. I designed BioScaffold parts, a family of variable regions that can be inserted into a DNA sequence so that at a later time another set of pieces can be substituted for each variable. The variable regions are selective so that a particular piece can be targeted to each region. I have used this technique to assemble protein domains, tune the expression levels of proteins and remove BioBrick scars. BioScaffold parts can be used in combination with BioBrick Standard Biological Parts to create and store devices with tunable components. I developed simplified methods to produce and examine SbpA, a protein that can either self associate into two-dimensional crystals or bring together fused enzymes when divalent cations such as calcium are added to the protein monomers. My fast and easy purification protocol allows SbpA to be produced under non-denaturing conditions as well as examination of the native state of the protein monomers before crystallization. The absence of a white precipitate when calcium is added to SbpA monomers concentrated to 1 mg/ml provides a simple visual screen that indicates that the protein has failed to crystallize. I also developed a protocol to embed SbpA crystallized on lipid monolayers in trehalose for electron microscopy, allowing creation of a 7 Å resolution map for SbpA. I created a cells-on-paper system to compose, isolate, and subsequently destack and examine different cell types grown in sheets of ordinary filter paper and maintained in a humidified incubation chamber. I found that E coli diluted in LB broth and then applied to filter paper grew at rates similar to the same culture spotted on agar plates. Track etch membranes could be used to isolate different cell types, while still allowing chemical communication between the layers. Use of plasmids that contain fluorescent proteins allowed the behaviour of cells to be tracked using a scanner after destacking of the layers. The cells-on-paper system can be used both to test and construct modular synthetic systems composed of bacterial ensembles and to create and examine the behavior of compositions of cell types typically found in biofilms.
by Julie Erin Norville.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 219-233).
Mechanism-based chemical kinetic models are increasingly being used to describe biological signaling. Such models serve to encapsulate current understanding of pathways and to enable insight into complex biological processes. Despite the growing interest in these models, a number of challenges frustrate the construction of high-quality models. First, the chemical reactions that control biochemical processes are only partially known, and multiple, mechanistically distinct models often fit all of the available data and known chemistry. We address this by providing methods for designing dynamic stimuli that can distinguish among models with different reaction mechanisms in stimulus-response experiments. We evaluated our method on models of antibody-ligand binding, mitogen-activated protein kinase phosphorylation and de-phosphorylation, and larger models of the epidermal growth factor receptor (EGFR) pathway. Inspired by these computational results, we tested the idea that pulses of EGF could help elucidate the relative contribution of different feedback loops within the EGFR network. These experimental results suggest that models from the literature do not accurately represent the relative strength of the various feedback loops in this pathway. In particular, we observed that the endocytosis and feedback loop was less strong than predicted by models, and that other feedback mechanisms were likely necessary to deactivate ERK after EGF stimulation. Second, chemical kinetic models contain many unknown parameters, at least some of which must be estimated by fitting to time-course data. We examined this question in the context of a pathway model of EGF and neuronal growth factor (NGF) signaling. Computationally, we generated a palette of experimental perturbation data that included different doses of EGF and NGF as well as single and multiple gene knockdowns and overexpressions. While no single experiment could accurately estimate all of the parameters, we identified a set of five complementary experiments that could. These results suggest that there is reason to be optimistic about the prospects for parameter estimation in even large models. Third, there is no standard formulation for chemical kinetic models of biological signaling. We propose a general and concise formulation of mass action kinetics based on sparse matrices and Kronecker products. This formulation allows any mass action model and its partial derivatives to be represented by simple matrix equations, which enabled straightforward application of several numerical methods. We show that models that use other rate laws such as MichaelisMenten can be converted to our formulation. We demonstrate this by converting a model of Escherichia coli central carbon metabolism to use only mass action kinetics. The dynamics of the new model are similar to the original model. However, we argue that because our model is based on fewer approximations it has the potential to be more accurate over a wider range of conditions. Taken together, the work presented here demonstrates that experimental design methodology can be successfully used to improve the quality of mechanism-based chemical kinetic models.
by Joshua Farley Apgar.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 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 (leaves 180-203).
The aim of this thesis is to consider how fundamental engineering principles might best be applied to the design and construction of engineered biological systems. I begin by applying these principles to a key application area of synthetic biology: metabolic engineering. Abstraction is used to compile a desired system function, reprogramming bacterial odor, to devices with human-defined function, then to biological parts, and finally to genetic sequences. Standardization is used to make the process of engineering a multi-component system easier. I then focus on devices that implement digital information processing through transcriptional regulation in Escherichia coli. For simplicity, I limit the discussion to a particular type of device, a transcriptional inverter, although much of the work applies to other devices as well. First, I discuss basic issues in transcriptional inverter design. Identification of key metrics for evaluating the quality of a static device behavior allows informed device design that optimizes digital performance. Second, I address the issue of ensuring that transcriptional devices work in combination by presenting a framework for developing standards for functional composition. The framework relies on additional measures of device performance, such as error rate and the operational demand the device places on the cellular chassis, in order to proscribe standard device signal thresholds. Third, I develop an experimental, proof-of-principle implementation of a transcriptional inverter based on a synthetic transcription factor derived from a zinc finger DNA binding domain and a leucine zipper dimerization domain. Zinc fingers and leucine zippers offer a potential scalable solution to the challenge of building libraries of transcription-based logic devices for arbitrary information processing in cells.
(cont.) Finally, I extend the principle of physical composition standards from parts and devices to the vectors that propagate those parts and devices. The new vectors support the assembly of biological systems. Taken together, the work helps to advance the transformation of biological system design from an ad hoc, artisanal craft to a more predictable, engineering discipline.
by Reshma P. Shetty.
Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.
Page 322 blank. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 295-321).
Living cells naturally use gene regulatory networks termed "genetic circuits" to exhibit complex behaviors such as signal processing, decision-making, and spatial organization. The ability to rationally engineer genetic circuits has applications in several biotechnology areas including therapeutics, agriculture, and materials. However, genetic circuit construction has traditionally been time- and labor-intensive; tuning regulator expression often requires manual trial-and-error, and the results frequently function incorrectly. To improve the reliability and pace of genetic circuit engineering, we have developed biomolecular and computational frameworks for designing genetic circuits. A scalable biomolecular platform is a prerequisite for genetic circuits design. In this thesis, we explore TetR-family repressors and the CRISPRi system as candidates. First, we applied 'part mining' to build a library of TetR-family repressors gleaned from prokaryotic genomes. A subset were used to build synthetic 'NOT gates' for use in genetic circuits. Second, we tested catalytically-inactive dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and synthetic sgRNAs to build transcriptional logic gates with high on-target repression and negligible cross-talk, and connected them to perform computation in living cells. We further demonstrate that a synthetic circuit can directly interface a native E. coli regulatory network. To accelerate the design of circuits that employ these biomolecular platforms, we created a software design tool called Cello, in which a user writes a high-level functional specification that is automatically compiled to a DNA sequence. Algorithms first construct a circuit diagram, then assign and connect genetic "gates", and simulate performance. Reliable circuit design requires the insulation of gates from genetic context, so that they function identically when used in different circuits. We used Cello to design the largest library of genetic circuits to date, where each DNA sequence was built as predicted by the software with no additional tuning. Across all circuits 92% of the output states functioned as predicted. Design automation simplifies the incorporation of genetic circuits into biotechnology projects that require decisionmaking, control, sensing, or spatial organization.
by Alec A.K. Nielsen.
Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 157-169).
A major effort of systems biology is the building of accurate and detailed models of biological systems. Because biological models are large, complex, and highly nonlinear, building accurate models requires large quantities of data and algorithms appropriate to translate this data into a model of the underlying system. This thesis describes the development and application of several algorithms for simulation, quantification of uncertainty, and optimal experimental design for reducing uncertainty. We applied a previously described algorithm for choosing optimal experiments for reducing parameter uncertainty as estimated by the Fisher information matrix. We found, using a computational scenario where the true parameters were unknown, that the parameters of the model could be recovered from noisy data in a small number of experiments if the experiments were chosen well. We developed a method for quickly and accurately approximating the probability distribution over a set of topologies given a particular data set. The method was based on a linearization applied at the maximum a posteriori parameters. This method was found to be about as fast as existing heuristics but much closer to the true probability distribution as computed by an expensive Monte Carlo routine. We developed a method for optimal experimental design to reduce topology uncertainty based on the linear method for topology probability. This method was a Monte Carlo method that used the linear method to quickly evaluate the topology uncertainty that would result from possible data sets of each candidate experiment. We applied the method to a model of ErbB signaling. Finally, we developed a method for reducing the size of models defined as rule-based models. Unlike existing methods, this method handles compartments of models and allows for cycles between monomers. The methods developed here generally improve the detail at which models can be built, as well as quantify how well they have been built and suggest experiments to build them even better.
by David Robert Hagen.
Ph. D.

