Thesis (Sc. D.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / Includes bibliographical references (leaves 138-160). / Peroxisome proliferator-activated receptors (PPARs) belong to a nuclear receptor superfamily; two major isoforms, PPARα and PPAR[gamma], are primarily involved in lipid and glucose homeostasis. However, evidence also suggests roles for PPARs in regulating inflammation and atherosclerosis, and prompted investigation into the efficacy of PPAR agonists in parenchymal rejection (PR) and transplantation-associated arteriosclerosis (TxAA). Four different PPAR agonists (fenofibrate and Wy14643 for PPARα; BRL69453 and 15-deoxy-[delta]¹⁴̓¹⁶ Prostaglandin-J₂ for PPAR[gamma]) in an in vitro model of the alloresponse all demonstrated a robust and substantial attenuation of IFN[gamma], a cytokine that critically affects both rejection and TxAA. This occurred in a dose-dependent manner, independent of known IFN[gamma]-inducing cytokines. At the same times, PPAR activation increased the overall expression of chemokines but substantially decreased expression of two relevant chemokine receptors. Of the four agonists tested, fenofibrate, a largely PPARα-specific agonist, had the best profile of IFN[gamma] production to chemokine and chemokine receptor expression. At early time points after cardiac transplantation, fenofibrate administration showed findings consistent with those seen in vitro, including a tendency to reduce IFN[gamma]. However, long-term fenofibrate treatment significantly increased graft IFN[gamma] expression and inflammatory cell infiltration, thereby augmenting PR without ameliorating TxAA. Congenital deficiency of PPARα confirmed that the receptor plays a regulatory role in IFN[gamma] expression but is not necessary for graft infiltration by inflammatory cells. The findings constitute the first examination of the efficacy of PPAR agonists in / (cont.) solid-organ transplantation and suggest that in order to fully realize the beneficial anti-inflammatory effects of fenofibrate, additional strategies must be employed to inhibit graft inflammatory cell infiltration. Finally, a novel immunologic research tool based on the boron neutron capture reaction is proposed. / by Emanuela Binello. / Sc.D.

Thesis (Sc.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2000. / Includes bibliographical references. / In the past decade, biodegradable polymers have become the materials of choice for a variety of biomaterials applications. In particular, poly(lactic-co-glycolic acid) (PLGA) microspheres have been extensively studied for controlled-release protein delivery. However, significant issues arise in formulating such delivery systems since few proteins have been successfully encapsulated and released from these microspheres. Here, methods are developed to determine the causes of protein instability and solutions are provided for overcoming these problems. A commonly used technique for protein encapsulation in PLGA microspheres is the double-emulsion method. The harsh processing associated with this method can cause denaturation of the encapsulated protein. Herein, we have used Fourier transform infrared (FTIR) spectroscopy to determine the secondary structures of two model proteins, bovine serum albumin (BSA) and chicken egg-white lysozyme, within PLGA microspheres. This is a novel technique for in situ evaluation of proteins within microspheres and potentially a powerful and quick method for assessment of formulations. Results for both proteins indicate changes in structure upon entrapment within the microspheres. However, addition of the stabilizing excipient trehalose prevents the denaturing effects incurred during processing. In addition, BSA released from microspheres is improved by incorporation of trehalose. With microspheres made by double emulsion, there is often a large, initial burst of drug release upon injection. This results in inefficient use of therapy. To prevent this loss, a modified spontaneous emulsification method was explored. / (cont.) With this procedure both protein and polymer are soluble in a single solvent system thus avoiding creation of a water/solvent interface. The process was optimized for microsphere size and protein loading. Addition of a charged surfactant served to improve protein solubility and thus increase protein loading. In vitro and in vivo release kinetics showed a minimal burst, lower than that found with double emulsion microspheres, followed by sustained release. Upon injection of the microspheres in vivo, the PLGA microspheres begin to degrade. Degradation of the polymer generates acidic monomers, and acidification of the inner polymer environment is a central issue in the development of these devices for drug delivery. To quantitatively determine the intraparticle acidity, pH-sensitive fluorescent dyes were entrapped within the microspheres and imaged with confocal fluorescence microscopy. The technique allows visualization of the spatial and temporal distribution of pH within the degrading microspheres. The data indicate the formation of a very acidic environment within the particles with the minimum pH as low as 1.5. The images show a pH gradient, with the most acidic environment at the center of the spheres and higher pH near the edges, which is characteristic of diffusion-controlled release of the acidic degradation products. Strategies to avoid the accumulation of acidic monomers involve decreasing the diffusion distance of the degradation products by either decreasing the overall diameter of the microspheres or creating porous particles. / by Karen Fu. / Sc.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (p. 127-138). / With the discovery of hearing disorders caused by mutations in proteins expressed in the tectorial membrane (TM), the importance of the TM in cochlear mechanics has never been clearer. However, the exact role of the TM in cochlear mechanics remains a mystery. In this thesis, I have investigated material properties of two mouse models of genetic hearing disorders that affect proteins found in the TM, a-tectorin and type XI collagen. The Tecta mutants had a missense mutation in ac-tectorin, a protein found exclusively in the TM in the organ of Corti. The effect of the mutation was to decrease the fixed charge concentration, which was found to be the primary determinant of the bulk modulus. However, the shear modulus was not affected. Collla2 is one of the genes that encodes for type XI collagen. Mutation in this gene causes no significant change in fixed charge concentration and, therefore, bulk modulus. However, the radial shear impedance is lowered. These measurements suggest that TM shear impedance is dominated by radially oriented collagen fibers and plays a key role in driving outer hair cell (OHC) bundle deflection. At the same time, the TM bulk modulus is dominated by the presence of fixed charge and may play a key role in coupling energy from outer to inner hair cells. / by Kinuko Masaki. / Ph.D.

