Thesis (S.M. in Molecular Systems Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 38-43). / Recent epidemiological data has indicated that chronic exposure to metals such as arsenic and cadmium increases the risk of cancer and other diseases. These metals may have negative biological effects on cells by disrupting homeostatic cellular processes and altering normal signal transduction. One possible mechanism for many of these negative effects may involve overproduction of reactive oxygen species that damage proteins, lipids, carbohydrates, and nucleic acids. To compound this oxidative damage, there is evidence consistent with the inhibition of repair of damaged DNA by these metals. As a result, there is an increase in mutagenicity and toxicity in the organisms. This thesis reviews the current literature relevant to the biochemistry and biology of arsenic and cadmium. / by Leslie Lai. / S.M.in Molecular Systems Toxicology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 86-92). / Endothelial cells show an unexpected behavior shortly after the onset of laminar flow: their crawling speed decreases ~40% within the first 30 min, but only in a confluent monolayer of endothelial cells, not in subconfluent cultures, where cell-cell interactions are limited. This led us to study early shear effects on cell-cell adherens junctions. We found a 30±6% increase in the number of VE-cadherin molecules in the junctions. The strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin protein also increased after laminar flow. These observations suggest that endothelial cell junction proteins respond to flow onset. The process of clustering may induce diffusion of monomers to the junction area, resulting in an overall increase in VE-cadherins in the junctions. To directly confirm the role of adherens junctions in the decrease in cell crawling speed, we used siRNA-knockdown technique to produce cells lacking VE-cadherin. These cells showed no decline in crawling speed under flow. Our interpretation is consistent with previous data on junction disassembly 8 hr after flow onset. The speed of endothelial cell crawling returns to the original level by that time, and junctional disassembly may explain that phenomenon. In order to understand better the change in VE-cadherin distribution under flow and during junction formation and remodelling, we developed a mathematical model of VE-cadherin redistribution in endothelial cells. This model allowed us to develop a quantitative framework for analysis of VE-cadherin redistribution and estimate the amount of protein in the junctions and on the apical surface. In addition to that, the model explains rapid junction disassembly in the leukocyte transmigration and junction formation in subconfluent cells. / (cont.) These studies show that intercellular adhesion molecules are important in the force transmission and shear stress response. Their role, however, is not limited to flow mechanotransduction. Intercellular force transmission has an important application - organ development and, specifically, angiogenesis. We studied the role of VE-cadherin in vessel development in HUVECs and showed that VE-cadherin-null cells do not form vessels in the in vitro assay. This observation confirms the important role of intercellular force transmission in response to external force caused by flow or exerted by other cells. / by Aleksandr Rabodzey. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 42-43). / Optical tweezers instruments use laser radiation pressure to trap microscopic dielectric beads. With the appropriate chemistry, such a bead can be attached to a single molecule as a handle, permitting the application of force on the single molecule. Measuring the force applied in real-time is dependent on detecting the bead's displacement from the trapping laser beam axis. Back-focal-plane detection provides a way of measuring the displacement, in two-dimensions, at nanometer or better resolution. The first part of this work will describe the design of a simple and inexpensive position sensing module customized for optical tweezers applications. Single molecule fluorescence is another powerful technique used to obtain microscopic details in biological systems. This technique can detect the arrival of a single molecule into a small volume of space or detect the conformational changes of a single molecule. Combining optical tweezers with single-molecule fluorescence so that one can apply forces on a single molecule while monitoring its effects via single molecule fluorescence provides an even more powerful experimental platform to perform such microscopic studies. Due to the enhanced photobleaching of fluorophores caused by the trapping laser, this combined technology has only been demonstrated under optimized conditions. / (cont.) The second part of this work will describe a straightforward and noninvasive method of eliminating this problem. The study of mechanotransduction in biological systems is critical to understanding the coupling between mechanical forces and biochemical reactions. Due to the recent advances in single molecule technology, it is now possible to probe such mechanisms at the single molecule level. The third and final part of this work will describe a basic mechanotransduction experiment using the well-studied ZIF268 protein-DNA system. An experimental assay and method of analysis will be outlined. / by Peter Lee. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / (cont.) kinetic studies. Moderate increases ([approximately]30%) in nucleobase deamination products were observed in the SJL mice bearing the RcsX tumor, but the biological meaning of these increases awaits studies of DNA repair kinetics. dO was not detected in any study at levels above 5 per 10⁸ nt, leading to the prediction that it will not be present at significant levels in inflamed tissues in humans. As a complement to the LC-MS method for the quantification of nucleobase deamination products, enzymatic probes were also developed for oxidative and nitrosative DNA lesions. These probes would not only allow differential quantification of the two types of DNA damage, but would also allow the lesions to be mapped in any DNA sequence by coupling their activity with the technique of ligation-mediated PCR. As an extension of the biomarker study, the effects of ONOO⁻ dose and dose-rate on the DNA damage and mutations induced in the supF gene were investigated. The observations suggest that both the dose and dose-rate at which a genetic target is exposed to ONOO⁻ substantially influence the damage and mutational response and these parameters will need to be considered in assessing the potential effects of ONOO⁻ in vivo. Finally, an extended study using the analytical method developed in this thesis yielded results in E. coli consistent with a new paradigm: perturbations of nucleobase metabolism may lead to incorporation of the purine precursors hypoxanthine (I) and xanthine (X) into DNA. This can be regarded as another endogenous process causing DNA damage that may lead to human diseases such as cancer ... / The objective of this thesis project was to develop nucleobase deamination products as biomarkers of inflammation and to study the role of these DNA lesions in the pathophysiology of inflammation-induced carcinogenesis. The basis of this research is the epidemiological evidence that chronic inflammation is associated with an increased risk of cancer, yet the link between the inflammatory process and the development of cancer has eluded definition. Biomarker development began with the establishment of a sensitive liquid chromatography-mass spectrometry (LC-MS) method to quantify four of the nucleobase deamination products: 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from the deamination of dG; 2'-deoxyuridine (dU) from dC; and 2'-deoxyinosine (dl) from dA. The analytical method was then validated and tested with both in vitro and in vivo studies involving quantification of nucleobase deamination products in isolated plasmid DNA and human lymphoblastoid cells exposed to nitric oxide (NO)̇ at controlled physiological concentrations. Finally, the formation of nucleobase deamination products was analyzed in SJL/RcsX mice, an established mouse model of NO ̇over production. This set of studies revealed several important features of the nitrosative chemistry of NO ̇derivatives. The in vitro formation of dX, dl and dU was found to occur at nearly identical rates (k = 1.2 x 10⁵ M⁻¹s⁻¹). Low levels of nucleobase deamination products were formed in cells exposed to NO,̇ which suggests that cellular factors significantly influence the nitrosative chemistry. However, cellular glutathione (GSH) was found to play a smaller role than expected, considering the effects of GSH on steady-state concentrations of N₂0₃ in in vitro / by Min Dong. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaf 43). / (cont.) electrodes. / Flexible multi-electrode arrays represent an evolving technology for chronic deep neuronal recording. We focus on developing a biocompatible method of implanting a novel 12-electrode device for long-term recording (weeks to months) in a deep target responsible for object identification, the inferotemporal cortex (IT). Numerous electrodes embedded in a bioerodible polymer matrix composed of 50/50 poly(D,L-lactide-co-glycolide) created an electrode bundle that was sufficiently stiff for insertion into brain tissue, but was expected to become more flexible after polymer degradation. Titanium electrode ports used to house these multi-electrode arrays were successfully designed, constructed, and implanted on a non-human primate as well. These ports were tested for mechanical durability over a course of 6-8 weeks. To evaluate the recording properties of the electrodes, many parameters of the electrode bundle tip were explored and evaluated, including gold-plating, tip cutting (flat cut, angle cut, or individual wire cut), and splaying. Gold-plating lowered electrode impedance and improved signal quality on originally high impedance electrodes. The angle cut and individually cut electrodes were superior to the flat cut ones; they recorded from 6-7 active channels at varying depths during advancement. Splaying, the removal of polymer some distance from the array tip to separate the bundle into individual electrodes, also improved the recording properties in acute experiments. Pending chronic experiments will further reveal 1) the true degradation rate of the array and thus the flexibility restored, 2) the electrode tip characteristics following polymer erosion, and 3) the long-term recording quality and stability of all the / by Yihvan Vuong. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 152-164). / Human Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae that consists of enveloped, partially double stranded DNA viruses that specifically target hepatocytes for viral replication. Although a vaccine has been available for more than 20 years chronic HBV infection afflicts 350-400 million worldwide. It is estimated that 0.5-1.2 million people die each year from HBV-attributable cases of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Significant disadvantages exist among currently available therapeutics (e.g. IFNca, lamivudine, adefovir, etc.) that include limited efficacy and the promotion of drug-resistant viral strains. These therapeutics are the research products of the HBV molecular biology that can be manipulated in the laboratory setting. Future antiviral drug therapy is dependent upon the development of better cell culture systems that will allow the study of the complete viral life cycle. The use of primary human and primate hepatocytes is restricted by multiple experimental limitations including a rapid loss of susceptibility to infection in culture, lot-to-lot variability inherent in primary cell culture, and the necessity of treatment with chemical agents such as DMSO for reproducible infection. / (cont.) Permissive cell lines are capable of supporting viral replication upon transfection with the HBV genome. These cell lines have helped to elucidate the later events in the viral life cycle. However, there is less understanding of the early stages that include virus attachment, internalization, uncoating, nuclear transport, and genome repair. Our group has developed an in vitro system that recreates many of the features of a perfused capillary bed structure. Various metrics (e.g. biochemical production, tissue morphology, liver-enriched mRNA expression, and drug metabolism) confirm that this system maintains a well-differentiated liver phenotype. Using DHBV as a surrogate model, this study has attempted to demonstrate that hepatocytes maintained in a more sophisticated culture system retain susceptibility to infection. This study has endeavored to establish the perfused three-dimensional culture system as potential tool to study early events of the viral life cycle. This research lays the foundation for the future development of a human HBV infection model in which early stages of the viral life cycle can be studied and therapeutic targets identified. / by Alexandria V. Sams. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division; and, (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, June 2007. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (p. 131-135). / The tongue is an intricately configured muscular organ, which undergoes a series of rapid shape changes intended to first configure and then transport the bolus from the oral cavity to the pharynx during swallowing. In order to observe the synergistic interactions of the eight different muscles in the tongue, MRI techniques were used to determine the three-dimensional fiber architecture, measure the mechanical function of the tongue during swallow, and relate the two quantitatively to identify fiber contraction. Diffusion Tensor Imaging was applied to the in vivo tongue of five subjects at rest to image the tongue myoarchitecture. The data revealed the complex relationships between extrinsic and intrinsic fibers. Phase Contrast MRI was applied to ten subjects to assess the complex array of mechanical events occurring during the propulsive phase of swallowing. Physiological strain rate data was acquired in 4 and 6 time point series during approximately 3 ml water bolus swallows. Data acquisition was gated to pressure from the tip of the tongue against the hard palate, indicating the beginning of the irreversible late accommodation. This method demonstrated that the propulsive phase of swallowing is associated with a precisely organized series of compressive and expansive strain rate events. Individual subject data sets from both of these methods were then related. The alignment between local fiber direction and the co-located strain rate tensor was quantified by the dot product between the two vectors. Using this technique, the sagittal muscle activity was observed over the course of the swallow. In the first 200 ms after gating, the verticalis and palatoglossus contract in order to form the bolus. / (cont.) From 300-400 ms, the genioglossus contracts, opening the bolus into the throat, while the verticalis and geniohyoid relax. at 500 ms, relaxation throughout the tongue occurs. These data support the concept that propulsive lingual deformation is due to complex muscular interactions involving both extrinsic and intrinsic muscles. / by Samuel M. Felton. / S.B. / M.Eng.

Thesis (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management; and, (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division; in conjunction with the Leaders for Manufacturing Program at MIT, 2008. / MIT Institute Archives copy: pages, 85-86, 87-88, 89-90 bound in reverse order. / Includes bibliographical references (p. 79-83). / The biopharmaceutical industry uses recombinant protein technologies to provide novel therapeutics to patients around the world. These technologies have presented exciting opportunities for breakthrough medical treatments while creating a host of challenges in the discovery, development and manufacture of these products. Protein aggregation is one of the challenges currently limiting the ability to bring new biopharmaceutical products into the market and to manufacture existing commercial products. The mechanisms of aggregation and subsequent particle formation are highly complex, incompletely understood, and difficult to measure quantitatively with currently available analytical tools. Aggregates, and their effect on product appearance, may compromise value to the patient (bioavailability, dose, therapeutic activity and immunogenicity) as well as value to the company (yield loss and performance in a competitive marketplace) and are therefore tightly regulated. This thesis is intended to explore the problem of protein particles through two main avenues: meeting current regulatory criteria and influencing future regulation. Process changes, analytical characterization, and organizational improvements are each addressed to achieve that goal. An experiment was designed and completed to jointly examine (1) changes to manufacturing processes using novel filtration applications intended to reduce or remove protein particles from solution and (2) analytical tools for improved characterization. Organizational dynamics and resource allocation add an extra layer of complexity and are discussed in relation to leveraging knowledge regarding particles. / (cont.) Additionally, three objectives are established to influence the direction of future regulation: the need for improved characterization, industry collaboration and a healthy interface with regulatory bodies. / by Leigh Anne Hunnicutt. / S.M. / M.B.A.

Thesis (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management; and, (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division; in conjunction with the Leaders for Manufacturing Program at MIT, 2009. / Includes bibliographical references. / The primary goal of the project was to make the machine maintenance process more efficient. In order to do this, we conducted a 3-day Value Stream Mapping workshop, during which we generated a map of the present value stream. We identified shortcomings and inefficiencies within this value stream, and then mapped out a future state Value Stream, creating a 90-day action plan to move towards that desired future state. This action plan relied heavily upon measuring and improving key performance indicators, which allowed us to make objective measures of our progress. A second internship objective was to reduce the number of clean room re-sanitizations. Currently, clean rooms undergo a costly and time-consuming re-sanitization whenever an air handler is shut down for a brief period of time. It is likely that some of these cleanings are unnecessary, and eliminating extraneous cleanings could reduce maintenance costs significantly. In order to reduce unnecessary clean room re-sanitizations, we designed and conducted a study to measure environmental standards during an air handler shutdown. Genzyme's Validation and Quality Control Departments approved this experimental plan prior to execution. We shut down an air handler that supplied a clean room, and monitored the air for viable and non-viable particles for several hours. Viable particulate levels did not exceed action limits during a four-hour shutdown, and non-viable particulates dropped to pre-experiment levels as soon as we re-started the air handler. Based on these results, we conclude that air-handler shutdowns should not require room re-sanitization. / by Dana Ari Levine. / S.M. / M.B.A.