A method of minimizing the total active disc area, required for the removal of soluble biochemical oxygen demand (SBOD), by a multi-stage rotating biological contactor (RBC) operating steady-state is proposed. Both deterministic and stochastic inputs are considered.
Total active disc area and the number of RBC stages are optimized for the deterministic case, and are then incorporated into a risk-based method of assigning per-stage active disc areas. The risk of the final stage SBOD exceeding a fixed effluent standard is evaluated by taking into account the variable nature of the influent flowrate and SBOD concentration. Bivariate normal, lognormal and shifted lognormal distributions are considered as models for the input random variables. The effluent SBOD probability density function is obtained according to the method of transformation of random variables. Illustrative examples are presented.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 226-238).
To date, there has been limited usage of biomass and agricultural residues in rural areas as a form of renewable energy, mainly due to the expensive costs involved in collecting and transporting raw biomass. A decentralized biomass torrefaction system has the potential to upgrade the quality and transportability of distributed biomass residues in situ, thereby creating additional localized economic values and mitigating the environmental consequences associated with open burning of the excess biomass residues. Nonetheless, most existing biomass torrefaction systems so far have been designed for large-scale, centralized deployment, and are unsuitable to be scaled down in decentralized applications due to their high level of sophistication and capital cost. We propose a biomass torrefaction system based on the concept of torrefaction in a low-oxygen environment. By eliminating the stringent requirements of an inert torrefaction environment, we demonstrated that we can greatly simplify the reactor design and derive a laboratory-scale system that is also scalable. We proceeded to build and validate this torrefaction system with respect to different operating conditions and types of biomass. Using a quantitative definition for torrefaction severity, we were also able to relate the various fuel user requirements in real life back to the fundamental reactor operations. By quantifying in detail the overall energy performance, pressure requirements, and transient timescales, we also demonstrated how such a reactor system can be operated at scale, as well as the various design improvements that can further boost the performance of a scaled-up system. Therefore, this work builds the foundation towards the development of a low-cost, small-scale, and portable torrefaction system that can potentially be widely deployed in rural areas.
by Kevin S. Kung.
Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The high mortality rates associated with uncontrolled bleeding motivate hemostatic material development for traumatic injuries. Uncontrolled, or hemorrhagic, internal bleeding requires hemostatic materials that can be directly delivered to or target the bleeding locations. To address these needs, injectable systems are being developed that: (1) generate artificial clots independent of the coagulation cascade or (2) interact with blood components to accelerate or otherwise improve coagulation processes. Hemostatic materials designed for internal bleeding can save lives in the battlefield, en route to emergency rooms, and in the operating room. This thesis first focuses on developing a shear-thinning hydrogel for injection onto bleeding surfaces and into ruptured vasculature. Based on in vitro assays of hydrogel performance, it was amenable to clinical delivery methods and reduced whole blood clotting times by 77%. In vivo bleeding models showed reduced blood loss and improved survival rates following a lethal liver injury. The hydrogel was also used as an embolic agent, where its occlusive potential in an anticoagulated model was demonstrated. Next, recombinant protein-based hemostatic materials were expressed to modulate clotting kinetics and performance. By incorporating clot interacting peptide sequences (CIPs) into a protein scaffold, a family of multifunctional fibrinogen like proteins (MFLPs) was developed and assayed. Clot turbidity, an indication of fibrin clot formation, was increased among enzyme-interacting CIPs. Mimicking the polymerization mechanism of fibrinogen, knob sequences were shown to be procoagulant at low concentrations by increasing clot turbidity, reducing clotting times, and inhibiting plasmin lysis. Finally, to understand the impact of hemostats on clot structure, imaging procedures were developed to systematically assess hemostatic materials and their influence on clot architecture. Static and dynamic approaches were developed to quantify the activity of hemostats based on the spatial distribution of fibrinogen, red blood cells, and platelets around hemostat surfaces. Quantification of these hemostat-blood component interactions resulted in a unique pattern of interactions for each hemostat studied. These techniques could serve as a screening technique for hemostats and improve characterization prior to in vivo assays. Taken together, the results highlight multiple approaches to address internal bleeding and opportunities to improve in vitro characterization of hemostats using microscopy.
by Reginald Keith Avery.
Ph. D.

Lyophilization is a common method used for long term stability of pharmaceutical and biopharmaceutical products that are unstable in the liquid state for a substantial period of time. Currently, formulation and cycle development are often determined empirically. Although this approach is gradually changing as scientific publications reveal more about the nature of protein stability, nevertheless the lack of material during early stage development prevents large screening investigations to identify optimum formulations. The use of high throughput methods coupled with factorially designed experiments enables a far more efficient and wider screening and optimisation of viable formulations for development. This thesis explores the use of micro titre plates for formulation development with emphasis on formulations for lyophilization. This is coupled with design of experiment methods to provide a powerful engineering tool for the formulation scientist. While much has been done to model freeze drying cycles and optimize cycle parameters, current models are generic and require system specific data which can be hard to collect. By applying design of experiment principles, a system specific model was developed to allow the optimisation of cycle development to identify key parameters and produce a product that would meet critical quality attributes. Such a platform would lend itself well to quality by design and its application in lyophilization development.

Thesis (Ph.D)--Mechanical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Rosen, David; Committee Member: Allen, Janet; Committee Member: Bras, Bert; Committee Member: Ku, David; Committee Member: Shofner, Meisha; Committee Member: Yen, Jeannette. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 151-162).
Multipotent stromal cells derived from bone marrow hold great potential for tissue engineering applications because of their ability to home to injury sites and to differentiate along mesodermal lineages to become osteocytes, chondrocytes, and adipocytes to aid in tissue repair and regeneration. One key challenge, however, is the scarcity of MSC numbers isolated from in vivo, suggesting a role for biomimetic scaffolds in the cells' ex vivo expansion before reintegration into target tissue. Toward this end, immobilized epidermal growth factor (tEGF) has recently been found to promote MSC survival and proliferation and is a prime candidate to be incorporated into scaffolds to control MSC behavior. To rationally and effectively design scaffolds to drive MSC responses of survival, proliferation, migration, and differentiation, we must first understand these responses and the underlying protein signaling pathways that mediate them. While our knowledge of MSC behavior is limited as a field, MSC migration is particularly less studied despite being critical for tissue and scaffold infiltration. In this thesis, we quantitatively investigate the effects of tEGF and extracellular matrix (ECM) on MSC migration response and signaling. We take a systems level computational view to show a combined biomaterials and small molecule approach to control MSC migration. Cell migration is a delicately integrated biophysical process involving polarization and protrusions at the cell front, adhesion and translocation of the cell body through contractile forces, followed by disassembly of adhesion complexes at the cell rear to allow detachment and productive motility. This process is mediated by a multitude of crosstalking signaling pathways downstream of integrin and growth factor activation. Using a poly(methyl methacrylate)-grafted-poly(ethylene oxide) (PMMA-g-PEO) copolymer base, we modify the PEO sidechains with immobilized epidermal growth factor (tEGF) as a model system for biomimetic scaffolds. We systematically adsorb fibronectin, vitronectin, and collagen ECM proteins to alter surface adhesiveness and measure MSC migration responses of speed and directional persistence alongside intracellular activities of EGFR, ERK, Akt, and FAK phosphoproteins. While tEGF and ECM proteins differentially affected signaling and migration, univariate correlations between signals and responses were not informative, prompting the need for multivariate modeling to identify key patterns. Using decision tree "signal-response" modeling, we predicted that inhibiting ERK on collagen-adsorbed tEGF polymer surfaces would increase cell mean free path (MFP) by increasing directional persistence. We confirmed this experimentally, successfully demonstrating a two-layer approach-"coarse" biomaterials followed by small molecules "fine-tuning"-to precisely and differentially control MSC migration speed and persistence, setting the stage for combination therapies for bone tissue engineering.
by Shan Wu.
Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, June 2017
"May 2017." Cataloged from PDF version of thesis.
Includes bibliographical references (pages 103-111).
Biological hydrogels exhibit complex properties that cannot be recapitulated by current synthetic materials. Examples include mucus, which acts as a barrier against toxins and pathogens while simultaneously hosting trillions of microbes within the gut; cartilage which resists repetitive compressive forces while maintaining highly lubricated layers for efficient movement; and nuclear pore matrices which act as selective barriers in the transport of proteins and nucleic acids. An underlying theme that gives biological hydrogels their unique mechanical and biological functions is the presence of long polymeric molecules. These polymers are typically comprised of repeating subunits that are essential for correct polymer function, such as the phenylalanine-glycine (FG) repeats in nucleoporin proteins of nuclear pore complexes (NPCs) and the proline-threonine-serine (PTS) domains in mucin polymers found in mucus.
Although these polymeric subunits are well-identified, to date their structural complexity has limited our understanding of how they contribute to the overall hydrogel function. In this thesis, we focus on two main biological hydrogels: the self-assembled matrix of the nuclear pore complex that controls the passage of molecules between the nucleus and the cytoplasm, and mucus, which protects against invading pathogens and toxins. As both hydrogels consist of functionally redundant polymers and associated factors, understanding the relationship between polymer sequence and hydrogel function is a significant technical challenge. To simplify the problem, we design structurally reduced peptides and polymers with targeted individual biological features such as amino acid identity, spatial localization of charge, and glycosylation identity. We then study the effect of one or a combination of these properties on the overall hydrogel function.
Using this technique, we first demonstrate that peptide charge type and amino acid placement are important features for regulating selective transport through NPCs. For mucins, we identify single glycans that are sufficient to recapitulate the biofilm inhibition properties of mucin, and present novel evidence that mucins modulate horizontal gene transfer rates for opportunistic and commensal bacteria.
by Wesley George Chen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Due to its common dysregulation in epithelial-based cancers and the extensive characterization of its role in tumor growth, epidermal growth factor receptor (EGFR) has long been an attractive target for monoclonal antibodies. Intense research has culminated in the approval of two antibody-based drugs against EGFR for cancer treatment, with numerous others in clinical trials. However, therapeutic efficacy of these drugs has been disappointingly low due to autocrine signaling, receptor mutation, and transport limitations, necessitating novel antibody designs and mechanisms of action. Recently, it was reported that treatment with combinations of antibodies can induce receptor clustering, leading to synergistic receptor downregulation and anti-tumor activity. The aim of this thesis is to elucidate the details of this phenomenon and to exploit this mechanism to design more effective therapeutic antibodies targeting EGFR. We first illuminate several key aspects of combination antibody-induced clustering. By screening a panel of pairwise combinations, we show that the most potently downregulating pairs consist of two non-competitive antibodies that target EGFR extracellular domain 3. We further find the mechanism underlying downregulation to be consistent with recycling inhibition. Lastly, in contrast to the agonism associated with ligand-induced downregulation, we demonstrate that combination mAb-induced downregulation does not activate EGFR or its downstream effectors and it leads to synergistic reduction in migration and proliferation of cells that secrete autocrine ligand. To enhance antibody binding and induced receptor clustering, we design multispecific antibodybased constructs that engage up to four distinct epitopes on EGFR. We engineer two classes of constructs: one consisting of a full EGFR-specific antibody fused to the variable domain of a second anti-EGFR antibody and the other consisting of a full EGFR-specific antibody fused to one or more EGFR-targeted tenth type three domains of human fibronectin. Both classes of constructs induce robust receptor clustering and downregulation in the absence of signal activation. In vitro downregulation correlates well with in vivo inhibition of tumor growth in several mouse xenograft tumor models and mutational analysis demonstrates that the efficacy of our fusions is attributable to both signaling effects and antibody-dependent cell-mediated cytotoxicity. Our multi-epitopic strategy may be readily applied to other receptor systems to form the basis for a new category of antibody-based therapeutics.
by Jamie Berta Spangler.
Ph.D.

