Thesis: Ph. D. in Biomedical Engineering, Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 149-166). / Accurate, timely, and effective diagnosis is the first step in appropriately treating disease. Many diseases have confusing symptoms, nonspecific biomarkers, or require invasive biopsy; these factors and others contribute to the low rates of early diagnosis for noncommunicable diseases like cancer, clotting disorders, or fibrotic diseases. A promising approach is the introduction of pro-diagnostic agents that interact with pathologic processes to produce a readout. In this vein, our group has developed responsive nanomaterials that, upon cleavage by disease-associated proteases, release reporters into the urine. This thesis sought to improve these tools by enabling the noninvasive quantification of disease-associated protease activity, deskilling complex diagnostic procedures, and developing a pipeline for extending these tools to additional diseases. Drawing inspiration from existing diagnostics, we modified our protease nanosensors to release ligand-encoded reporters compatible with clinical ELISA and paper-based lateral flow assays. These detection techniques enable simple and inexpensive quantification of our synthetic disease reporters by ensuring compatibility with existing diagnostic resources and infrastructure. To demonstrate our platform's versatility, we adapted it to a highly sensitive single molecule array (SiMoA) assay and validated disease detection in mice using 1000-fold lower doses of nanosensors. We next used disease-specific protease expression data to develop an inhalable formulation of our protease nanosensors and investigated direct tissue delivery. Finally, we built a pipeline to improve protease substrate sensitivity and specificity. Using liver fibrosis as a model, we identified target proteases, designed a peptide-screening assay, and nominated peptide candidates that efficiently classify diseased tissue. The protease nanosensors developed here provide a noninvasive, quantitative, and otherwise unavailable glimpse of the complex proteolytic milieu of disease and health. These tools form a framework for developing new diagnostics that simply, rapidly, and inexpensively identify protease-driven diseases without complex equipment or specialized personnel. / by Andrew David Warren. / Ph. D. in Biomedical Engineering

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (p. 183-198). / In this thesis we address the whole-brain tractography segmentation problem. Diffusion magnetic resonance imaging can be used to create a representation of white matter tracts in the brain via a process called tractography. Whole brain tractography outputs thousands of trajectories that each approximate a white matter fiber pathway. Our method performs automatic organization, or segmention, of these trajectories into anatomical regions and gives automatic region correspondence across subjects. Our method enables both the automatic group comparison of white matter anatomy and of its regional diffusion properties, and the creation of consistent white matter visualizations across subjects. We learn a model of common white matter structures by analyzing many registered tractography datasets simultaneously. Each trajectory is represented as a point in a high-dimensional spectral embedding space, and common structures are found by clustering in this space. By annotating the clusters with anatomical labels, we create a model that we call a high-dimensional white matter atlas. / (cont.) Our atlas creation method discovers structures corresponding to expected white matter anatomy, such as the corpus callosum, uncinate fasciculus, cingulum bundles, arcuate fasciculus, etc. We show how to extend the spectral clustering solution, stored in the atlas, using the Nystrom method to perform automatic segmentation of tractography from novel subjects. This automatic tractography segmentation gives an automatic region correspondence across subjects when all subjects are labeled using the atlas. We show the resulting automatic region correspondences, demonstrate that our clustering method is reproducible, and show that the automatically segmented regions can be used for robust measurement of fractional anisotropy. / by Lauren Jean O'Donnell. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references. / Otitis media (OM) is the most common specifically-treated childhood disease in the United States. The widespread use of systemic antibiotics against a disease of such high incidence is believed to be a driving force behind the observed increase in adaptive resistance among pathogenic bacteria in the nasopharynx. Local, sustained delivery of antimicrobial agents to the site of infection allows for higher drug concentrations and optimized release profiles than are permitted by systemic administration. Higher antimicrobial concentrations sustained for longer periods of time also allow for a faster and more complete eradication of OM bacteria (e.g., H. influenzae, S. pneumoniae), and minimize antibiotic exposure to other bacteria and natural flora in the nasopharynx