Thesis: S.M., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 36-39). / The need for new antibiotics is great as bacterial strains with single and multiple drug resistance have continued to grow more prevalent since the 1980's. At the same time, the rate of approval of new antibiotics has dropped precipitously. Existing antibiotics commonly target the bacterial ribosome. A or cell wall synthetic pathways: two targets that are essential for bacterial survival. However, another option is to target a pathway which is more intimately connected to bacterial pathogenesis: protein secretion. In bacteria, most secreted polypeptides are pushed across the membrane, via the SecYEG channel, by the SecA ATPase. Relatively little is understood of how SecA couples ATP hydrolysis to polypeptide translocation. X-ray crystallography and many biochemical studies support a model in which the two-helix finger (2HF) of SecA pushes the polypeptide through the SecYEG channel, however some evidence is contradictory. We aim to directly measure conformational changes of the 2HF by utilizing single-molecule Fbrster resonance energy transfer (smFRET). Directly measuring conformational changes in an ATPase will also provide further insight into the guiding principles of ATPase function. First, we will build a smFRET microscope and assemble a software package to analyze the data it collects. We will then validate these tools by reproducing results currently in the literature from Holden et al. and McKinney et al.. Next, we will assess the potential limitations of current tools for smFRET data analysis, especially as applied to ATPases. We will propose a new approach that may be useful in these systems. Finally, we will use the smFRET microscope to measure ATP-dependent conformational dynamics of the 2HF. This evidence will help differentiate between three proposed models: the 2HF (1) is not directly involved in polypeptide translocation, (2) moves unidirectionally, directly driving translocation, or (3) moves back and forth but in a way that is coordinated by ATP hydrolysis with progress capture elsewhere in SecA. / by Jacob D. Sargent. / S.M.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages [119]-139). / RNA-sequencing is a sensitive method for inferring gene expression and provides additional information regarding splice variants, polymorphisms and novel genes and isoforms. Using this extra information greatly increases the complexity of an analysis and prevents novice investigators from analyzing their own data. The first chapter of this work introduces a solution to this issue. It describes a community-curated, scalable RNA-seq analysis framework for performing differential transcriptome expression, transcriptome assembly, variant and RNA-editing calling. It handles the entire stack of an analysis, from downloading and installing hundreds of tools, libraries and genomes to running an analysis that is able to be scaled to handle thousands of samples simultaneously. It can be run on a local machine, any high performance cluster or on the cloud and new tools can be plugged in at will. The second chapter of this work uses this software to examine transcriptome changes in the cortex of a mouse model of tuberous sclerosis with a neuron-specific knockout of Tsc1. We show that upregulation of the serotonin receptor Htr2c causes aberrant calcium spiking in the Tsc1 knockout mouse, and implicate it as a novel therapeutic target for tuberous sclerosis. The third chapter of this work investigates transcriptome regulation in the superior colliculus with prolonged eye closure. We show that while the colliculus undergoes long term anatomical changes with light deprivation, the gene expression in the colliculus is unchanged, barring a module of genes involved in energy production. We use the gene expression data to resolve a long-standing debate regarding the expression of dopamine receptors in the superior colliculus and found a striking segregation of the Drd1 and Drd2 dopamine receptors into distinct functional zones. / by Rory Kirchner. / Ph. D.

