Nanoparticles (NPs) are progressively being investigated for use in biomedical applications, including biological agents delivery like gene delivery, drug and protein delivery. Activation of complement pathways and interactions with immune recognition subcomponents can modulate the clearance of the NPs and subsequent inflammatory response. Such modulation could affect the intended translational applications either in the development as tissue-specific drug delivery platform or in the treatment of pulmonary diseases such as tuberculosis and lung cancer thus, poses challenges to develop them for in vivo applications. Here, we set out to study the interaction between innate immune components such as properdin, a small fragment of properdin, TSR4+5, a key lung pattern recognition molecule, surfactant protein D (SP-D), and carbon nanotubes (CNTs), and potential downstream effects on the immune response via macrophages. We report, that human properdin, an up-regulator of the complement alternative pathway and stabilizer of C3 convertase can opsonize CNTs via its thrombospondin type I repeat (TSR) 4 and 5. Uptake of properdin bound CNTs was enhanced by a macrophage cell line, THP-1, surging a robust pro-inflammatory immune response. In addition, recombinant TSR4+5 on CNTs, inhibited complement consumption, suggesting that TSR4+5, can be potentially used as a complement inhibitor in a number of pathological circumstances arising due to unintentional complement activation. Similarly, a recombinant fragment of human SP-D (rfhSP-D) bound to CNTs via its C-type lectin domain and augmented phagocytosis by THP-1 monocytic cell lines, together with an increased pro-inflammatory response. Furthermore, rfhSP-D opsozined CNTs continued to activate complement pathway via the classical pathway. Complement deposition on the rfhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response. Furthermore, like CNTs, Iron oxide nanoparticles are also recognized by complement pathway, but mainly by alternative complement pathway. Complement deposition enhanced their uptake by activated THP-1 macrophages and dampened the pro-inflammatory responses. These studies emphasise the significance of understanding the interaction between innate immune humoral factors including complement in developing nanoparticle-based drug delivery strategies.

Varicella-zoster virus causes chicken pox (Varicella) primary infection, which becomes latent in the dorsal root ganglia and trigeminal ganglia, and may reactivate to cause shingles, the most serious complication of which is post-herpetic neuralgia occurring in 50% of individuals over 60 years. Severity of lesions depends on host's immune response. Like many viruses, varicella-zoster virus appears to have evolved escape mechanisms from host immune surveillance by downregulating cell surface major histocompatibility complex class 1 expression and may delay of resolution of infection. Major histocompatibility complex class 1 is processed and transported to the cell surface through the Golgi apparatus. Varicella-zoster virus genome encodes a membrane gene, open reading frame type 1, which is localised in the enoplasmic reticulum and Golgi apparatus. Given the cellular localisation of open reading frame type 1 in the Golgi apparatus, this study investigated the expression of major histocompatability complex class 1 in human immortalised keratinocytes transfected with only empty vector. As a control, the expression of major histocompatability complex class 1 human immortalised keratinocytes parental cells were also examined using current molecular biology techniques. The results of this thesis demonstrate that the expression of varicella-zoster virus-open reading frame type 1 prevents the transport of major histocompatibility complex class 1 complexes to the cell surface and causes its retention in the Golgi apparatus, which is compensated by treatment with IFN-[alpha]. Varicella-zoster virus (VZV)-open reading frame type 1 does not affect the synthesis of human leukocyte antigen class 1 heavy chains or the expression of the transporter associated with antigen processing. Additionally, we determined that varicella-zoster virus-open reading frame type 1 impedes the surface expression of human leukocyte antigen class-A and human leukocyte antigen class-B, which present viral peptides to major histocompatibility complex class I-restricted cytotoxic T lymphocytes, but not the natural killer cell inhibitory ligands human leukocyte antigen class-C and non-classical human leukocyte antigen class-E. This selective downregulation of cell surface human leukocyte antigen class 1 molecules may allow the virus to establish infection by avoiding immune clearance of virus-infected cells by both cytotoxic T lymphocytes and natural killer cells. However, it remains to be seen if open reading frame type 1 expressing cells evade cytotoxic T lymphocytes killing, through downregulation of classical major histocompatibility complex class I, and natural killer killing, through lack of downregulation of non-classical major histocompatibility complex class I. The study outcome will be a valuable attempt to elucidate factors involved in varicella-zoster virus-related lesion progression, contribute to existing knowledge, and importantly allude to further investigations on the pathogenesis of this virus on human disease. The data obtained may also offer novel means of therapeutic intervention.

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

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

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

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

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

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

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: M. Eng., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages. 63-65). / Microbial pathogens are becoming a pressing global health issue due to the rapid appearance of resistant strains, accompanied by slow development of new antibiotics. In order to improve these treatments and engineer novel therapies, it is crucial that we increase our understanding of how these antibiotics interact with cellular metabolism. Evidence is increasingly building that the efficacy of antibiotics relies critically on downstream metabolic effects, in addition to inhibition of primary targets. Here we present a novel computational pipeline to expedite investigation of these effects: we combine computational modelling of metabolic networks with data from experimental screens on antibiotic susceptibility to identify metabolic vulnerabilities that can enhance antibiotic efficacy. This approach utilizes genome-scale metabolic models of bacterial metabolism to simulate the reaction-level response of cellular metabolism to a metabolite counter screen. The simulated results are then integrated with experimentally determined antibiotic sensitivity measurements using machine learning. Following integration, a mechanistic understanding of the phenotype-level antibiotic sensitivity results can be extracted. These mechanisms further support the role of metabolism in the mechanism of action of antibiotic lethality. Consistent with current understanding, application of the pipeline to M. tuberculosis identified cysteine metabolism, ATP synthase, and the citric acid cycle as key pathways in determining antibiotic efficacy. Additionally, roles for metabolism of aromatic amino acids and biosynthesis of polyprenoids were identified as pathways meriting further investigation. / by Sarah Natalie Wright. / M. Eng.