and upper respiratory tract. We have developed in situ-forming hydrogels to serve as sustained release reservoirs for noninvasive trans-tympanic treatment of OM. A hydrogel that includes potentially synergistic chemical penetration enhancer (CPE) combinations and an antimicrobial sufficiently increases antimicrobial flux such that therapeutic levels can traverse the tympanic membrane (TM) within 12 hours, in vitro. We compare excised chinchilla TMs treated with ciprofloxacin (fluoroquinolone antibiotic) alone and with different combinations of sodium lauryl sulfate, limonene, and bupivacaine, with respect to resultant changes in TM electrical resistance and trans-TM ciprofloxacin flux. We also investigate the interactions of CPEs and local anesthetics with respect to both permeability enhancement and changes in nerve block potency and efficacy. Finally, we evaluate our hydrogel formulations in an in vivo chinchilla model of OM, and demonstrate early success in their ability to safely and effectively eradicate middle ear bacteria. / by Emmanuel J. Simons. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (p. 139-142). / In recent years, research in human speech communication suggested that the inventory of sound units that are observed in vowels across languages is strongly influenced by the acoustic properties of the human subglottal system. That is, there is a discrete set of possible vowel features that are constrained by the interaction of the acoustic/articulatory properties of the vowels and a small set of attributes that are observed in the subglottal region. This thesis tests the hypothesis that subglottal resonances govern vowel feature boundaries for three populations: adult speakers of English; adult speakers of Korean; and children learning English. First, we explored the relations among F1 of vowels, the first subglottal resonances (SubF1) and the feature [low] in English. For the diphthong [??], F1 peaks for vowels showed an acoustic irregularity near the speaker' s SubF1. For monophthongs, analysis of F1 frequency distributions shows a boundary between [+low] and [-low] vowels at the speakers' SubF1. Second, we studied the relations among F2 of Korean vowels, SubF2 and the feature [back], to test whether the relation between subglottal resonances and the feature boundary, demonstrated earlier for English, also can be applied to other languages. Results show that the F2 boundary between [back] and [front] vowels was placed near SubF2 in Korean, as in English. Third, we explored the development of vowel formants in relation to subglottal resonances for 10 children in the age range of 2;6-3;9 years using the database of Imbrie (2005). Results show that at the earlier ages, formant values deviated from the expected relations, but during the six month period in which the measurements were made, there was considerable movement toward the expected values. / (cont.)The transition to the expected relations appeared to occur by the age of 3 years for most of these children, in a developmental pattern that was inconsistent with an account in terms of simple anatomical increase. These three sets of observations provide evidence that subglottal resonances play a role in defining vowel feature boundaries, as predicted by Stevens' (1972) hypothesis that contrastive phonological features in human languages have arisen from quantal discontinuities in articulatory-acoustic space. / by Youngsook Jung. / Ph.D.

Thesis (Ph. D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2001. / Includes bibliographical references (p. 137-151). / Deep vein thrombosis (DVT) of the lower extremity and induced pulmonary embolism are common complications resulting from prolonged periods of bed-rest or immobilization of the limbs. One of the most effective methods of prophylaxis against DVT is external pneumatic compression (EPC). In spite of its wide acceptance as an effective means of prophylaxis, its mechanism remains poorly understood and optimal compression conditions have not been defined. Understanding the biological consequences of EPC is an important goal for optimizing the performance of compression device and providing guidance for clinical use. In the first part of this thesis, a computational model of the leg was developed to simulate hemodynamic conditions under EPC and the influence of different modes of compression were analyzed and compared. Then, a new in vitro cell culture system was developed that can be used to examine the effect of hemodynamic conditions during EPC on endothelial cell (EC) function. The biologic response was assessed through changes in cell morphology and the expression of various pro-thrombotic and anti-thrombotic factors related to EC. / (cont.) The results show that intermittent flow associated with EPC up-regulates EC fibrinolytic potential and vasomotor function. Using DNA microarray technology, the data of thrombo-regulatory factors indicates that EC gene expression shifts toward anti-thrombotic vs. pro-thrombotic under EPC. Finally, Nitric Oxide (NO), an important regulator of vasomotor and platelet functions was studied in detail under various cycles of EPC. The results show that NO production and eNOS mRNA respond differentially to modes of EPC. Further exploration using the system can potentially reveal the optimum combination of forces to better regulate thromboresistant effects desired for DVT prophylaxis. / by Guohao Dai. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. / 164 / Page 130 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 121-129). / The BMP signaling pathway is essential for embryonic development and the maintenance of tissue homeostasis. Dysregulated BMP signaling, both loss and gain-of-function, has been demonstrated in the pathogenesis of diseases including cancer, atherosclerosis, anemia and particularly hereditary disorders such as pulmonary arterial hypertension, hereditary hemorrhagic telangiectasia, and fibrodysplasia ossificans progressiva (FOP). FOP is a rare and disabling condition caused by a highly recurrent mutation in the ACVR1 gene encoding the BMP type I receptor activin-like kinase 2 (ALK2), characterized by the progressive heterotopic ossification (HO) of skeletal muscle and connective tissue leading to widespread joint immobilization, with significant morbidity and premature mortality. There are currently no effective treatments for FOP. The goal of this thesis is to develop and characterize highly selective BMP type I receptor inhibitors targeting ALK2 for the treatment of FOP. Despite the high degree of structural homology between all the BMP and TGF-[beta] type I receptors, I hypothesized that potent and selective inhibitors targeting a single BMP type I receptor, ALK2, could be developed based on a previously identified pyrazolo[1,5-a]pyrimidine core scaffold. I screened a library of pyrazolo[1,5-a]pyrimidine derivatives in a high throughout sensitive radiometric assay of BMP and TGF-[beta] type I receptor kinase activities. I identified a derivative with a unique chemical moiety (5-quinoline) that demonstrated high selectivity for ALK2, but with lower potency than the parent molecule. We synthesized a new 5-quinoline derivative with increased potency and selectivity for ALK2 over the other BMP type I receptors and greatly improved selectivity against the TGF--[beta] type I receptors. I used this highly selective compound to examine ALK2-mediated BMP signaling in vitro and demonstrated in vivo efficacy in two mouse models of HO. In a complementary approach, we generated a library of novel BMP type I receptor inhibitors based on the 2-aminopyridine core scaffold. I developed a structure activity relationship to determine the key structural elements responsible for potency and selectivity. We identified a several novel derivative compounds with improved potency and selectivity for ALK2 over the parent. We successfully used this set of derivatives to address a specific question in FOP biology, of whether ATP-competitive kinase inhibitors exert differential activity against wild-type or diverse FOP-causing ALK2 mutants. Finally, in our SAR of pyrazolopyrimidine compounds, we identified a highly potent inhibitor of both BMP and TGF-[beta] type I receptor activity. I characterized the ability of this compound to inhibit ligand-induced BMP and TGF-[beta] signaling in a variety of cell culture models, as well as inhibit the activity of individual type I receptors. We then used this compound to examine the contribution of individual BMP and TGF-[beta] receptors to signal transduction. We used the broad activity of this inhibitor to limit signaling of all endogenous BMP and TGF-[beta] type I receptors in cells, while reconstituting the activity of specific type I receptors using engineered, inhibitor-resistant mutant receptor kinases which we developed by modifying gatekeeper residues critical for interactions with inhibitor. These mutant receptor kinases demonstrated preserved basal and ligand-mediated signaling functions which were unaffected by inhibitor. These results demonstrate proof-of-principle of a system for examining the function of individual receptors of this pathway in isolation. The work presented in this thesis advances the development of novel BMP type I receptor kinase inhibitors of high selectivity and potency which could serve as important tools for the study of BMP signaling and as therapies for diseases of excessive BMP signaling such as FOP. Development of highly potent and selective inhibitors of ALK2 offers the hope of rational disease modifying therapy for the treatment of FOP. / by Agustin Humberto Mohedas. / Ph. D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2003. / Includes bibliographical references (leaves 38-40). / A recent analysis by the Tufts Center for the Study of Drug Development estimates that the cost of developing a single new chemical entity (NCE) into a successful therapeutic agent is $802 million. This figure is largely dependent on the expense of investigating NCEs that ultimately fail to be approved for use: between 70 - 90% of NCEs do not achieve New Drug Application (NDA) approval, and many of these failures are identified during the later, more costly phases of drug