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
A billion years of evolution has crafted a diverse set of proteins capable of complex and varied functionalities. Within recent decades, scientists have applied both rational design and directed evolution to accelerate development of high-value proteins, including biotherapeutics. While computational modelling increasingly facilitates protein design, empirically screening large collections of protein variants remains an essential component of protein engineering. This process requires generating protein variation, partitioning variants with a selection pressure, and identifying highly functional proteins. This thesis presents computational tools for initial protein library design, leverages high-throughput sequencing for phage display screenings, and reports biotemplating of an inorganic phase-change material onto the filamentous M13 phage surface.
Designing ensembles of protein variants involves optimizing library size and quality with constraints on screening capacity, cost, and experimental complexity. Incorporating degenerate codons during oligonucleotide synthesis enables residue-specific protein randomization with a known amino acid scope. However, this widely adopted method often generates uneven variant abundances that diverge exponentially with additional randomized residues. The first section of this work presents tools for the design and assessment of degenerate-codon-based protein libraries. This framework facilitates incorporating an arbitrarily large number of randomized sites, non-standard genetic codes, and non-equimolar nucleotide mixtures. In addition to library size and coverage calculations, whole-population diversity metrics and abundance quantiles are reported.
An evolutionary solver to optimize non-equimolar base compositions to match amino acid profiles, as well as mutational profiling for spike-in oligonucleotides is also presented. The second section of this thesis develops an experimental and data analysis pipeline for integrating high-throughput DNA sequencing with M13 phage display biopanning. Deeply sequencing naïve M13 peptide libraries elucidated censorship patterns for both p3 and p8 coat protein fusions. Streptavidin panning recapitulated the HPQ binding motif after a single panning round, and additional biopannings pursued M13 p8 variants that interact with both gold films and carbon nanotubes. Furthermore, this thesis explores the effect of M13 p8 surface charge on the biotemplating of an inorganic phase-change material. An ambient temperature synthesis for modulating the atomic composition of germanium-tin-oxide nanomaterials is reported.
by Griffin James Clausen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

The aims of this project were: 1) the synthesis of a range of new polyether-based vinylic monomers and their incorporation into poly(2-hydroxyethyl methacrylate) (poly(HEMA)) based hydrogel networks, of interest to the contact lens industry. 2) the synthesis of a range of alkyltartronic acids, and their derivatives. These molecules may ultimately be used to produce functionalised poly(-hydroxy acids) of potential interest in either drug delivery or surgical suture applications. The novel syntheses of a range of both methoxy poly(ethylene glycol) acrylates (MPEGAs) and poly(ethylene glycol) acrylates (PEGAs) are described. Products were obtained in very good yields. These new polyether-based vinylic monomers were copolymerised with 2-hydroxyethyl methacrylate (HEMA) to produce a range of hydrogels. The equilibrium water contents (EWC) and surface properties of these copolymers containing linear polyethers were examined. It was found that the EWC was enhanced by the presence of the hydrophilic polyether chains. Results suggest that the polyether side chains express themselves at the polymer surface, thus dictating the surface properties of the gels. Consequentially, this leads to an advantageous reduction in the surface adhesion of biological species. A synthesis of a range of alkyltartronic acids is also described. The acids prepared were obtained in very good yields using a novel four-stage synthesis. These acids were modified to give potassium monoethyl alkyltartronates. Although no polyesterification is described in this thesis, these modified alkyltartronic acid derivatives are considered to be potentially excellent starting materials for poly (alkyltartronic acid) synthesis via anhydrocarboxylate or anhydrosulphite cyclic monomers.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 96-104).
Identification of effective drug targets to intervene, either as single agent therapy or in combination, is a critical question in drug development. As complexity of disease like cancer is revealed, it has become clear that a holistic network approach is needed to identify drug targets that are specially positioned to provide desired leverage on disease phenotypes. In this thesis we develop a computational framework to exhaustively evaluate target behaviors in biochemical network, either as single agent or combination therapies. We present our single target therapy work as a problem of identifying good places to intervene in a network. We quantify a relationship between how interventions at different places in network affect an output of interest. We use this quantitative relationship between target inhibited and output of interest as a metric to compare targets. In network analyzed here, most targets show a sub-linear behavior where a large percentage of targeted molecule needs to be inhibited to see a small change on output. The other key observation is that targets at the top of the network exerted relatively small control compared to the targets at the bottom of the network. In the combination therapy work we study how combination of drug concentrations affect network output of interest compared to when one of the drugs was given alone at equivalent concentrations. By adapting the definitions of additive, synergistic, and antagonistic combination behaviors developed by Ting Chao-Chou (Chou TC, Talalay P (1984), Advances in enzyme regulation 22: 27-55) for our system and systematically perturbing biochemical pathway, we explore the range of combination behaviors for all plausible combination targets. This holistic approach reveals that most target combinations show additive behaviors. Synergistic, and antagonistic behaviors are rare. Even when combinations are classified as synergistic or antagonistic, they show this behavior only in a small range of the inhibitor concentrations. This work is developed in a particular variant of the epidermal growth factor (EGF) receptor pathway for which a detailed mathematical model was first proposed by Schoeberl et al. Computational framework developed in this work is applicable to any biochemical network.
by Nirmala Paudel.
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, Biological Engineering Division, 2007.
MIT Science Library copy: printed in leaves.
Also issued printed in leaves.
Includes bibliographical references (p. 127-141).
Utilizing molecular recognition and self-assembly, material-specific biomolecules have shown great promise for engineering and ordering materials at the nanoscale. These molecules, inspired from natural biomineralization systems, are now commonly selected against non-natural inorganic materials through biopanning random combinatorial peptide libraries. Unfortunately, the challenge of studying the biological inorganic interface has slowed the understanding of interactions principles, and hence limited the number of downstream applications. This work focuses on the facile study of the peptide-inorganic interface using Yeast Surface Display. The general approach is to use combinatorial selection to suggest interaction principles followed by rational design to refine understanding. In this pursuit, two material groups-II-VI semiconductors and synthetic sapphire (metal oxides)-are chosen as inorganic targets due to their technological relevance and ease of study. First, yeast surface display (YSD) was established as a broadly applicable method for studying peptide-material interactions by screening a human scFv YSD library against cadmium sulfide (CdS), a II-VI semiconductor. The presence of multiple histidine residues was found to be necessary for mediating cell binding to CdS. As a follow-up, a systematic screen with yeast displayed rationally designed peptides was performed on a panel of II-VI semiconductors and gold. Cell binding results indicated that peptide interaction was mediated by a limited number of amino acids that were influenced by locally situated residues. Interpretation of the results facilitated design of new peptides with desired material specificities. Next, the nature of peptide/metal oxide binding interface was interrogated using sapphire crystalline faces as model surfaces.
(cont.) Biopanning a random peptide YSD library and subsequent characterization of the identified binding partners revealed the importance of multiple basic amino acids in the binding event. Study of rationally designed basic peptides revealed a preference for those amino acids to be spaced in such a manner that maximized simultaneous interaction with the surface. Fusing peptides to maltose binding protein (MBP) allowed for quantitative affinity measurement with the best peptides having low nanomolar equilibrium dissociation constants. Finally, peptides were demonstrated as facile affinity tags for protein immobilization in micro-patterning and biosensor assays.
by Eric Mark Krauland.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
Includes bibliographical references.
Recent studies have developed preliminary wiring diagrams for a number of important biological networks. However, the design principles governing the construction and operation of these networks remain mostly unknown. To discover design principles in these networks, we investigated and developed a set of computational tools described below. First, we looked into the application of optimization techniques to explore network topology, parameterization, or both, and to evaluate relative fitness of networks operational strategies. In particular, we studied the ability of an enzymatic cycle to produce dynamic properties such as responsiveness and transient noise filtering. We discovered that non-linearity of the enzymatic cycle allows more effective filtering of transient noise. Furthermore, we found that networks with multiple activation steps, despite being less responsive, are better in filtering transient noise. Second, we explored a method to construct compact models of signal transduction networks based on a protein-domain network representation. This method generates models whose number of species, in the worst case, scales quadratically to the number of protein-domain sites and modification states, a tremendous saving over the combinatorial scaling in the more standard mass-action model was estimated to consist of more that 10⁷ species and was too large to simulate; however, a simplified model consists of only 132 state variables and produced intuitive behavior. The resulting models were utilized to study the roles of a scaffold protein and of a shared binding domain to pathway functions.
by Bambang Senoaji Adiwijaya.
Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (leaves 57-62).
Implantable, controlled release drug delivery devices offer several advantages over systemic oral administration routes and immediate drug release treatments including direct therapy to target organ, more continuous maintenance of plasma and tissue drug levels and the potential for reduced side effects or toxicity. Urology has emerged as a unique field in which minimally invasive implantation techniques are available and such devices could provide improved beneficial therapies over conventional treatments. Urological indications for which localized drug therapy is already being advocated and investigated are highly suitable for treatment with implantable controlled release devices. This thesis describes the in vitro performance evaluation of an implantable, bio-resorbable device that can provide localized drug therapy of ciprofloxacin (CIP) to the seminal vesicle and nearby prostate gland for treatment of chronic prostatitis (CP). The device functions as an elementary osmotic pump (EOP) to release CIP for a period of 2-3 weeks after implantation in the seminal vesicle (SV) through transrectal needle injection or cystoscopic methods. The device is composed of an elastomeric, resorbable polymer cast in a tubular geometry with solid drug powder packed into its core and a micromachined release orifice drilled through its wall. Drug release experiments were performed to determine the effective release rate from a single orifice and the range of orifice size in which osmotic-controlled zero-order release was the dominant mechanism of drug delivery from the device. Device stability and function in an alkaline environment of similar pH to that of the SVs and infected prostate gland was also assessed in vitro. The device was found to function well in both de-ionized water and NaOH pH-8 solution with a sustained zero-order release rate of 2.47 ± 0.29 jtg/hr when fabricated with an orifice of diameter 100-150pm.
by Irene S. Tobias.
M.Eng.

