Thesis (M. Eng.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / Includes bibliographical references (p. 76-77). / The focus of this study was to investigate the histology of tissue formed when fetal (16-20 days gestation) and neonatal (2 days old) rat lung cells were grown in a collagen-glycosaminoglycan scaffold. This project employed a collagen-GAG scaffold specifically developed for tissue engineering and investigated the effect of this substratum on the formation of lung histotypic structures in vitro. A cell isolation procedure was developed whereby 19-days gestation type II alveolar cells reaggregated to form alveolar-like structures. The effects of selected scaffold design variables including pore diameter and degradation rate of the substratum on lung tissue regeneration were explored. Lung cell behavior revealed as the cells interact with an analog of the extracellular matrix was also examined. Differences in fetal and neonatal lung cell behavior were identified using histological analysis. Lung cells were obtained from Sprague-Dawley rats after 16-, 19-, and 20-days of gestation and at 2 days after term. These cells were seeded into type I collagen-GAG matrices, sized 8mm in diameter by 2mm in thickness. The medium used, F12K and Ham's nutrient mixture, was supplemented with 10% fetal bovine serum. A seeding density between 1 to 5 million cells per sponge sample was used. Histology studies were performed at termination periods of 2, 14, and 28 days. This paper describes the in vitro formation and long-term maintenance of alveolar-like structures from enzymatically dissociated 19-days gestation fetal rat lung cells cultured on a collagen sponge substrate as a model system for lung tissue engineering. / by Patty P. Chen. / M.Eng.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Brain injuries acquired from hemorrhage, ischemic strokes and trauma affect millions worldwide each year and often cause irreversible loss of neural tissue that disrupts vital neurological functions. Cell transplantation has traditionally been the strategy for achieving neuroregeneration but endogenous regenerative responses such as neurogenesis and neovascularization are increasingly recognized as an elegant alternative. These responses are directed toward injured tissues via chemokine signaling such as the stromal cell-derived factor-I a (SDF-1 [alpha])/CXCR4 pathway and offer the potential to replace lost neurovascular elements. Endogenous regenerative responses are, however, not fully effective in the injured brain. Two prominent barriers are the loss of chemokine expression and disappearance of stroma following tissue loss in the brain lesion, which lead to sub-optimal engagement of endogenous regenerative responses and inability of recruited cells to infiltrate the lesion. The overall goal of this thesis was therefore to develop an injectable lesion-filling matrix that could re-establish chemokine release and stroma, thereby enhancing endogenous regenerative responses. Toward this goal, we demonstrated injectable gelatin-hydroxylphenylpropionic acid (Gtn-HPA) hydrogels as an appropriate scaffolding material that was permissive for proliferation, migration and differentiation of adult neural progenitor cells (aNPCs). We also synthesized dextran sulfate/chitosan polyelectrolyte complex nanoparticles (PCN), which could encapsulate SDF-1[alpha] efficiently and sustain its release for 4 weeks. When used in an in vitro migration assay to fill a core region that was surrounded by an aNPC-laden hydrogel construct, the resulting Gtn-HPA/SDF- 1[alpha]-PCN matrix recruited aNPCs to accumulate around and migrate into the core region. When injected into the brain lesion in a rat model of intracerebral hemorrhage, Gtn-HPA/SDF-1[alpha]-PCN matrix successfully increased the migration of endogenous neuronal precursors into the injured striatum and amplified neovascularization. Gtn-HPA/SDF-l[alpha]-PCN matrix also led to a newly formed vasculature within the lesion and supported infiltration by endogenous cells that included neutrophils expressing CXCR4 and VEGF. The neutrophil infiltrate did not spread to surrounding tissue or induce necrosis and compelled further investigation for their role in the injured brain. Importantly, Gtn-HPA/SDF-l[alpha]-PCN matrix reduced brain tissue loss and improved behavioral recovery. Overall, Gtn-HPA/SDF-l[alpha]-PCN matrix offered an opportunity to enhance endogenous regenerative responses and confer benefits to the injured rat brain. / by Teck Chuan Lim. / Ph. D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 147-152). / Optical coherence tomography (OCT) allows label-free, three-dimensional imaging of tissue structure. Current implementations of OCT can either image over long depth ranges at slow imaging speeds, or over limited depth ranges at high speeds. Here, we describe a new OCT paradigm that supports simultaneous high speed and long depth range imaging through subsampling bandwidth compression. We show that this requires replacing the conventional wavelength-swept OCT laser source with a wavelength-stepped laser. First we validated this concept by modifying a slow, conventional wavelength-swept source with an intra-cavity Fabry-Perot etalon to provide a wavelength-stepped output. Using this source in an existing OCT system, we show that we can passively compress signals across a large depth range into a limited RF bandwidth. Next, to demonstrate high-speed optical domain subsampled imaging, we developed a novel wavelength-stepped laser source based on intra-cavity pulse compression/stretching; this source provided an A-line rate of ~19 MHz. We then built a polarization-based quadrature interferometer to remove imaging artifacts induced by subsampling and comple-conjugate ambiguity. A calibration and error compensation method was developed to fully remove residual artifacts in the image. We combined the high speed laser and the interferometer to demonstrate the first OCT camera-like imaging across several centimeters of depth range. The optically subsampled OCT technology developed in this work may offer a new three-dimensional camera platform for endoscopic and intraoperative imaging applications. / by Meena Siddiqui. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007. / Includes bibliographical references (leaves 152-157). / There is a pressing need in minimally-invasive surgery for novel imaging methods that can rapidly and accurately localize the surgical instrument and its target. We have developed two novel localization methods for the guidance of cardiac ablation and other minimally-invasive therapies. The first method, the Inverse Solution Guidance Algorithm (ISGA), is for the non-invasive and rapid localization of the site of origin of an arrhythmia and an ablation catheter tip from body-surface ECG signals. We have substantially developed ISGA to provide accurate catheter guidance even in the presence of significant electrical inhomogeneities, and we have evaluated the method in numerical simulations and phantom studies. Due to the rapidity of arrhythmic origin localization, ISGA may prove a highly effective means of guiding the ablative therapy of hemodynamically-unstable VT. The second method, the Bioelectrical Image Guidance (BIG) Method, is a novel algorithm for the accurate and inexpensive guidance of a wide-range of minimally-invasive surgeries, from cardiac ablation to breast cancer biopsy. / (cont.) The surgical instrument is localized within a detailed 3-D MRI or CT image by applying currents to the body surface and comparing the potentials measured at the instrument tip with potential distributions simulated prior to the surgery. We have developed and evaluated this method in numerical simulations. We have also built an experimental guidance system and tested it in a phantom model. Our results indicate that the BIG Method may one day provide an accurate and convenient means by which to guide minimally-invasive surgery within a highly detailed anatomical image. / by Maya E. Barley. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references (leaves 152-166). / Controlled-release drug-delivery systems enable efficient and defined administration of therapeutic agents to target tissues. However, ultimate drug distribution and pharmacologic effect are determined by target tissue pharmacokinetics. In muscular tissues, complex architecture that is further augmented by dynamic motion and contraction can alter the pharmacokinetics and deposition of locally delivered macromolecules. We developed a system and applied a quantitative schema to investigate the impact of controlled mechanical loads applied to skeletal and cardiac muscle tissue on intramuscular transport of locally delivered drug. In a series of studies, we examined how the interaction between architectural configuration and functional mechanics alters the transport of drugs across both physicochemical and binding properties. We correlated these pharmacokinetic effects with characteristic parameters in the physiologic range of the tissue to derive mechanistic insight into the fundamental structural and dynamic elements that underlie these effects. While previous studies have revealed the unilateral scaling of substrate uptake with mechanical influences, we elucidated an architecturally defined pharmacokinetic setpoint whereby maximal drug penetration corresponds with optimal muscle function. Our findings elucidate basic biologic design in muscle that optimizes the interface between tissue and its physical environment. The unique insights from our investigations have broad impact on current understanding of the pharmacokinetic influences of biologic form and function, and elucidate new clinical strategies for controlled release and local delivery of a wide range of therapeutic compounds to mechanically active tissues. / by Peter I-Kung Wu. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 51-58). / It has become increasingly important for companies in the biopharmaceutical industry to maximize the clinical, commercial and economic impact of their products on a global scale. In this context, both Western and Asian firms have been engaging in international merger and acquisition (M&A) activities to improve global capabilities and competiveness. The M&A activities in the sector are driven by near-term expiration of blockbuster drug patents and marketplace pricing constraints, resulting in a perceived need to attain improved economies of scale. Across the industry, one can see an increased emphasis on biotechnology medicines (or biologics). Recent large business deals that have seen Pfizer acquire Wyeth, Merck acquire Schering-Plough and Roche complete the acquisition of Genentech all have some element of positioning around the exploitation of biologics for future growth. These trends are thought to put pressure on medium-/small-sized R&D firms to come up with competitive strategies in the global biologics market. Furthermore, the biologics market faces the threat of biosimilars (biogenerics or follow-on biologics). With the advent of expected changes in the US government healthcare policy, a number of companies will be facing competition from biosimilars in the near term. Mitigating the impact of the threat of biosimilars, to some extent, is the fact that manufacturing of most non-vaccine biologics is challenging because of the structural and biological complexity of the commercial product as well as the significant differences in the manufacturing process from one product to the next. Technical capacity and the ability to respond to shifting demands are likely to be one of the critical determinants for the success of individual companies