development. The exponential growth in the number of putative NCEs as a result of combinatorial chemistry and high-throughput screening has only confounded this problem by significantly increasing the number of early-phase NCEs under consideration for further costly development in human clinical trials. It is widely agreed upon that there are 3 major categories of reasons for drug failure: safety (toxicity), efficacy, and economics. This thesis is concerned with developing a Bayesian domain-knowledge probabilistic model (called Pharminator) to address the first two of these categories, with a goal of predicting clinical success of an NCE. Pharmacoeconomic modeling is a vastly different domain compared to Pharminator's clinical trial domain, and is beyond the scope of this thesis. While several clinical predictive models have been described in the literature over the past 10 years, the ongoing costly failure rate in drug development warrants developing more reliable predictors of NCE clinical success. The number of NDA approvals in 2002 fell to a 5-year low of 18, compared to 30, 35, 27, and 24 in 1998, 1999, 2000, and 2001 respectively, despite rapidly increasing numbers of NCEs as a result of high-throughput screening and combinatorial chemistry. / (cont.) Therefore, previous decision models have had no apparent impact on this problem. The Pharminator model combines knowledge of drug development logistics, existing data on NCE attrition rates, and Bayesian decision theory in a manner that may improve upon the performance of previously described models. The product of this model is an application to be used by drug development teams at the Phase I/Phase IIa time point for a given NCE that has passed the FDA Investigational New Drug (IND) screening process. The users are prompted to answer several key questions about the NCE. and conditional probability The tables are to be used in the model, as well as the observed data upon which prediction will be made. The output is a numeric and graphical (distribution plot) report of the prior and posterior probability distributions for clinical success, safety and efficacy. The application is demonstrated on one fictional agent and one real agent designed to demonstrate key behaviors of the model. Retrospective and prospective testing and validation will continue beyond the completion of this thesis in order to optimize the performance of this model. / by Asher Daniel Schachter. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 137-154). / Neovascularization is an essential process to repair ischemic tissues following myocardial infarction, stroke, diabetic complications, or transplant procedures. Blood vessels are generated by distinct vasculogenic and angiogenic processes. Although multiple proangiogenic factors have been identified, limited success has been achieved translating these as clinical therapeutics. Furthermore, recent studies have shown that vasculogenesis contributes to adult neovascularization in multiple settings. Harnessing the vasculogenic potential of embryonic stem cells is an emerging concept to generate neovasculature. The differentiation of embryonic stem cells into endothelium has been well documented, however most studies focus on genetic or chemokine regulation. Limited information exists which implicates the role of the extracellular microenvironment in stem cell differentiation. Heparan sulfate glycosaminoglycans (HSGAG) are a crucial part of the dynamic extracellular matrix and have been shown to regulate multiple signaling cascades, including vasculogenic specific growth factors VEGF and FGF. The goal of this thesis is to elucidate the role of HSGAG in vasculogenesis. An embryonic stem cell embryoid body model was used to establish the necessity of sulfated HSGAG for endothelial differentiation. We identified that the chemical composition of HSGAG sulfation patterns change with differentiation. Perturbation of HSGAG structure by chemical, enzymatic, or genetic modification effectively inhibited vasculogenesis. Genetic silencing of HSGAG modifying enzyme, N-deacetylase/N-sulfotransferase-1, translated to inhibition of HSGAG sulfation and resulted in impaired blood vessel development in zebrafish embryos. Interestingly, vessel formation in both embryonic stem cell and zebrafish models was restored by the addition of exogenous HSGAG, opening the door for engineering glyco-based microenvironments for controlling vascular development. To explore novel mechanisms of vasculogenesis modulated by HSGAG perturbation, we performed a global transcriptome analysis of N-deacetylase/N-sulfotransferase-1 mutant zebrafish embryos. Several novel pathways were identified that regulate vascular differentiation, including Foxo3A and Insulin-Like Growth Factor (IGF) pathways. We explored the role of IGFs in vasculogenesis specifically and determined for the first time that IGF1 and IGF2 promote mesoderm and endothelial differentiation, mediated through HIFl[alpha] stabilization, in embryonic stem cells. In summary, we've identified several mechanisms by which HSGAG regulate neovascularization, laying the groundwork for incorporating HSGAG in strategies for ischemic tissue regeneration. / by Stephanie Marie Piecewicz. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, June 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 170-192). / Spinal cord injury (SCI) results in sudden life-altering paralysis with chronic medical consequences. Although no clinical therapy is currently available to reverse paralysis, a number of biomacromolecule drug candidates in the form of proteins, enzymes and monoclonal antibodies have demonstrated restoration of function in studies with SC animal models. However, the inability to address drug formulation stability issues and overcome delivery barriers has limited the clinical translation of these promising drugs. To address this inadequacy, we designed a versatile injectable hydrogel platform and investigated its utility for local delivery of biomacromolecules to SCI contusion lesions. To develop this hydrogel platform we synthesized a library of different tri-thiol-functionalized ethoxylated polyol esters (TEPE) and combined these entities with PEG diacrylates (PEGDA) of various molecular weights to form crosslinked materials with diverse physiochemical properties using Michael addition thiol-ene chemistry. This hydrogel platform afforded unprecedented temporal control over both material degradation and the triphasic release of model biomacromolecule drugs over a 5 to 35 day period. Favorably, these materials display fast and controllable gelation kinetics under physiological conditions as well as non-swelling hydrolytic degradation profiles making them amendable for use in volume constrained anatomical sites within the spinal cord. Many of the biomacromolecule drugs of interest for SCI are complex and fragile making them susceptible to aggregation, denaturation and loss of activity during biomaterial encapsulation and controlled release. We devised a strategy to improve the long-term functional stability of biomacromolecules within hydrogels by covalently incorporating trehalose, a non-reducing disaccharide, into hydrogel networks by reacting trehalose diacrylate monomers with TEPEs and PEGDA. The covalent incorporation of trehalose within hydrogels afforded prolonged stabilization and controlled release of model enzymes in vitro and in vivo via a proposed mechanism of strong and ordered hydrogen bonding interactions. There is currently limited information pertaining to the performance of hydrogel therapies in non-penetrating contusive SCI, which represents the dominant injury mode observed clinically. Therefore, in the final part of this thesis we evaluated biomacromolecule drug delivery outcomes following intrathecal and intraparenchymal injection of hydrogel within rat thoracic SCI contusion lesions. Intraparenchymal hydrogel injection but not intrathecal administration afforded prolonged release of biomacromolecules locally within spinal cord parenchymal tissue over a two week period. Using a fluorescently labeled model drug, FITC-Dextran, we observed localized diffusion of drug within the neuropil as well as evenly distributed punctated deposits within the extracellular space that appeared to drain into local perivascular spaces. Overall, this work validates novel strategies for improved localization, temporal delivery and long term functional stability of promising biomacromolecule drug candidates in SCI. / by Timothy Mark O'Shea. / Ph. D.

Thesis (S.M.)--Harvard-MIT Program in Health Sciences and Technology, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 88-92). / Patient-centered, disease-focused nonprofits are playing an increasingly prominent role in accelerating the development of new diagnostics, drugs, and therapies. They are engaging in a variety of complex venture philanthropic activities as they seek to bridge the "valley of death" gap between basic and clinical research. Examples of such activities include developing preclinical research tools, supporting clinical trials infrastructure, and investing in private biotechnology companies. In this thesis, 1: 1) quantify the financial contribution of US-based nonprofits to biomedical research and development (R&D) and the allocation to therapeutic areas; and 2) propose a framework for understanding the core functions of biomedical venture philanthropies. I find that US-based nonprofits contributed $3.7 billion to biomedical R&D in 2011, and that within certain disease areas nonprofit spending is comparable to or exceeds spending by the National Institutes of Health (NIH). I catalogue nonprofit activities and place them in a framework of five core functions: bridging gaps, enabling research, directing pipelines, informing stakeholders, and shaping markets. I present several case studies via this framework, discuss opportunities, and point out challenges such as a lack of conflict of interest standards. Methods included recording and analyzing publically available financial data from over 400 biomedical nonprofits, and conducting a series of in depth interviews with nonprofit executives and other related professionals. / by Jordanna Polis Schutz. / S.M.