Thesis (M. Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2004.
Includes bibliographical references (p. 83-85).
The next generations of both biological engineering and computer engineering demand that control be exerted at the molecular level. Creating, characterizing and controlling synthetic biological systems may provide us with the ability to build cells that are capable of a plethora of activities, from computation to synthesizing nanostructures. To develop these systems, we must have a set of tools not only for synthesizing systems, but also designing and simulating them. The BioJADE project provides a comprehensive, extensible design and simulation platform for synthetic biology. BioJADE is a graphical design tool built in Java, utilizing a database back end, and supports a range of simulations using an XML communication protocol. BioJADE currently supports a library of over 100 parts with which it can compile designs into actual DNA, and then generate synthesis instructions to build the physical parts. The BioJADE project contributes several tools to Synthetic Biology. BioJADE in itself is a powerful tool for synthetic biology designers. Additionally, we developed and now make use of a centralized BioBricks repository, which enables the sharing of BioBrick components between researchers, and vastly reduces the barriers to entry for aspiring Synthetic Biologists.
by Jonathan Ari Goler.
M.Eng.and S.B.

Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 213-223).
Within synthetic biology, significant progress in creating networks using transcriptional and translational control has been made, but a network comprised solely of protein-protein interactions has not yet been built. To realize this goal, new design rules for assembling and connecting protein devices into circuits are required. In this thesis, a framework for rapid assembly and delivery of genetic networks in Saccharomyces cerevisiae is described. Utilizing this scheme, design principles of modular chimeric proteins, engineered pathway convergence, and phosphorylated activated localization are developed and subsequently applied to create phosphoin/phospho-out composable protein devices in yeast. The devices implement BUFFER, NOT and OR logical operations and collectively form a functionally complete set whereas multiple instances of the devices can be connected to implement any logical expression. Furthermore, bridge devices to interface small molecule sensors and transcriptional networks with protein devices are created. To illustrate composability, two systems are engineered in yeast, the first of which interfaces a phosphorylation load driver within flanking transcriptional regulatory modules to mitigate retroactivity, exemplifying time-scale separation as a means of realizing functional modularity in biological networks. The second system, a fast bistable toggle switch, is the first synthetic network based solely on protein interactions and can be interfaced endogenously to allow controllable abrogation of yeast budding. Work in this thesis provides a set of useful design principles for engineering protein networks in eukaryotes and may improve understanding of natural signaling motifs, allow modulation of existing networks, or foster new synthetic biology applications.
by Deepak Mishra.
Sc. D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
Includes bibliographical references (p. 77-80).
This study aims to investigate the relationships between growth parameters (agitation, glycerol concentration, salt concentration) and responses (biomass, growth rate, protein expression), by a 3-factor-3-level central composite factorial design. This experimental design involved running shake flask culture at 15 different experimental conditions with duplicates. Optical density (OD600), dry cell weight (DCW), and BCA Protein Assays were done on each experiment. Mathematical models in terms of these parameters' effects and their interactions were proposed for each of the responses. The significance of each effect and interaction, as well as the goodness-of-fit of mathematical models to data were examined by analysis of variance. It was found that biomass (with R²Adj=0.951) is a strong function of glycerol concentration (higher glycerol concentration leads to higher biomass), but it varies much less with agitation, and it is completely independent of salt concentration. Growth rate (R²Adj=0.901), however, varies strongly with agitation and salt concentration, but much more weakly with glycerol concentration. Protein production has a low R²Adj value of 0.746, implying that higher-order terms, e.g. x₁² and x₂², should be tested for significance in the model.
(cont.) Collected data were fitted to the proposed models by response surface regression, after which surface and contour plots of responses were generated to identify trends in them. High agitation (300 rpm in shaker) gave rise to both highest biomass and growth rate. In addition, biomass at high glycerol concentration (3% v/v) was almost twice as much as biomass at low glycerol concentration (1% v/v) at high agitation rate (19 g/L compared to 11 g/L). At the same agitation rate, growth rate shows the largest increase of 20.5% with increasing salt concentration from 0.7% to 2.1%. Protein production reached maximum of 7.3 mg/mL at medium agitation rate (250 rpm), high salt and glycerol concentrations.
by Joyce Chan.
M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (leaves 244-249).
This thesis utilizes modeling approaches rooted in statistical physics and physical chemistry to investigate several aspects of cellular signal transduction at both the modular and global levels. Design principles of biological networks and cell signaling processes pertinent to disease progression emerge from these studies. It is my hope that knowledge of these principles may provide new mechanistic insights and conceptual frameworks for thinking about therapeutic intervention into diseases such as cancer and diabetes that arise from aberrant signaling. Areas of interest have emphasized the role of scaffold proteins in protein kinase cascades, modeling relevant biophysical processes related to T cell activation, design principles of signal transduction focusing on multisite phosphorylation, quantifying the notion of signal duration and the time scale dependence of signal detection, and entropy based models of network architecture inferred from proteomics data. These problems are detailed below. The assembly of multiple signaling proteins into a complex by a scaffold protein guides many cellular decisions. Despite recent advances, the overarching principles that govern scaffold function are not well understood. We carried out a computational study using kinetic Monte Carlo simulations to understand how spatial localization of kinases on a scaffold may regulate signaling under different physiological condition. Our studies identify regulatory properties of scaffold proteins that allow them to both amplify and attenuate incoming signals in different biological contexts. In a further, supplementary study, simulations also indicate that a major effect that scaffolds exert on the dynamics of cell signaling is to control how the activation of protein kinases is distributed over time[2].
(cont.) Scaffolds can influence the timing of kinase activation by allowing for kinases to become activated over a broad range of times, thus allowing for signaling across a broad spectrum of time scales. T cells orchestrate the adaptive immune response and are central players in maintenance of functioning immune system. Recent studies have reported that T cells can integrate signals between interrupted encounters with Antigen Presenting Cells (APCs) in such a way that the process of signal integration exhibits a form of memory. We carried out a computational study using a simple mathematical model of T cell activation to investigate the ramifications of interrupted T cell-APC contacts on signal integration. We considered several mechanisms of how signal integration at these time scales may be achieved. In another study, we investigated the role of spatially localizing signaling components of the T cell signaling pathway into a structure known as the immunological synapse. We constructed a minimal mathematical model that offers a mechanism for how antigen quality can regulate signaling dynamics in the immunological synapse These studies involving the analysis of signaling dynamics led us to investigate how differences in signal duration might be detected. Signal duration (e.g. the time scales over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. We present an analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales[3].
(cont.) Topological features of these networks that allow for different frequency dependent signal processing properties are identified. One role of multisite phosphorylation in cell signaling is also investigated. The utilization of multiple phosphorylation sites in regulating a biological response is ubiquitous in cell signaling. If each site contributes an additional, equivalent binding site, then one consequence of an increase in the number of phosphorylations may be to increase the probability that, upon disassociation, a ligand immediately rebinds to its receptor. How such effects may influence cell signaling systems is not well understood. A self-consistent integral equation formalism for ligand rebinding, in conjunction with Monte Carlo simulations, was employed to further investigate the effects of multiple, equivalent binding sites on shaping biological responses. Finally, this thesis also seeks to investigate cell signaling at a global scale. Advances in Mass Spectrometry based phosphoproteomics have allowed for the real-time quantitative monitoring of entire proteomes as signals propagate through complex networks in response to external signals. The trajectories of as many as 222 phosphorylated tyrosine sites can be simultaneously and reproducibly monitored at multiple time points. We develop and apply a method using the principle of maximum entropy to infer a model of network connectivity of these phosphorylation sites. The model predicts a core structure of signaling nodes, affinity dependent topological features of the network, and connectivity of signaling nodes that were hitherto unassociated with the canonical growth factor signaling network. Our combined results illustrate many complexities in the broad array of control properties that emerge from the physical effects that constrain signal propagation on complex biological networks.
(cont.) It is the hope of this work that these studies bring coherence to seemingly paradoxical observations and suggest that cells have evolved design rules that enable biochemical motifs to regulate widely disparate cellular functions.
by Jason W. Locasale.
Ph.D.