Thesis: Ph. D. in Biomedical Engineering, Harvard-MIT Program in Health Sciences and Technology, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 165-191). / Cancer metastasis is the underlying mechanism of 90% of cancer-related deaths, yet few therapeutics exist that directly target it. Part of this scarcity is attributable to a general lack of knowledge with regards to the underlying mechanisms that mediate traversal of the sequential steps required for malignant dissemination. Recently, biologists and clinicians have gained appreciation for the role that the microenvironment plays in promoting or inhibiting metastasis. This thesis sought to expand our understanding of the involvement of extracellular matrix (ECM) interactions during metastasis through the development and use of a novel ECM microarray screening platform. This platform consists of 768 unique single and pairwise combinations spotted in quintuplicate as 150pm features onto polyacrylamide coated glass slides. Cells are seeded onto these arrays and queried for their adhesion, proliferation, and marker expression using automated fluorescence microscopy in conjunction with automated cell counting and image analysis. In the first part of this thesis, this platform is used in conjunction with a mouse model of lung adenocarcinoma metastasis (KraLSL-G12D/+; p53flox/flOx) where distinct stages of metastasis are defined by characteristic cell lines derived from these mice. Hierarchical clustering of the adhesion profiles revealed conserved alterations in ECM adhesion signatures that correlate with metastasis. Additionally, they identify a role for combinations of ECM composed of fibronectin with any of galectin-3, galectin-8, or laminin. In the next part of this thesis, these molecules are investigated for their involvement in mice bearing the autochthonous tumors or in humans with lung cancer. The integrin Q3p1 is found to mediate adhesion to the fibronectin-galectin combinations in vitro and promote metastasis in vivo. Subsequently, this thesis investigates the role of carbohydrate-mediated interactions in promoting galectin adhesion. The oncofetal T-Antigen glycan motif is found to be increasingly expressed on cells with elevated metastatic potential, and is found to be the result of aberrant glycosyltransferase activity. Finally the role of galectin-3 in the metastatic niche and its presentation on bone marrow derived cells recruited to tumors is investigated. The results of this thesis suggest a role for novel phenotypic screening platforms in investigating regulation of the cancer microenvironment. Additionally, we extend these studies to the role of ECM in the epithelial-mesenchymal transition (EMT) and lay the groundwork for the development of nanoparticle-based therapeutics targeting the conserved glycan-ECM interactions. Such technologies will likely prove useful to study other disease mechanisms as well as identify novel biomarkers. / by Nathan E. Reticker-Flynn. / Ph. D. in Biomedical Engineering

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 173-174). / Since the FDA Modernization Act of 1997, recent legislation has sought to establish performance goals and consistency in the regulatory review and clearance of new medical devices. Despite these goals, regulatory review times have varied dramatically for devices seeking clearance through the 510(k) pathway; devices that are by definition "substantially equivalent" to other devices previously cleared by the FDA and currently on the market. The goal of this thesis is to investigate the factors that impact the ability for a company to obtain a 510(k) clearance in a predictable timeframe and assess the most important factors that influence time to clearance. The Medical Device industry is a dynamic and rapidly evolving sector that presents complex challenges to regulatory oversight. The 510(k) program and larger device regulatory process has endured the test of time and is well structured to address these challenges. This research focuses on the 7,481 510(k) "substantially equivalent" clearances during the period from October 1, 2007 through June 30, 2010 (FY08 - Q3FY10) in order to analyze the variables that impact clearance times. By testing various hypotheses about the most important factors I aim to provide the device community with the data and suggestions for improving confidence around a particular 510(k) submission's likely approval time. I performed subset analyses on the dataset, segmenting clearances by the type of 510(k) (traditional, abbreviated, and special), clinical category, fiscal year of clearance, use of 3rd party review, submission volume, and product diversity according to clinical category. The results show a distinct increase in clearance time from FY08 to FY10 and suggest that recent scrutiny of the FDA premarket review process may be affecting the efficiency of the Agency FDA review process for medical devices. Further, the 3rd party review program is under-utilized with only 14%, on average, of eligible devices gaining clearance through this process. Through a discussion of two specific critiques of the 510(k) process, namely a recent Government Accountability Office study and an article by the Public Citizen (a public advocacy group), the potential implications of regulatory reform is explored from both a policy and industry perspective. / by Andrew J Koert. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2003. / Includes bibliographical references. / Cardiovascular disease is the primary cause of mortality in developed nations. Current diagnostic techniques are not able to provide the information that is related to the majority of associated complications. Raman spectroscopy, which is capable of providing a detailed chemical analysis of biological tissue, has previously been shown to be a useful method for diagnosing atherosclerosis. However, widespread clinical implementation has been prohibited by the lack of optical fiber probes which can provide remote access to small diameter vessels. To this end, we have developed a new spectroscopic model and a novel Raman probe. The model interprets Raman spectra of intact tissue in terms of its morphology. The spectrally identifiable morphological structures include collagen and elastin fibers, cholesterol crystals, calcium mineralizations, adipocytes, crystals containing 3-carotene, foam cells, necrotic core, and smooth muscle cells. This model prospectively classifies atherosclerotic tissue into three categories: non-atherosclerotic, non-calcified plaque, and calcified plaque, with >94% accuracy. Furthermore, this model has the potential ability to identify the vulnerable atherosclerotic plaques whose rupture accounts for the majority of myocardial infarctions. By studying the distribution of Raman light in tissue, we have designed, constructed, and tested small diameter, high throughput optical fiber Raman probes which employ a modular micro-optical filter configuration to remove the intense background generated in the fibers. The probes have been extensively tested during peripheral vascular surgery, providing real-time disease diagnosis for the first time. These in vivo investigations demonstrate the clinical applicability of Raman spectroscopy and have also provided the first identification of vulnerable plaques with this technique. / (cont.) We have shown that plaque vulnerability is spectrally identified by determining the collagen, foam cell/necrotic core, calcification, cholesterol, and hemoglobin content of the lesion. This type of analysis may eventually lead to a diagnostic technique capable of staging atherosclerotic lesions, thereby providing a method to optimize therapeutic measures. In addition, the small diameter Raman probes have applicability for studying other diseases such as breast, oral, and gastrointestinal cancer. / by Jason Taylor Motz. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2004. / Includes bibliographical references. / This thesis integrates hardware development, data analysis, and mathematical modeling to facilitate our understanding of brain cognition. Exploration of these brain mechanisms requires both structural and functional knowledge to (i) reconstruct the spatial distribution of the activity, (ii) to estimate when these areas are activated and what is the temporal sequence of activations, and (iii)to determine how the information flows in the large-scale neural network during the execution of cognitive and/or behavioral tasks. Advanced noninvasive medical imaging modalities are able to locate brain activities at high spatial and temporal resolutions. Quantitative modeling of these data is needed to understand how large-scale distributed neuronal interactions underlying perceptual, cognitive, and behavioral functions emerge and change over time. This thesis explores hardware enhancement and novel analytical approaches to improve the spatiotemporal resolution of single (MRI) or combined (MRI/fMRI and MEG/EEG) imaging modalities. In addition, mathematical approaches for identifying large-scale neural networks and their correlation to behavioral measurements are investigated. Part I of the thesis investigates parallel MRI. New hardware and image reconstruction techniques are introduced to improve spatiotemporal resolution and to reduce image distortion in structural and functional MRI. Part II discusses the localization of MEG/EEG signals on the cortical surface using anatomical information from AMTRI, and takes advantage of the high temporal resolution of MEG/EEG measurements to study cortical oscillations in the human auditory system. Part III introduces a multivariate modeling technique to identify "nodes" and "connectivity" in a / (cont.) large-scale neural network and its correlation to behavior measurements in the human motor system. / by Fa-Hsuan Lin. / Ph.D.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2016. / "June 2016." Cataloged from PDF version of thesis. / Includes bibliographical references (pages 131-154). / Fluid volume status is a physiologic parameter that currently lacks a reliable diagnostic tool. Volume control becomes an issue during sickness and/or stress (physical and mental) in a wide range of populations. Unfortunately, current diagnostics suffer from being imprecise, invasive, and/or easily confounded and cannot unambiguously and practically inform volume status. There exists a need for a tool that can inform individuals and clinicians of fluid status in a noninvasive, rapid, and reliable manner. Drawing on the molecular sensitivity of IH nuclear magnetic resonance (NMR), we explored the ability of NMR methods to quantitate physiologic fluid volume changes. We first proved that NMR methods could detect volume changes in an animal model of dehydration. Correlation between NMR value changes in specific tissues and clinical tools used to assess dehydration validate NMR as a viable tool. We then proceeded to design and fabricate practical NMR sensors that could be easily integrated into the clinic. New methods of magnetic instrument design optimized for both field strength and spatial resolution were developed resulting in compact device prototypes with signal fidelity rivaling those of impractical commercial systems. Finally, we explored the ability of these devices to detect intravascular fluid changes during hemodialysis. Our methods and devices were able to detect intravascular blood property changes associated with blood dilution, in addition to overall fluid volume changes due to hemodialysis therapy. These results, methods, and devices provide the foundation and framework for the integration of NMR-based personalized fluid volume assessment into standard clinical practice. / by Matthew Li. / Ph. D. in Medical Engineering and Medical Physics