in the biologics (and biosimilars) market. To meet the perceived needs, companies have either expanded their manufacturing capacity and capabilities by building inhouse facilities or by striking long-term supply deals through contract manufacturing organizations (CMOs). Utilizing highly efficient and cost-effective overseas biologics, CMOs could be a value-added business model for Western participants. The most dramatic cost-saving strategy would likely result from outsourcing operations to firms in emerging Asian countries like India and China. However, intellectual property protection and quality control issues have been considered problematic in these countries. In this context, other relatively well developed Asian countries-Japan, South Korea (referred to as Korea) and Singapore, which have relatively strong intellectual property protection and sophisticated manufacturing environments, might be strategic partners for Western firms in the contract biomanufacturing markets. In this research study, the current biopharmaceutical industry trends and global strategies of companies in Japan, Korea and Singapore were explored. As a sub-segment of the biopharmaceutical industry, the geographical features and defining characteristics of the biologics CMO market were examined. The framework for analysis was based on an assessment of the key contributing factors: capacity, capital and cost. The potential capabilities among emerging Asian participants in the global biologics CMO markets were assessed through personal interviews with senior/executive corporate managers of Asian domestic biopharmaceutical companies (principally Japanese and Korean firms). As the results indicate, the biopharmaceutical industry of each country has been influenced both by corporate strategy and government policy. The quantitative analyses show that the current biologics CMO market in these countries is underdeveloped with a few existing participants focusing on high-tech biomanufacturing of commercial products. In addition, the macro-and micro-environment of the biotechnology industry in these countries appears to be unfavorable for the development of a global biologics CMO market. Through individual interviews, it was found that biopharmaceutical corporate managers believe that the opportunities for growth/development of an Asian emerging CMO at the global level are modest, expressing the view that their direct presence in the biologics markets as global-scale CMO participants was unlikely to take place because of financial concerns (high risk investment and profit margin sharing), absence of a global network (no proven track record) and existence of an R&D-intensive corporate culture. In conclusion, while the capabilities of large and established domestic biopharmaceutical firms in these Asian countries certainly can meet regulatory, legal and technical requirements as emerging global CMO participants, the possibilities for development of a global CMO capability in these countries are likely to be small. Strategic considerations for the possible/likely development paths of the CMO market in these Asian countries are provided. / by Soo Jin Chun. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Metastasis, the migration of cancer cells from the primary site of tumorigenesis and the subsequent invasion of secondary tissues, causes the vast majority of cancer deaths. To spread, metastatic cells dramatically rearrange their shape in complex, dynamic fashions. Genes encoding signaling proteins that regulate cell shape in normal cells are often mutated in cancer, especially in highly metastatic disease. To study these key signaling proteins in locomotion and metastasis, we develop and validate statistical methods to extract information from highthroughput morphological data from genetic screens. Our contributions fall into three major categories. 1) To define and apply robust statistical measures to identify genes regulating morphological variability. We develop and thoroughly test methods for measuring morphological variability of single-cells populations, and apply these metrics to genetic screens in yeast and fly. We further apply these techniques to subsets of genes involved in cellular processes to study genetic contributions to variability in these processes. We propose new roles for genes as suppressors or enhancers of morphological noise. We validate our findings on the basis of known gene function and network architecture. 2) To perform inference of protein signaling relationships by utilizing high-throughput morphological data. We apply machine-learning techniques to systematically identify genetic interactions between proteins on the basis of image-based data from double-knockout screens. / (cont.) Next, we focus on RhoGTPases and RhoGTPase Activating Proteins (RhoGAPs) in Drosophila., where by using basic knowledge of network architecture we apply our techniques to detect signaling relationships. 3) To integrate expression data with high-throughput morphological data to study the mechanisms for determination of cell morphology. We utilize morphological and microarray data from fly screens. By comparing expression data between control treatment conditions and treatment conditions displaying morphological phenotypes (e.g. high population variability), we identify genes and pathways correlated with this class distinction, thereby validating our previous studies and providing further insight into the determination of morphology. A key challenge in systems biology is to analyze emerging high-throughput image-based data to understand how cellular phenotypes are genetically encoded. Our work makes significant contributions to the literature on high-throughput morphological study and describes a path for future investigation. / by Oaz Nir. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 211-225). / The cambium cells of the periosteum, which are known osteoprogenitor cells, have limited suitability for clinical applications of bone tissue engineering due to their low cell number (2-5 cells thick). Extracorporeal shock waves (ESWs) have been reported to cause thickening of the cambium layer and subsequent periosteal osteogenesis. This work proposes that ESW-therapy can be used as a non-invasive, inexpensive, and rapid method for stimulating cambium cell proliferation, and investigates the use of these cells for orthotopic bone growth. The response of periosteal cells to ESWs was evaluated using two different energy densities applied to either the intact femur or tibia of the rat. Just four days after application of ESWs, there was a significant 3- to 6-fold increase in cambium cell number and thickness. The most effective treatment of those tested was high dose ESW applied to the tibia. Immunohistochemical staining of the proliferated cells demonstrated osteoblasts and bone formation (osteocalcin stain); it also demonstrated extensive vonWillebrand factor expression, which reveals the vascular contribution to the proliferating cambium layer. In a rabbit model, ESW-thickened cambium layer cells were overlaid in situ on a porous calcium phosphate scaffold. At two weeks post-surgery, there was a significant increase in all outcome variables for the ESW-treated group when compared with controls: a 4-fold increase in osteoprogenitor tissue in the scaffold upper half, a 10- fold increase in osteoprogenitor tissue above the scaffold, and a 2-fold increase in callus size. The results successfully demonstrated the efficacy of ESW-stimulated periosteum for bone tissue engineering. / by Cathal John Kearney. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Cancer is a Darwinian evolutionary process in which rounds of mutation and selection lead to increasingly fit clones. Understanding how cancers evolve and in particular how they form lethal metastases is critical to informing the design of new therapies. In the first part of this thesis, we performed whole-exome sequencing of paired endometrial cancer primaries and metastases to explore how tumors sample the genetic landscape. We find that mutations of PTEN, and TP53 occur early in the evolution of endometrial cancers, whereas BAF complex alterations occur late. We identified novel recurrent alterations in primary tumors, including mutations in the estrogen receptor cofactor NRIPJ in 12% of patients. Phylogenetic analyses in cases with multiple metastases indicated these metastases typically arose from one lineage of the primary tumor. We observed subclones within the sequenced part of the primary tumor that seeded metastases. We document extensive heterogeneity and genomic disruption across the various clinical stages in endometrial cancers. In the second part of this thesis we explore how the widespread genomic disruption observed in tumors can generate therapeutic opportunities. We use data from genome-scale shRNA screens to perform an unbiased analysis of all copynumber: gene dependency interactions. We identify a class of interactions called CYCLOPS interactions in which genomic loss of essential genes sensitizes cancer cells to their further suppression. We explore the properties of CYCLOPS genes and show that the splicing factor SF3B1 is one of them. Biochemical analyses showed that cancer cells harboring hemizygous loss of SF3BI lack a buffer of SF3BI present in cells whose SF3BI locus is intact. These data provide evidence for the utility of developing nononcogene targeted therapies as a means of advancing cancer therapeutics. / by William J. Gibson. / Ph. D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 35-38). / Background: The optimal long-term antiplatelet therapy (APT) that balances the benefit of preventing myocardial infarction (MI) with the risk of severe bleeding is unknown in patients greater than one year after drug-eluting stent (DES) placement. Methods: We modeled life expectancy (LE) using published data by building a Markov model to compare several APT strategies composed of aspirin and clopidogrel, both as monotherapy and in various clinically plausible combinations. The base case examined a 65-year old person treated with a DES then continuous aspirin plus clopidogrel (Dual-Rx) for one year without complications. We considered risk of mortality from myocardial infarction and severe bleeding. We used a lifetime horizon and projected LE without quality-adjustment. Results: In the base-case analysis, APT yielding greatest LE was a toss-up between Dual-Rx indefinitely (LE of 13.48 years), clopidogrel indefinitely (LE of 13.45 years), and aspirin indefinitely (LE of 13.42 years); of the strategies considered, no APT was least preferred (LE of 13.36 years). All parameters were varied over plausible ranges in sensitivity analyses, including the duration of future treatment with clopidogrel (base-case, life long). The choice of APT remained a toss-up unless: the annual probability of MI fell below 0.0087 (base-case, 0.013) or the relative risk of systemic bleeding exceeded 1.52 (base case, 1.00), in which case clopidogrel indefinitely was preferred; or the efficacy of clopidogrel to prevent MI fell below 0.09 (base case, 0.20) or the relative risk of clopidogrel for severe gastrointestinal hemorrhage exceeded 3.33 (base case, 2.01), in which case aspirin indefinitely was preferred. Conclusions: For patients with a drug-eluting stent placed greater than one year ago, the antiplatelet therapy which yields the greatest life expectancy is a toss-up between dual antiplatelet therapy (clopidogrel plus aspirin indefinitely), clopidogrel indefinitely, and aspirin indefinitely. However, additional research (including a clinical trial, subgroup analysis, and modeling) is needed. / by J. Stewart Evans. / S.M.