Thesis (S.M. in Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references (leaves 45-47).
Design and fabrication of a microfluidics system capable of generating reproducible and controlled micro-biochemical environments that can be used as a diagnostic assay and microreactor is important. Here, a simple technique was developed to create a robust microfluidics system capable of generating precise gradients of biochemical properties within its channels. Through this approach, it is possible to create a gradient generator with mammalian cells patterned and seeded under its poly(dimethylsiloxane) (PDMS) channels. Cells that were seeded and patterned under the PDMS channels remained viable and capable of performing intracellular reactions. Using the gradient generator within the PDMS microfluidic device, a gradient of specific and controlled biochemicals can be flowed on seeded cells allowing for high-throughput molecular interaction analysis. The microfluidics system provides a way to study and analyze cell response in the presence of a combination of biochemical signals.
by Guan-Jong Chen.
S.M.in Toxicology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
Includes bibliographical references.
A multi-faceted approach was applied to the molecular design of substrates for the selective adhesion, proliferation and differentiation of connective tissue progenitors (CTPs) from human bone marrow aspirates. The basic premise of the thesis is that integrin-specific adhesion peptides, when presented in a biophysically appropriate spatial arrangement against an inert background, allow enrichment of CTPs in vitro. Comb copolymer comprising a methyl methacrylate backbone with 10-mer poly(oxyethylene) sidechains was selected as the vehicle to present small adhesion peptides at the surface. This polymer shows excellent performance in cell resistance studies and offers sufficient functionalizable sites to create high local densities of ligands. Methods for preparing comb copolymer substrates with peptides in [approx.] 300 nm² clusters with inter-ligand spacings closer than integrins were developed. This nanometer-scale clustered presentation was favorable to integrin binding. Cells were more spread on RGD peptide substrates with a higher degree of nanoscale clustering but of the same overall peptide surface density as comparable substrates with lower degree of peptide clustering. We evaluated adhesion peptides for their ability to support CFU formation of marrow-derived CTPs using colony forming unit (CFU) assay.
(cont.) The results, analyzed with a statistical model implemented to capture characteristics of CFU assay, showed that while RGD substrates supported a moderate amount of alkaline phosphatase -positive CFU (CFU-AP) formation, the bone sialoprotein peptide FHRRII(A, and two [alpha]4 [beta]1 peptides demonstrated the best performance in promoting CFU-AP formation. Patient variability in CFU data could be partially explained by the variations in marrow aspirate cell integrin expression, particularly [alpha]5 and [alpha]v [beta]3. The high level of ECM protein association seen with aspirate cells, as revealed by immunoblotting, may inhibit cell adhesion and account for the fairly low CFU counts observed. Treatments of marrow aspirate with phosphate saline buffer (PBS) and RGD solution reduced a significant amount of protein association. A comprehensive study showed that patients' marrow aspirates were naturally partitioned into two groups of very different colony formation behavior and integrin and AP expressions but consistency was observed within each group. Upon treatment of marrow cells with divalent ion-free PBS, CFU-AP formation on RGD substrates drastically increased in one group of patients. The designs and assays developed in this thesis could be applied for the further understanding of marrow aspirates, such as their interaction with a high-affinity [alpha]5 [beta]1 peptide, and with that knowledge, further optimize the surface design of bone marrow grafts.
by Ada Au.
Ph.D.

Thesis (S.M. in Molecular and Systems Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.
Vita.
Includes bibliographical references (leaves 60-62).
The purpose of this work is to probe the dTDP-L-rhamnose pathway in an effort to develop small molecule inhibitors that could act as therapeutics for Mycobacterium tuberculosis. The necessity for newer, more effective treatments for tuberculosis is growing, as the bacteria evolve resistance to traditional treatments. In an effort to develop more effective and perhaps more abbreviated courses of treatment, a plan was developed to investigate a pathway involved in cell wall biosynthesis as a promising target: the dTDP-L-rhamnose pathway. This pathway plays an essential role in linking the peptidoglycan and arabinogalactan portions of the mycolic acid-arabinogalactan-peptidoglycan complex, a significant part of the mycobacterial cell wall. The mounting level of biochemical understanding of this pathway and its importance in bacterial cell wall biosynthesis indicates that it is not only a relevant target but also an accessible one. Of the four enzymes crucial to this biosynthetic pathway, one was chosen as the primary focus: dTDP-D-glucose-4,6- dehydratase (RmlB). There are 3 steps in the reaction mechanism of RmlB: oxidation of the C4 position of dTDP-D-glucose to form a 4-keto structure, dehydration of the C6 position via the elimination of water and a subsequent reduction to result in a 6-deoxy product. Crystal structures of this particular enzyme, dTDP-D-glucose 4,6-dehydratase (RmlB), complexed with single substrates or substrate analogs have provided a foundation for these studies, enabling the rational design of a small library of potential inhibitors. Twelve mechanism-based inhibitors of RmlB are proposed. These compounds reflect the current understanding of the mechanism and mimic the sugar portion of the sugar-nucleotide substrate at various steps throughout the reaction mechanism. Each of the proposed inhibitors is designed to inhibit one of the specific steps of the mechanism. While the intention of this project is to synthesize each compound in this library from commercially available starting materials in 15 steps or less, the primary goal of this particular dissertation is to synthesize 3 of the 12 proposed inhibitors from the commercially available starting material 1,5-anhydro-D-glucitol. The long term goal of this work is to produce these compounds in significant amounts in order to test their efficacy in an animal model of mycobacterial infection.
by Neena Sujata Kadaba.
S.M.in Molecular and Systems Toxicology

In-house windrow composting of broiler litter has been studied to reduce microbial populations between flocks. Published time-temperature goals are used to determine the success of the composting process for microbial reductions. Spatial and temporal density of temperature measurement can influence the ability to determine what portion of a windrow pile has achieved specified time-temperature goals. Based on this motivation, an investigation of the heating profile in windrowed litter and the identification of the effects of spatial and temporal sampling densities on the prediction of the heating profile in windrowed broiler litter were executed. Likewise, an investigation of the effects of moisture content on heat generation during composting of broiler litter was conducted. Ultimately, the research projects were designed with the goal of determining the efficacy of windrow composting as a treatment method for reducing microbial populations in broiler litter and to produce recommendations for the implementation of future windrow temperature monitoring investigations. While past investigations have reported success of windrow composting for microbial population reductions, a lack of intense spatial and temporal temperature monitoring has likely mis-represented the pile heating profile and resultant effects on microbial populations.

The design of a delivery system that specifically delivers anticancer drug to the tumour site avoiding normal tissues damage has always been a challenge. In this thesis we describe the engineering and biological performance of novel temperature-sensitive liposomes (TSL) that have both a substantial in vivo stability and an efficient content-release by mild hyperthermia (HT). First, we explain the development of novel lipid-peptide hybrids (Lp-Peptide) by anchoring leucine zipper temperature-sensitive peptide within the liposomal lipid bilayer. We characterized this system by studying its physicochemical properties and the interaction of the peptide with the lipid bilayer. Then we examined its potential to retain and trigger the release of the anticancer drug, doxorubicin, in vitro at physiological temperatures and after exposure to mild HT. In addition, the blood kinetics, tumour and other tissues accumulation were explored when we studied the system in vivo. Our data suggested that Lp-Peptide hybrids can increase both immediate and long-term drug accumulation in the tumour. Therefore, we studied their therapeutic activity comparing two different heating protocols to mimic intravascular and interstitial drug release. The last chapter of this thesis explored the opportunities of increasing the therapeutic specificity of TSL by designing anti-MUC-1 targeted vesicles based on the traditional TSL (TTSL) to trigger drug release after specific uptake into cancer cells. The system was evaluated by studying the in vitro cellular binding, uptake and therapeutic efficacy. Taking this system a step further, its biodistribution and therapeutic potential were also examined. Different protocols were applied to explore the effect of HT on the accumulation of targeted TTSL into the tumour and their therapeutic efficacy. In summary, our studies demonstrate the critical factors to consider in the design of clinically relevant TSL and the importance of matching the heating protocol to their physicochemical and pharmacokinetic parameters to maximise therapeutic benefits.

Dietary supplementation with fibre has been shown to ameliorate features of the metabolic syndrome and inhibit malignant growth in certain types of cancer. These effects have been linked to short-chain fatty acids (SCFA), mostly acetate. However, the ubiquitous role of SCFAs in metabolism, combined with a short tissue half-life and the non-targeted nature of oral and peripheral administrations make achieving phenotypically relevant levels of SCFA by standard delivery techniques challenging and limit their therapeutic potential. Liposomal encapsulation of a therapeutic agent overcomes these issues by protecting against degradation, increasing circulation time and passively targeting both the liver and tumour tissue. In this research project, I have designed a bifunctional liposome formulation to transport SCFA, monitored their distribution and uptake utilising visualisation by MRI, PET/CT and fluorescence microscopy. These bifunctional liposomes were useful for effectively encapsulating small molecules within their aqueous core, which in this case was acetate, and capable of acetate delivery into cells while also being amenable to cellular imaging. I have shown that preferential delivery of liposome encapsulated acetate (LITA) nanoparticles to key sites of metabolic control provide beneficial therapeutic effects in animal models of both obesity and cancer. Chronic administration of LITA nanoparticles in an obeseogenic model led to a significant reduction in adiposity, intrahepatocellular lipid, inflammatory tone and genetic indication of a decrease fatty acid synthesis in the liver. Application of LITA in a murine xenograft model caused an inhibition of tumour growth in three colorectal cancer cell lines: HT-29, HCT116 p53+/+ and HCT116 p53-/-. The mechanisms for these two outcomes are not fully defined; however cellular energy homeostasis of both scenarios was restored. These results indicate that LITA nanoparticles can be used to improve multiple metabolic pathways, in vivo.

An in silico design algorithm has been developed to design binding ligands for protein targets of known three-dimensional structure. In this method, the binding energy of a candidate ligand is used to ascribe it a probability of binding. A sample of a virtual library of candidate ligands is then used to ascribe implicit weights to all the ligands in the library. These weights are used to obtain virtual sub-libraries which collectively carry a greater probability to bind to the target. This algorithm is presented along with validation studies on the different algorithmic components, demonstrating how optimization of the design method can be best achieved.

The differentiation of myosin into the respective heavy chain isoforms has shown a correlation with high mechanical stress. Aortic valve myosin expression has been reported; however, the characterization of the pressure response has yet to be fully developed. Thus, a cyclic pressure bioreactor was developed to elucidate the á/â-myosin heavy chain (MHC) expression in aortic valve leaflets subject to physiological and pathological transvalvular pressure loads. The pressure bioreactor achieved the desired pressure modulation via LabVIEW controlled solenoid valves. Results showed á/â-MHC expression on the fibrosal endothelium and minimal dispersal in the subendothelium, indicating the presence of smooth muscle cells. Endothelial layer denudation was evident with time progression while protein expression was limited to sites of excision or injury, indicating a causal relationship with high shear stress. In conclusion, á/â-MHC expression is limited by endothelium detachment and lack of smooth muscle cells, possibly on account of insufficient mechanical stimuli.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Structural materials in nature integrate classical materials selection rules with morphometry (geometry or shape-based rules) to create high-performance, multi-functional structures that exhibit tunable properties through extraordinary complexity, hierarchy, and precise structural control. This thesis explores the use of morphometry as a materials design parameter through the development of bio-inspired, flexible composite armor based on the articulated exoskeleton of an armored fish, Polypterus senegalus, which achieves uniform coverage and protection from predatory threats without restricting flexibility. First, the functional implications of shape and shape variation are examined as materials design parameters within the biological exoskeleton using a new method that integrates continuum strain analysis with landmark-based geometric morphometric analysis in 2D and 3D. Bioinspired flexible composite prototypes are fabricated using multi-material 3D printing and tested under passive loading (self-weight) and active loading (bending) to examine how the shape of scales contributes to local, interscale mobility mechanisms that generate anisotropic, global mechanical behavior. With one prototype design scheme, a wide array of mechanical behavior is generated with stiffness ranging over several orders of magnitude, including 'mechanical invisibility' of the scales, showing how morphometry can tune flexibility without varying the constituent materials. Finally, finite element models simulating the bending experiments are created to establish a computational framework for analyzing the mechanical response of the prototypes. The finite element models are then extended to examine the effect of different loading conditions, scale morphometry, multi-material architecture, and constituent material properties. The results show how morphometric-enabled materials design, inspired by structural biological materials, can allow for tunable behavior in flexible composites made of segmented scale assemblies to achieve enhanced user mobility, custom fit, and flexibility around joints for a variety of protective applications.
by Swati Varshney.
Ph. D.

A new instrument system for the non-invasive assessment of biological systems has been designed, built and validated. This system utilizes the optical characteristics of a biological system to assess both its static and dynamic response over time. The changes in the optical properties can then be used to quantify the response of the biological system to external or internal stimuli. This new system incorporates an efficient light delivery system, as well as a novel light collection system to provide high repeatability, reproducibility, sensitivity, and detection thresholds. The system incorporates simultaneous measurement of transmitted light, forward scattered light, specularly reflected light, and non-specularly reflected light in across the visible spectrum, to provide flexibility in the biological systems that can be measured. It is shown that typical values are 0.02% for repeatability and 0.15% for reproducibility. Sensitivity in transmission and backscatter measurements was improved by a factor of 20 compared to a previously built instrument, which was the basis for the new design. The system is capable of detecting light attenuated down to 0.0063% of the maximum intensity, resulting in a superior threshold. The system was then validated by measuring the optical properties of various biological samples, both natural and engineered. These samples include bovine, porcine, and human corneas and lenses, as well as synthetic materials. Biological sample testing were consistent with published results on the back vertex distance (BVD) test (bovine lenses), the bovine corneal opacity test (BCOP, bovine corneas), and published results obtained with the precursor to the new device.

Tissue sealants of a liquid based formulation are widely studied in biomedical research with many starting to gain FDA approval. To date, little investigation has been put toward methods of application for tissue sealant materials, more specifically a powder based formulation. The focus of this research was to develop and prototype a powder spray device capable of administering powder based formulations with a long-term goal of integrating the device within the clinical setting. Powders can be administered in a variety of dry forms. These forms can range from non-homogenous nanoscale particles to homogeneous micro and nano-scale spheres. Incorporation of therapeutics within the powder makes this method of application favorable for the prevention or maintenance of disease. Pneumatic conveying is the transport of granulated solids using gas and is the principal basis from which the powder spray gun was designed. Fluidization aids were added to the device in order to increase powder flow properties. Analysis of spray field, spray rate, characterization of powder and ex-vivo testing was performed. All results suggest that the powder spray device is applicable for the deposition of powder based tissue sealants in a clinical setting.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.
This project chronicles the design and construction of a small scale recirculating water tank for the purpose of studying biological hydrodynamics. Currently available systems were analyzed and studied prior to developing a new, cost effective design that provides laminar flow through a two foot test section. Construction details provide the information for duplicating a low cost water tank for fluid flow and visualization.
by Thomas V. Hennessey, III.
S.B.

Thesis: Ph. D. in Chemical Engineering Practice, Massachusetts Institute of Technology, Department of Chemical Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
Adoption of non-mammalian host organisms for biologic drug manufacturing could lead to step-changes in cost of manufacturing and volumetric productivity, increasing access to these life-saving drugs for large patient populations. One promising alternative host is Pichia pastoris, a methylotrophic yeast that is currently used to manufacture ten approved drugs worldwide. Its fast growth rate and ability to grow to high cell densities can enable fast production cycles, agile process development, and potentially low production costs. While P. pastoris has already been engineered to produce antibodies with human-like glycoforms, titers are still lower than those typically achieved with CHO cells. While standard fermentation processes for P. pastoris have been designed, several areas have limited investment to date. Few chemically-defined cultivation media have been reported for P. pastoris fermentation and all are minimal salt solutions.
While several studies have demonstrated that addition of complex nutrients improves growth and productivity, defined compounds with similar effects have not been identified. Also, methanol feeding protocols for P. pastoris have only been developed for fed-batch operation and have not been studied for perfusion cultivation. In this thesis, we describe the design of a rich defined medium (RDM) for cultivation of P. pastoris through systematic screening and gene expression analysis. The use of RDM for expression of three proteins yields titers comparable to, or higher than, those in standard complex medium. We then outline a similar methodology for the optimization of individual amino acids and fatty acids in the medium. We also describe how a transcriptomic analysis of methanol feeding strategy in perfusion mode enabled the identification and alleviation of limiting biological processes.
This work demonstrates how combining traditional process development strategies with genome-wide sequencing for P. pastoris leads to rapid improvement of fermentation processes. Continued progress in this area could lead to a new model for low-cost production of high-quality biologic drugs.
by Catherine Bartlett Matthews.
Ph. D. in Chemical Engineering Practice
Ph.D.inChemicalEngineeringPractice Massachusetts Institute of Technology, Department of Chemical Engineering

Els glicolípids són producte d’alt valor degut a les seves propietats amfipàtiques que els doten d’un ampli rang d’aplicacions en els sectors químic (ex., biosurfactants) o biomèdic (ex., adjuvant de vacunes). Depenent de la unitat lipídica que els forma aquests compostos poden ser classificats en diferents famílies. Si la unitat lpídica és una ceramida o diacilglicerol, el glicolípid resultant es coneixerà com a glicoesfingolípid o glicoglicerolípid (GGL) respectivament. Mentre que els glicoesfingolípids han demostrat jugar un paper clau en diversos processos biològics, els glicoglicerolípids són interessants degut al seu ús potencial per a ser usats com a adjuvants de vacunes o supressors tumorals. Tot i que l’interès per aquests compostos és alt, la seva aplicació es veu obstaculitzada per la seva baixa disponibilitat i alt cost de producció. La síntesi química requereix de complexes passos de protecció i desprotecció per tal d’aconseguir la desitjada regio- i estereoespecificitat de l’enllaç glicosídic que, conseqüentment, comporta una reducció del rendiment i eficiència del procés. Per això, vam considerar l’enginyeria metabòlica com a estratègia potencial per a la producció de glicolípids i ens vam centrar en l’obtenció d’una plataforma d’enginyeria metabòlica en E. coli per tal d’obtenir aquests complexes productes d’interès. En estudis previs, el nostre grup va reportar que la sintasa de glicolípids MG517 de Mycoplasma genitalium era funcional i que s’obtenien glicoglicerolípids a partir de UDP-glucosa (UDP-Glc) i diacilglicerol (DAG). Addicionalment, una primera generació de soques modificades va demostrar que la disponibilitat de DAG era limitant per a la producció de GGL (Mora-Buyé et al., 2012). En el present projecte, cinc estratègies diferents d’enginyeria metabòlica van ser proposades per tal d’augmentar la producció de GGL utilitzant E. coli. Les primeres quatre estratègies tenien com a objectiu incrementar el pool del precursor lipídic, DAG. Sent així, la primera estratègia es basà en augmentar la disponibilitat de DAG a través de l’eliminació de reaccions competitives. Per aconseguir-ho, es van knockejar diferents gens involucrats en la ß-oxidació i l’activació d’àcids grassos (∆tesA i ∆fadE) reportant un increment en la producció de casi el doble. La segona estratègia es va basar en incrementar la disponibilitat d’àcids grassos mitjançant la modulació de factors de transcripció (fabR i fadR). Aquesta estratègia no va reportar un increment de la producció però si un canvi en el perfil lipídic amb un increment d’àcids grassos insaturats. La tercera estratègia es basava en incrementar la conversió dels donadors d’acils a àcid fosfatídic, precursor del DAG, sobreexpressant les aciltransferases PlsC i PlsB. La quarta estratègia es centrà en augmentar la disponibilitat del diacilglicerol per la sobreexpressió de la proteïna de fusió PlsCxPgpB, capaç de redirigir el flux cap a DAG, o CDH promovent la hidròlisi de fosfolípids. D’entre les diferent soques modificades, ∆tesA co-expressant MG517 i la proteïna de fusió PlsCxPgpB va ser la soca més productora, amb un 350% d’increment en la producció de GGL comparant-la amb la soca parental expressant únicament MG517. Especialment interessant és que les soques coexpressant CDH van presentar un canvi en el perfil de GGL cap al lípid diglucosilat (representant al voltant d’un 80% del total de GGLs). Finalment, es va proposar una estratègia metabòlica per incrementar la disponibilitat de l’altre precursor, UDP-Glc. Aquesta cinquena estratègia es va basar en sobre expressar l’enzim GalU, responsable de la biosíntesi d’UDP-Glc, i eliminant l’enzim codificant per la UDP-sucre difosfatasa ushA. No obstant, cap d’aquestes modificacions va aconseguir millorar els nivells de GGLs. Per últim, tal i com va ser reportat pel nostre grup que la fosfatidiletanolamina era intercanviable en les membranes d’E. coli pels nous producte GGL, una llibreria de promotors i RBS va ser dissenyada per tal de disminuir la producció d’aquest fosfolípid, augmentant al mateix temps de la producció de glicolípids.
Los glicolípidos son productos de alto valor debido a sus propiedades amfipáticas, que los dota en un amplio rango de aplicaciones en los sectores químicos (ej., biosurfactantes) o biomédicos (ej., adyuvante de vacunas). Dependiendo de la unidad lipídica que los forma, los glicolípidos son clasificados en diferentes familias. Si la unidad lipídica es una ceramida o diacilglicerol, los glicolípidos son conocidos como glicoesfingolípidos o glicoglicerolípidos (GGL) respectivamente. Mientras que los glicoesfingolípidos han demostrado jugar papeles clave en diversos procesos biológicos, los glicoglicerolípidos son compuestos interesantes debido a su potencial uso como adyuvantes de vacunas o supresores tumorales. Aunque el interés por estos compuestos es muy alto, su aplicación se ve obstaculizada por su baja disponibilidad y altos costes de producción. La síntesis química requiere de complejos pasos de protección y desprotección para conseguir la deseada regio- y estereoespecificidad del enlace glicosídico, que conlleva a una reducción del rendimiento y eficiencia del proceso. Por ello, consideramos la ingeniería metabólica como estrategia potencial para la producción de glicolípidos y nos centramos en construir una plataforma de ingeniería metabólica en E. coli para conseguir estas complejas estructuras de interés. En previos estudios, nuestro grupo reportó que la sintasa de glicolípidos MG517 de Mycoplasma genitalium era funcional y que glicoglicerolípidos podían ser obtenidos a partir de UDP-glucosa (UDP-Glc) y diacilglicerol (DAG). Adicionalmente, la primera generación de cepas modificadas demostró que la disponibilidad de DAG era limitante en la producción de GGL (Mora-Buyé et al., 2012). En el presente proyecto, cinco estrategias diferentes de ingeniería metabólica fueron propuestas para aumentar la producción de GGL en E. Coli. Las primeras cuatro estrategias se centraron en aumentar el pool del precursor lipídico, DAG. Para ello, la primera estrategia se basó en incrementar la disponibilidad de DAG a través de la eliminación de reacciones competitivas. Para lograrlo, se knockearon diferentes genes relacionados con la ß-oxidación y la activación de ácidos grasos (∆tesA y ∆fadE) reportando un incremento de casi el doble. La segunda estrategia se basó en incrementar la disponibilidad de ácidos grasos mediante la modulación de factores de transcripción (fabR y fadR). Aunque estrategia no reportó una mejora en el rendimiento de GGL, sí mostró un cambio en el perfil de los ácidos grasos con un incremento de los ácidos grasos insaturados. La tercera estrategia se basó en incrementar la conversión de los donadores de acilos a ácido fosfatídico, precursor del DAG, mediante la sobreexpresión de las aciltransferasas PlsC y PlsB. La cuarta estrategia se centró en aumentar la disponibilidad de diacilglicerol mediante la sobreexpresión de la proteína de fusión PlsCxPgpB capaz de redirigir el flujo de DAG, o CDH promoviendo la hidrólisis de fosfolípidos. Entre las diferentes cepas modificadas, la cepa ∆tesA coexpresando MG517 y la proteína de fusión PlsCxPgpB fue la mayor productora, con un incremento de los niveles de GGL del 350%, comparándola con la cepa parental expresando únicamente MG517. Interesantemente, las cepas coexpresando CDH mostraron un cambio en el perfil de GGL hacia el lípido diglucosilado (hasta el 80% del total del GGLs). Finalmente, una estrategia metabólica fue propuesta para aumentar la disponibilidad del otro precursor, UDP-Glc. La quinta estrategia se basó en la sobreexpresión de la enzima GalU, responsable de la biosíntesis de UDP-Glc, y la eliminación de la UDP-azúcar difosfatasa codificada por el gen ushA. Sin embargo, ninguna de estas modificaciones mejoró los niveles de GGL. Por último, tal y como reportó nuestro grupo que la fosfatidiletanolamina era intercambiable en las membranas de E. coli por los nuevos compuestos GGL, una librería de promotores y RBS fue diseñada para disminuir la producción de este fosfolípido intentando al mismo tiempo aumentar la producción de glicolípidos.
Glycolipids are products of high-added value due to their amphipathic properties, which endow them with a broad range of applications in the chemical (i.e., biosurfactants) and biomedical sectors (i.e., vaccine adjuvants). Depending on their lipidic moiety, glycolipids are classified in different families. If the lipid moiety is a ceramide or diacylglycerol, the glycolipids are known as glycosphingolipids or glycoglycerolipids respectively. While glycosphingolipids have shown to play essential roles in many biological processes, glycoglycerolipids (GGL) are interesting compounds due to their potential use as vaccine adjuvants or tumor suppressors. Although the interest of these compounds is very high, their applications are hampered by their low availability and high productions costs. Chemical synthesis requires complex protection and deprotection steps to achieve the desired regio- and stereospecificity of the glycosidic linkage, which consequently lower the yield and efficiency of the process. Therefore, we considered metabolic engineering as a potential strategy for the production of glycolipids and we aimed at building up a metabolic engineering platform in E. coli to achieve these complex structures of interest. In previous studies, our group reported that the glycolipid synthase MG517 from Mycoplasma genitalium was functional and glycoglycerolipids were obtained from UDP-glucose (UDP-Glc) and diacylglycerol (DAG). In addition, the first generation of engineered strains demonstrated that the availability of the DAG was the key bottleneck in GGL production (Mora-Buyé et al., 2012). In the present project, five different metabolic strategies were proposed to increase the production of GGL using E. coli. The first four strategies were aimed at increasing the available pool of the lipidic precursor, DAG. Thus, the first strategy was based on increasing DAG availability by removing competing reactions. To achieve so, different genes involved in the ß-oxidation and activation of fatty acids were knocked out (ΔtesA y ΔfadE) reporting an almost 2-fold production increase. The second strategy was based on increasing fatty acids availability by modulating different transcriptional factors (fabR y fadR). Although this strategy did not report an improvement of GGL yield, showed a change in the fatty acid profile with an increase of unsaturated fatty acids. The third strategy was based on increasing the conversion of acyl donors to phosphatidic acid, precursor of DAG, by overexpressing PlsC and PlsB acyltransferases. The fourth strategy was based on increasing diacylglycerol availability by overexpressing the fusion PlsCxPgpB protein that could redirect the flux to DAG or CDH promoting the hydrolysis of phospholipids. Among the different engineered strains, the ∆tesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Interestingly, the strains co-expressing CDH showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs). Finally, a metabolic strategy was proposed to increase the availability of the other precursor, UDP-Glc. This fifth strategy was based on overexpressing GalU enzyme, which is responsible for the biosynthesis of UDP-Glc, and by removing the UDP-sugar diphosphatase encoding gene ushA. However, none of these modifications further improved the GGL titers. Finally, as it was also reported by our group that phosphatidylethanolamine was exchangeable in the membranes of E. coli by the new GGL compounds, a library of promoters and RBS was designed to decrease the production of this phospholipid trying at the same time to increase the production of glycolipids.

The mechanical properties of biological tissues are of increasing research interest to disciplines as varied as designers of protective equipment, medical researchers and even forensic Finite Element Analysis (FEA). The mechanical properties of biological tissue such as skin are relatively well known at low strain rates and strains, but there is a paucity of data on the high rate, high strain behaviour of skin - particularly under biaxial tension. Biaxial tensile loading mimics in vivo conditions more closely than uniaxial loading [1, 2], and is necessary in order to characterise a hyper-elastic material model[3]. Furthermore, biaxial loading allows one to detect the anisotropy of the sample without introducing noise from inter-sample variability - unlike uniaxial tensile testing. This work develops a high strain rate bulge test device capable of testing soft tissue or polymer membranes at high strain rates. The load history as well as the full field displacement data is captured via a pressure transducer and high speed 3D Digital Image Correlation (DIC). Strain rates ranging from 0.26s −1 to 827s −1 are reliably achieved and measured. Higher strain rates of up to 2500s −1 are achieved, but are poorly measured due to equipment limitations of the high speed cameras used. The strain rates achieved had some variability, but were significantly more consistent than those achieved by high rate biaxial tension tests found in the literature. In addition to control of the apex strain rate, the bi-axial strain ratio is controlled via the geometry of the specimen fixture. This allowed for strain ratios of up to 2 to be achieved at the apex 1 . When testing anisotropic membranes, the use of full field 3D DIC allowed for accurate and efficient detection of the principal axis of anisotropy in the material. No skin is tested, but instead three types of polydimethylsiloxane (PDMS, ”silicone') skin simulant are tested. These simulants were chosen to fully encapsulate the range of mechanical behaviour expected from skin - they were chosen to have stiffness's, strain hardening exponents and degrees of anisotropy significantly above or below the behaviour exhibited by skin. This ensured that the device was validated over a wider range of conditions than expected when testing skin. A novel approach to specimen fixation and speckling for silicone membranes is developed, as well as a fibre reinforced skin simulant that closely mimics the rate hardening and anisotropic behaviour of skin. In addition to bulge tests, uniaxial tensile tests are conducted on the various simulant materials in order to characterise their low strain rate behaviour. The composite skin simulant is characterised using a modified version of the anisotropic skin model developed by Weiss et al (1996) [4], and the pure silicone membranes are characterised using the Ogden hyper-elastic model.

Current tobacco baling technology utilizes hydraulic power to press tobacco into bales. The high system pressures at which hydraulic systems operate pose a risk to workers. Hydraulic systems are costly and hydraulic oil leaks contaminate baled tobacco. A pneumatically driven, vertically oriented, multi-stroke baler was designed as an affordable alternative to current hydraulic balers. Pneumatics was chosen due to the lower system operating pressure and absent risk of tobacco bale contamination. The transmission of power was achieved through a reversible pneumatic gearmotor turning left and right hand acme threaded rods coupled together to form a powerscrew. The plunger was driven by a scissor-jack design and was used to take advantage of the non-linear force response of tobacco. The scissor-jack was driven by acme nuts traveling along the acme rod of the powerscrew. The baler was tested with burley tobacco grown during the 2007 season at the Central Crops Research Station in Clayton, NC. The compressive force and plunger displacement was measured for each bale produced. These readings were used to determine the compressive force as a function of plunger travel and the compressive force as a function of bale density. The baler required 3-4 presses to produce burley bales roughly 42 inches cubed and weighing approximately 500-600 pounds.

Arroyo Chico Wash, an important drainage for central Tucson, Arizona, runs through numerous neighborhood and industrial areas before emptying into the Santa Cruz River. Within four suburban neighborhoods along the wash, direct observations, personal interviews, survey questions, and historical documents are used to describe design and management influences on the biological and human aspects of the wash. Plants along the wash are identified by 2-meter wide belt transects run at 100-meter intervals over the 4 kilometer length of the study area. Wild animal and bird lists are based on observation and information given by residents. Relationships between design, maintenance and neighborhood attitudes toward the wash are assessed using a survey questionnaire given to people living adjacent to the wash. Results show the wash in the Colonia Solana neighborhood has the greatest biological diversity, highest neighborhood satisfaction and highest recreational use. In neighborhoods where the wash is a "backyard easement", satisfaction and use are the lowest.

This thesis presents the design and the fabrication process of a three-dimensional (3D) neural interface consisting of a bundle of parallel micro-channels with (100μmx100μm) cross-sectional area and embedded micro-electrodes. This is a regenerative implant that is able to stimulate and record extracellular neural signals in the peripheral nervous system as demonstrated by the \(in-vivo\) experiments conducted in collaboration as part of this project. These implants have the potential to be developed into long-term neural interfaces capable of extracting neural signals from stumps of severed peripheral nerves to use as control inputs for muscles simulators or artificial limbs for amputees. The skeleton of the device is entirely made of flexible polyimide films. Gold micro-electrodes and micro-channels of photosensitive polyimide are patterned directly on polyimide substrates. After fabrication, the 2D electrode micro-channel array is rolled into a 3D structure forming concentric rolls of closed micro-channel arrays with a Swiss-roll like arrangement. Microflex Interconnection technique (MFI) was incorporated successfully into the implant. The performance of the implant microelectrodes was characterised \(in-vitro\) through impedance spectroscopy and \(in-vivo\) via implantation in animals for three months. The ability of the electrodes to stimulate and capture action potentials from regenerated tissue was also assessed.

Metalworking fluids (MWFs) are coolants and lubricants, which are widely employed in metal cutting works. They are designed to be a long lasting product. Manufacturers have designed MWFs with lack of awareness of end-of-life disposal by including biocides, which make biological treatment challenging. Here, Syntilo 9913 was used as a case study to develop a cradle-to-grave product that was biologically stable in use but amenable to sustainable hybrid biological treatment at end-of-life. The product was reverse engineered employing factorial design approach based on a priori knowledge of the product components. From the combinatorial work, it was observed that chemical interactions can results in synergistic and antagonistic effects in terms of the toxicity and biodegradability. One of the major components of most MWFs are amines such as Triethanolamine (TEA). TEA does not biodeteriorate in single compound screening, but in combination with many other components TEA was found to cause "softening" of MWF formulations. Octylamine was found to be best for "bio-hardening" but it was not economically sustainable. Hence, the modified biocide-free synthetic MWF, Syntilo 1601, was reformulated with TEA, isononanoic acid, neodecnoic acid, Cobratec TT50S, and pluronic 17R40, which were resistant to biological treatment. Although, no change in the overall oxidation state of the MWF, metabolic activity did occur as breakdown products were observed. This suggested that both raw materials and metabolic breakdown products were recalcitrant. Thus, immobilisation agents were applied to aid further biodegradation by removing toxic bottleneck compounds. It was found that hybrid nano-iron and kaffir lime leaf performed similarly in removing chemical oxygen demand and ammonium from the system. Work in this Thesis demonstrated that the combined use of biological treatment and immobilisation agents effectively overcome the limitations of biological treatment alone by removing bottleneck compounds, which allowed greater COD reduction. This laboratory scale is a proof of principle, which needs to be tested at full scale.

The ability to inject DNA and other foreign particles into cells, both germ cells (e.g. to produce transgenic animals) and somatic cells (e.g. for gene therapy), is a powerful tool in genetic research. Nanoinjection is a method of DNA delivery that combines mechanical and electrical methods. It has proven to have higher cell viability than traditional microinjection, resulting in higher integration per injected embryo. The nanoinjection process can be performed on thousands of cells simultaneously using an array of microneedles that is inserted into a monolayer of cells. This thesis describes the needle array design requirements and the fabrication process used to meet them. The process uses unpassivated and passivated deep reactive ion etching (DRIE) to create needles with a constant diameter shaft and a pointed tip. The needle diameter and height are about 1 µm and 8 µm, respectively. A buckling analysis and physical testing show that the needles can withstand the force required to penetrate the cells. The chip is attached to a plastic suspension with a counter electrode and electrical connections to a voltage source. The suspension's motion is defined by two compliant orthoplanar springs that have been vertically and rotationally offset for added stability. The base of the suspension is designed to exactly fit in the bottom of a cell culture dish, where the needle array can be pushed into the cell monolayer. Injection protocol was created and followed to perform tests with needle insertion only, voltage application only, and the full nanoinjection process. The average cell viability for the full injection process was 98.2% compared to an average control viability of 99.5%. Zero volt injections with a high concentration of propidium iodide, a cell impermeable dye with two positive charges, resulted in dye uptake from diffusion, proving that the needles are penetrating the cells. Tests comparing injections with and without voltage had high variability in dye uptake. Therefore, glass cover slips were placed in the culture dishes to provide more consistent injection conditions. This reduced variation in zero voltage tests. It is recommended that this procedure be followed for performing injections with voltage.

Engineers often view variability as undesirable and seek to minimize it, such as when they employ transistor-matching techniques to improve circuit and system performance. Biology, however, makes no discernible attempt to avoid this variability, which is particularly evident in biological nervous systems whose neurons exhibit marked variability in their cellular properties. In previous studies, this heterogeneity has been shown to have mixed consequences on network rhythmicity, which is essential to locomotion and other oscillatory neural behaviors. The systems that produce and control these stereotyped movements have been optimized to be energy efficient and dependable, and one particularly well-studied rhythmic network is the central pattern generator (CPG), which is capable of generating a coordinated, rhythmic pattern of motor activity in the absence of phasic sensory input. Because they are ubiquitous in biological preparations and reveal a variety of physiological behaviors, these networks provide a platform for studying a critical set of biological control paradigms and inspire research into engineered systems that exploit these underlying principles. We are directing our efforts toward the implementation of applicable technologies and modeling to better understand the combination of these two concepts---the role of heterogeneity in rhythmic networks of neurons. The central engineering theme of our work is to use digital and analog platforms to design and build Hodgkin--Huxley conductance-based neuron models that will be used to implement a half-center oscillator (HCO) model of a CPG. The primary scientific question that we will address is to what extent this heterogeneity affects the rhythmicity of a network of neurons. To do so, we will first analyze the locations, continuities, and sizes of bursting regions using single-neuron models and will then use an FPGA model neuron to study parametric and topological heterogeneity in a fully-connected 36-neuron HCO. We found that heterogeneity can lead to more robust rhythmic networks of neurons, but the type and quantity of heterogeneity and the population-level metric that is used to analyze bursting are critical in determining when this occurs.